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Recruiting Mechanism and Functional Role of a Third Metal Ion in the Enzymatic Activity of 5′ Structure-Specific Nucleases
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2020, 142, 6, 2823–2834
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https://doi.org/10.1021/jacs.9b10656
Published January 15, 2020

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Abstract

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Enzymes of the 5′ structure-specific nuclease family are crucial for DNA repair, replication, and recombination. One such enzyme is the human exonuclease 1 (hExo1) metalloenzyme, which cleaves DNA strands, acting primarily as a processive 5′-3′ exonuclease and secondarily as a 5′-flap endonuclease. Recently, in crystallo reaction intermediates have elucidated how hExo1 exerts hydrolysis of DNA phosphodiester bonds. These hExo1 structures show a third metal ion intermittently bound close to the two-metal-ion active site, to which recessed ends or 5′-flap substrates bind. Evidence of this third ion has been observed in several nucleic-acid-processing metalloenzymes. However, there is still debate over what triggers the (un)binding of this transient third ion during catalysis and whether this ion has a catalytic function. Using extended molecular dynamics and enhanced sampling free-energy simulations, we observed that the carboxyl side chain of Glu89 (located along the arch motif in hExo1) flips frequently from the reactant state to the product state. The conformational flipping of Glu89 allows one metal ion to be recruited from the bulk and promptly positioned near the catalytic center. This is in line with the structural evidence. Additionally, our simulations show that the third metal ion assists the departure, through the mobile arch, of the nucleotide monophosphate product from the catalytic site. Structural comparisons of nuclease enzymes suggest that this Glu(Asp)-mediated mechanism for third ion recruitment and nucleic acid hydrolysis may be shared by other 5′ structure-specific nucleases.

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Introduction

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Recent structural data have shown the recurring presence of a third metal ion close to the two-metal-ion center of nucleic-acid-processing enzymes. (1−4) This third ion has been captured during different stages of catalysis of vital enzymatic reactions involved in DNA repair, recombination, and replication processes. (5−9) These reactions are often related to cancer progression. (10−13) Indeed, over the past few years, a third ion has been observed, or hypothesized, close to the two-metal-ion catalytic site of polymerases, (4) nucleases, (14−18) and topoisomerases. (19−21) This suggests that the third metal ion may be actively involved in catalysis. (1,4,22) However, there is debate over how this ion is recruited from the bulk and transiently binds the enzyme and how it could play a role in catalysis.
In this context, recent time-resolved in crystallo reaction intermediates (23) have elucidated how human exonuclease 1 (hExo1) exerts its catalytic function, with sequential structures showing how the enzyme/DNA complex evolves during catalysis. hExo1 is an essential hydrolytic enzyme for genome maintenance. Belonging to the RAD2/XPG family, (24−30) hExo1 is a 5′ structure-specific metallonuclease, which carries out a primary exonucleolytic activity on the 5′ recessed end and a secondary endonucleolytic cleavage on 5′-flap of the substrate DNA strand. (31,32)
The structures show the enzymatic mechanism for DNA hydrolysis in hExo1, which starts in the precatalytic state with the intact double-strand DNA (dsDNA) recognized and bound (tethered) to the helix-two-turn-helix (H2TH) motif and to a monovalent (K+/Na+) ion state in hExo1 (Figure 1). Then catalysis begins with formation of the assembled active site, where the dsDNA bifurcates into the 5′ and 3′ single strands (i.e., the dsDNA “junction”). At this point, the scissile phosphate of the processed single 5′ strand is properly located at the reactive metal center at the N-terminal domain. In this state, the catalytic residues Lys85 and Arg92 interact with the scissile phosphate, (33) after a rotation (clamped conformation) of the mobile helical arch formed by two α-helices (α4-α5) located near the junction. Here, the side chain of the guide residues Tyr32 and His36 are also rotated (Figure 1). These structural motifs contribute to the “threading mechanism”, whereby the 5′-flap DNA passes through the helical arch. (34) In this way, basic residues steer the phosphate of the 5′ strand, promoting the proper location for hydrolysis of the scissile phosphate on top of the two catalytic ions, as expected for the recognized two-metal-ion mechanism. (34−40)

Figure 1

Figure 1. Catalytic domain of hExo1 in complex with DNA substrate and the two catalytic metal ions (PDB ID 5V06). (Left) hExo1 in cartoon and with colors for different structural motifs. (Right) Closer view of the active site. Three metal ions (MgA, MgB, MgC) are in orange, nucleophilic water molecule (Nu) is in red, two guide residues (Tyr32, His36) in yellow, and residues of the catalytic pocket (Gly2, Asp30, Asp78, Asp152, Asp171, Asp173) are in cyan. Scissile phosphate is correctly positioned for the nucleophilic attack, and MgC is coordinated by the 5′ terminal phosphate.

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At this point, hydrolysis of the 5′ recessed end or the 5′-flap DNA substrate occurs. (41−43) In hExo1, this is proposed to be favored by a structured network of interactions involving Arg95, Arg96, Arg121, and Asn124, which are located along the mobile arch. In particular, a key role in phosphate steering is proposed for the Arg96 and Asn124, which interact with the phosphate next to the scissile one (i.e., the terminal 5′ phosphate in the 5′ recessed-end substrate). This is similar to what has been observed in the enzyme hFEN1. (34) After DNA hydrolysis, the nucleotide monophosphate group can leave the active site, with the “free” enzyme that now has Tyr32 and His36 back in their initial conformation.
Remarkably, these structural data show a transient third metal ion that is intermittently located close to the catalytic site during exonuclease catalysis (Scheme 1). This suggests that the transient third ion may play a role in substrate hydrolysis and/or leaving group departure. (44) Indeed, during hExo1 catalysis, four different structures of the assembled active site were solved in the presence of a second-shell and solvent-exposed third metal ion, preserved close to the two-metal ion center (Figure S1). This ion is not found in the structure of the cleaved product, demonstrating its transient nature during catalysis. (1,23)

Scheme 1

Scheme 1. Schematic Representation of the Motion of the Transient Third Metal Ion, Which We Found to Be Intermittently Recruited/Released by Glu89 (switching its inner/outer conformations) during Exonuclease Catalysisa
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aThis structural evidence suggests that the transient third ion may be crucial for substrate hydrolysis and/or leaving group departure.

Here, we used force-field-based molecular dynamic (MD) simulations coupled to enhanced sampling free-energy calculations to investigate the role of the third ion during hExo1 catalysis. We compared several systems of wild-type and mutated hExo1 from the reactant state to the product state. We found that the second-shell and conserved Glu89 residue selects, recruits, and places the third ion close to the two-metal-ion catalytic site. We show that this negatively charged residue is functional and conserved among Exo1 belonging to different organisms and that this enzymatic mechanism is likely shared by other nucleases.

Results

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Glu89 Selects, Recruits, and Places a Third Ion Close to the Catalytic Site of hExo1

First, we ran multiple unbiased force-field-based molecular dynamic (MD) simulations (∼700 ns in total) of the wild-type (wt) reactant state (Figure S2). We considered the enzyme in the reactive state, replacing the nonreactive Mn ions in the prereactive crystal structure (PDB ID 5V06) (23) with native Mg ions. (31) Notably, this structure features a third Mg ion (MgC) close to the two-metal-ion catalytic site. This ion is bound to the 5′ phosphate of the processed strand.
Residues Tyr32 and His36 are thought to guide substrate binding through interactions with their flexible side chain. (23) Located above the reaction center, these residues maintain the crystallographic conformation, in which their side chain points “down” toward the two catalytic ions MgA and MgB beneath it (Figure S3). These ions maintain an octahedral coordination (45) throughout the simulated time scale (i.e., 360 ns). As a result, the nucleophilic water molecule remains optimally positioned for nucleophilic attack, sitting on top of MgA, in front of the substrate’s scissile phosphodiester bond, at 3.88 ± 0.11 Å (Figure S3).
Despite the overall stability of the protein–DNA complex, the side chains at the base of the mobile arch (i.e., at the gateway) sample different conformations. In particular, the initial clamped conformation shows some flexibility over time, with Lys85 moving slightly further away from the scissile phosphate (∼6 Å vs 4 Å in PDB ID 5V06 see Figure S4). Moreover, we observed an amplified mobility of MgC, reflected in its enhanced RMSD of 1.63 ± 0.42 Å, as compared to the two catalytic ions and its anchor 5′ nucleotide, which are highly stable with an RMSD of 0.54 ± 0.17 and 0.82 ± 0.18 Å, respectively. Indeed, MgC moves from its initial position, forming new interactions with the flexible Glu89 carboxylate along the α-helix of the gateway. As a result, the mobile MgC alternates between a bidentate and a monodentate binding coordination with the 5′ phosphate and Glu89 (Figure S5), always maintaining an octahedral shell. In this regard, the motions of the Glu89 side chain are described by the pseudodihedral angle ϕ (taken along the N, Cα, Cδ, Cγ bonds; see Figure S5), which oscillates from ∼120° to ∼−10° (Figure S5), never reaching a conformation of the product state (−36°, PDB ID 5V0A). (23) Concomitantly, we observed that Arg96, which is located along the mobile arch, shortens its distance from the 5′ phosphate, reaching a value of ∼4.5 ± 0.19 Å (compared to an initial distance of 6 Å in PDB ID 5V06). This distance corresponds well to the value in the crystal structure of the product state (5.1 Å, PDB ID 5V0A) (Figure S6). Taken together, these results support the hypothesis of a gradual removal and possible departure of the third ion during catalysis, as suggested by comparing the structural data of the reactant (MgC present, PDB ID 5V06) and product states (MgC missing, PDB ID 5V0A).
To further characterize the structural impact of MgC bound close to the two-metal-ion catalytic site in the reactant state, we manually removed it and ran multiple simulations of the solvated system (∼1 μs, in total). This protein–DNA complex showed no major difference in the overall backbone stability (see Figure S2) compared to the three-ion reactant state (see above). However, in these replications, the catalytic residues Lys85 and Arg92 maintained their native H-bond pattern with the scissile phosphate (as in the crystallographic structure) for the whole simulation. Thus, the catalytic residue Lys85 behaved differently to the three-ion system. Also, in these simulations, at times Glu89 interacted transiently with Arg93, located along the mobile arch. This interaction was observed only when Glu89 adopted the outer conformations, in the absence of MgC. Concurrently, Arg96 maintained its starting orientation and never interacted with 5′ phosphate (Figure S6). Again, this differs from the observations in the presence of MgC (see previous paragraph).
Intriguingly, during the initial equilibration phase (∼10 ns), the side chain of Glu89 undergoes a marked rearrangement from the initial inner conformation to an outer conformation toward the bulk water. This rotation is captured well by the Glu89 pseudodihedral angle ϕ, which changes from positive values of ∼+100° (inner) to negative values of ∼−95° (outer) (Figure S7). Importantly, in this new solvent-exposed conformation, we observed that Glu89 carboxylate transiently recruits and binds monovalent ions from the bulk (either K+ or Na+, freely diffusing in solution). This result is also confirmed by the radial distribution function, g(r), calculated as the variation of the density of the ions from the center of mass of the 5′ phosphate group (Figure 2), which displays two peaks at ∼2.7 and ∼3.3 Å.

Figure 2

Figure 2. Radial distribution function, g(r), calculated for ions around 10 Å from the center of mass of the 5′ phosphate group. Plot shows the presence of ions ∼3 Å from the 5′ phosphate group for the RS2M system. In this system, a K+ ion approached the negatively charged group. For the RSGlu89Ala and PSGlu89Ala systems, there are no ions within ∼5.5 Å of the 5′ phosphate, as indicated by the g(r) values of ∼0. In the upper right corner, the 5′ phosphate group and Glu89Ala residues are shown in licorice (taken from the PSGlu89Ala simulations).

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After metal binding (∼10 ns), Glu89 flips back into its initial inner conformation, carrying the coordinated metal closer to the 5′ phosphate, at ∼3.25 Å. This metal ion is thus brought into a very similar location compared to MgC in the crystal structure of the prereactive state (PDB ID 5V06). After a few hundred nanoseconds (e.g., ∼200 ns for K+ ion), the third metal ion departs spontaneously from the catalytic site. Glu89 then flips again into its outer conformation toward the bulk. These unprompted metal binding and release events, synchronized with the flipping of the Glu89 side chain, were observed multiple times in our extended simulations (Figure S7). This indicates that Glu89 may recruit a third metal ion, bringing it transiently closer to the catalytic site. (46)
To further test Glu89’s role as metal-ion recruiter, we ran multiple MD simulations (∼1 μs in total), inserting the Glu89Ala mutation in the reactant state in the absence of MgC. The overall stability of the enzyme–DNA complex was maintained, with a low RMSD value of 1.27 ± 0.15 Å (see Figure S2). The prereactive state at the active site was also maintained throughout the simulations, that is, the two catalytic metal ions, MgA and MgB, stably maintained their internuclear distance. The nucleophilic water molecule remained properly positioned in front of the scissile phosphodiester bond and the catalytic residues. Finally, Lys85 and Arg92 maintained their initial interaction network with the scissile phosphate. Notably, in this mutated system, we did not see any ion approaching the terminal 5′ phosphate from the bulk solvent. This result is supported by g(r), which confirms that no ion is located within ∼6 Å of the center of mass of the 5′ phosphate group (Figure 2), further suggesting that Glu89 recruits a third metal ion from the bulk.

Third Ion Promotes Leaving Group Departure after DNA Hydrolysis

Here, we used MD simulations (∼1 μs in total) of the products of the wild-type (wt) native state. Thus, we inserted a native aspartate at the Asp225Ala mutation and replaced Mn with native Mg ions in the postreactive crystal structure (PDB ID 5V0A). Notably, at this catalytic stage, the DNA’s processed strand is enzymatically cleaved, with the consequent generation of the leaving group, i.e., the adenosine 5′-monophosphate (AMP) nucleotide, which is now detached from the newly formed 5′ recessed-end substrate. Importantly, the third ion is not present at the catalytic active site in the crystallographic structure.
In the postreactive crystal structure, the Glu89 side chain adopted an intermediate conformation (ϕ of Glu89 was −36°, Figure S1) between the inner (∼+100°) and the outer (∼−95°) conformations. Then during the MD simulations, the Glu89 side chain stably adopted an outer conformation (ϕ of Glu89 becomes ∼−100°). However, after ∼50 ns, we observed the unprompted approach of a third Mg2+ ion from the bulk water (Mgbulk), which came close to the Glu89 side chain. This transient ion thus reached a position close to the 5′-monophosphate of the AMP at a distance of ∼3.3 Å, which was equivalent to that in the prereactive simulations and crystal structure (PDB ID 5V06). In this position, the third metal ion interacted with the 5′ phosphate and Glu89 for the remaining simulation time (Figure 3A). During this event, the two catalytic metal ions moved apart slowly, reaching a distance of ∼5.5 Å (compared to 3.9 Å in the starting model PDB ID 5V0A). The drifting of the internuclear two-metal-ion distance was coupled to a shift in the leaving AMP. This is described well by the collective variable CV1, which measures the distance between the center of mass (COM) of the heavy atoms of AMP and the COM of the Cα of the aspartates in the first coordination shell of the two-metal-ion center (i.e., Asp152, Asp171, Asp173) (Figure S8A). During our simulations, CV1 increased by ∼2 Å, from 9 to 11 Å, reflecting the partial exit of the leaving AMP (Figure S8B and S8C). Moreover, His36, which was initially in the down orientation, immediately rotated into the up conformation (Figure S9), forming a π–π interaction with AMP. This interaction helps the initial displacement of the leaving AMP. Notably, the up conformation of His36 was found in the structure of the enzyme after AMP departure (PDB ID 5V0B), (23) which further suggests the need of this rotation during leaving group release. We also noted a gradual and slight opening in the gateway region at the bottom of the mobile arch, which however maintained an ordered secondary structure (Figure S10). This event is described well by the increase of ∼1 Å of the two distances d1 and d2, which reflect the opening of the α4/α5 interhelix passage (calculated using the Cα of Glu89 and Arg92 along the α4 helix and the Cα of Asn124 and Ile125, located at the bottom of α5 helix; see Figure 4). The probability density function of ϕ, calculated for two states (Mgbulk > 4 Å or Mgbulk < 4 Å from the 5′ phosphate), shows the relative peaks of the two conformations assumed by Glu89, i.e., inner and outer (Figure 3B).

Figure 3

Figure 3. (A) Distance (dMG in yellow) between the third Mg2+ ion, from the bulk (Mgbulk), and the phosphorus of the 5′ phosphate group of AMP. Inset, representation (snapshot from the PS2M simulations) of Mgbulk approaching the terminal 5′ phosphate as well as the pseudo-dihedral angle ϕ of Glu89 side chain (defined by the N–Cα–Cδ−-Cγ atoms). (B) Probability density of the pseudo-dihedral angle ϕ in Glu89 during the simulation: (blue) probability density for dMG values > 4 Å shows the outer conformation as the most populated; (red) probability density for dMG values < 4 Å shows the inner conformation is the most populated.

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Figure 4

Figure 4. (A) Graphic representation of d1 and d2 distances (PDB ID 5V0A). (B) Probability density of the distances d1 and d2 calculated during simulations of the systems PS2M (in red), PS3M (in orange), and PSGlu89Ala (in green).

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In the products, Arg96 invariantly interacts with the 5′ phosphate in AMP (Figure S6), as also reported for the reactant state simulations in the presence of the MgC. Moreover, after ∼450 ns, we noted a second arginine residue located along the α4 helix, Arg93, which approached the same 5′ phosphate of the AMP, at ∼5 Å. These interactions thus favor the positional shift and partial departure of the AMP from the catalytic site, as indicated by CV1, with consequent destabilization of the two-metal-ion site. We compared these results with an additional postreactive model, which initially contained a third ion at the catalytic site (∼1 μs in total, see SI). These simulations confirmed the enhanced instability of the prone-to-escape leaving group with a partial shift from its starting position during the simulations (Figure S8). Taken together, these results suggest that complete leaving group departure is eventually expected, although this would require longer simulations and would also likely implicate the overtaking of an energetic barrier. (47)
As with the reactant state (see above), we also ran simulations (∼1 μs in total) in the product state of a system with the Glu89Ala mutation in the absence of MgC. The overall stability of the enzyme–DNA complex was maintained (Figure S2). Importantly, in the absence of the Glu89 recruiter, no ion approached the 5′ phosphate of the leaving group, as shown by g(r) (Figure 2). As a result, the leaving group also showed higher stability in its position, as highlighted by the low RMSD value of 1.71 ± 0.18 Å. Moreover, Arg93 almost never interacted with the 5′-phosphate of AMP, and Arg96 stably maintained its initial interaction with AMP. In addition, we did not observe any opening in the gateway region, with the distances d1 and d2 remaining unchanged during the simulations (Figure 4). These results support the hypothesis that Glu89 recruits MgC before (or during) DNA cleavage. In return, MgC seems to promote the release of the leaving group after the chemical step for phosphodiester bond hydrolysis, acting as a shuttle for the AMP departure. (47)

Energetics of the Glu89 Flipping and Leaving Group Departure via Metadynamics Simulations

To sample and determine the semiquantitative energetics of the inner ↔ outer conformational switch of Glu89, we used the pseudodihedral angle ϕ as the collective variable to run multiple metadynamics simulations, with and without the third metal ion at the catalytic site, in the reactant and product states (for a total of ∼920 ns).
In the reactant state with the third ion, Glu89 tended to adopt inner conformations located in an energy minimum at ϕ ≈ 70°, while outer conformations were not visited due to their high energy (Figure 5, red profile). In the inner conformation, MgC stayed close to the reactive center. However, in the absence of the third metal ion, Glu89 could be found in two isoenergetic minima, i.e., inner and outer conformations, separated by a barrier of only ∼3 kcal mol–1 (Figure 5, blue profile). This explains the fact that both Glu89 conformations were similarly populated in our unbiased MD simulations of the system without the third metal ion.

Figure 5

Figure 5. (Bottom) Free energy surface obtained through well-tempered metadynamics simulations for RS3M (red), RS2M (blue), PS2M (green), and PS3M (light purple) systems. Results show three conformations (outer, intermediate, inner). (Top) Graphic representations, taken from PS3M simulaitons, of the three conformations are shown in licorice.

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In the product state, regardless of the presence or absence of the third ion, Glu89 visited both the inner and the outer conformations (Figure 5, green and light purple profiles). The conformational switch showed a barrier of ∼4.5 kcal mol–1, with or without MgC. We also located a metastable conformation of Glu89 bound to MgC, at ϕ ≈ −40°, in which the glutamate’s side chain adopted an intermediate orientation between the two minima (inner and outer). Interestingly, this metastable state corresponds well to the crystallographic conformation (ϕ = −36°, PDB ID 5V0A) in which MgC is missing. This is likely because, at this point, Glu89 is already solvent exposed.
Then we evaluated possible pathways and energetics for the release of the leaving group from hExo1 in the presence and absence of MgC. We used confined metadynamics, (48) which enhances the sampling of transversal and often slow degrees of freedom of complex (rare) events, such as the exit of the adenosine monophosphate (AMP) nucleotide from the catalytic site (see SI for further information). Here, the collective variable was CV1 (Figure 6), which captures the degree of departure of the leaving AMP from the reactive site (see definition of CV1, above).

Figure 6

Figure 6. Free energy surface obtained through confined well-tempered metadynamics simulations for PS2M (blue) and PS3M (yellow) systems. (Top) Schematic representation of the CV1, exemplified using a snapshot from PS3M simulations. It represents the distance between the center of mass (COM) of the heavy atoms of the nucleotide leaving group and the COM of the Cα of the aspartates (Asp152, Asp171, Asp173) in the first coordination shell of MgA, MgB. Two different minima, at 8.8 and 12.4 Å, agree with the MD results, in which a partial exit of the leaving group (CV1 ≈ 12.4 Å) was seen only in the presence of MgC (Figure S8).

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In the presence of MgC, AMP fell into a minimum at CV1 = ∼12 Å, showing that the leaving group is already shifted out of the catalytic site (CV1 = ∼8.5 Å in the uncleaved prereactive state). This is in line with the plain MD simulations, where it was only in the presence of the third metal ion that the leaving group partially exited from the active site, increasing the CV1 value of ∼2 Å, reaching a value of ∼12 Å (Figure S8). At this point, to allow the full departure of the leaving group, the freed AMP stayed complexed with MgC. In this way, the AMP/MgC complex exited from the catalytic site, passing through the aperture under the mobile arch formed only when MgC is present (due to MgC-mediated AMP drifting out from the catalytic center). At this point, the Glu89 sampled the inner and outer conformations until the leaving group overcame the gateway region. At this point, Glu89 stably adopted the inner conformation, in agreement with the crystallographic structure of the complex after release of the leaving group (PDB ID 5V0B). The physical step for AMP/MgC unbinding occurred with a barrier of ∼16.5 kcal/mol (Figure 6).
In the absence of the third ion, the energy barrier for the overall unbinding process was much higher at ∼35 kcal/mol. Indeed, the system behaved quite differently. In the initial configuration, the leaving AMP fell into an energy minimum where it was poorly solvated, at CV1= ∼9 Å (compared to the case where it was solvated and complexed with MgC, CV1= ∼12 Å, Figure 6). From this state, the exit of the AMP alone had to overcome a first barrier of ∼20 kcal/mol, which is already higher than the barrier in the presence of MgC. Then the exit path showed metastable states (relative energy minima at CV1 = ∼13.5 Å and CV1 = ∼17 Å, Figure 6) where the leaving AMP seemed to be transiently trapped by the formation of short-lived interactions with the enzyme. This may slow the AMP unbinding kinetics. Notably, these transient interactions were not formed for the AMP/MgC leaving complex. It is also worth noticing that in both systems PS3M and PS2M we observed the exit of MgB from the catalytic pocket, which occurred concertedly with the exit of the AMP leaving (see Movie in SI). In detail, the MgB catalytic ion remained coordinated to the oxygen of the OH in C3 position of the sugar of the AMP leaving group. Interestingly, the concerted departure of MgB, MgC, and the leaving group AMP agrees with previous studies showing that MgB dissociates from the catalytic site ∼200 times faster compared MgA. (49) Thus, the concerted exit of both MgB and MgC is found here to promote AMP departure by stabilizing the newly formed negative charge on the leaving group after substrate hydrolysis. (47)

Discussion

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Recently, a time series of structural intermediates captured during human exonuclease1 (hExo1) catalysis has revealed the presence of a third metal ion (MgC) close to the active site. (23) Intriguingly, a transient metal ion was recently observed in a few DNA/RNA-processing enzymes. (1,2,4,50) The role of this third additional metal ion at the catalytic center is still unclear. (44) Here, we used force-field-based molecular dynamics (MD) simulations and free-energy calculations to investigate the recruiting mechanism and functional role of a third metal for hExo1 catalysis. We simulated and compared several model systems built with recent hExo1 structures of the wild-type (wt) hExo1/DNA complex in the reactant and product states with and without MgC.
During our multiple and extended MD simulations (∼6 μs in total) we first observed that Glu89 sometimes oscillated but mostly maintained its starting conformation. In this inner conformation, the Glu89 carboxylate group points toward the 5′ phosphate. However, at times and only in the absence of MgC the Glu89 carboxylate group switched its orientation, adopting outer conformations that pointed toward the bulk solvent. Free-energy calculations confirmed that in the reactant state and in the presence of MgC Glu89 tended to adopt inner conformations, which are located in an energy minimum at ϕ ≈ 70°. Outer conformations are not visited due to their high energy. However, in the absence of MgC, the inner and outer conformations become isoenergetic, with a barrier of only ∼3 kcal/mol in between.
Importantly, following this conformational switch in equilibrium MD simulations in the absence of MgC, we observed that transient monovalent ions were freely recruited from the bulk by the outer conformation of Glu89. This conformation therefore seems to act as an anchor point for (third)metal–enzyme complexation. Then Glu89 could switch back and adopt the inner conformation, bringing the bound metal ion (either K+ or Na+, from these simulations) close to the terminal 5′ phosphate (∼3.5 Å) (Figure S7). This third metal was spontaneously recruited and located in the same position as the third ion captured in the prereactive crystal structure (PDB ID 5V06). This Glu89-mediated mechanism for metal recruitment was further validated by simulations of mutated Glu89Ala systems. These simulations confirmed that in the absence of Glu89 no metal ion from the bulk was spontaneously recruited close to the catalytic center. Interestingly, the role of Glu89 in hExo1 is similar to the role previously proposed for Glu188 in Bacillus halodurans ribonuclease H (BhRNase H), where MD simulations suggested that this residue attracted a transient third ion. (46) Intriguingly, a transient third solvent-exposed cation was found close to the two-metal-ion active site of D. mobilis homing endonuclease, I-DmoI. (51)
In the unbiased MD simulations of the product state we observed the unprompted entry of the third metal ion MgC from the bulk, reconstituting the three-metal-ion system (Figure 3). This happened concomitantly to the rotation of the Glu89 side chain from outer to inner, thus destabilizing the geometry of the catalytic active site. Notably, during this process with the double-strand DNA already bound to hExo1, the enzyme maintained an ordered secondary structure of the mobile arch. After the initial DNA binding and subsequent DNA hydrolysis, the enzyme will have to position the “next” scissile bond into the active site. An ordered structure may thus favor the processivity of the exonuclease activity of this enzyme. At this stage the system evolved toward the final catalytic step, i.e., the exit of the leaving group from the catalytic site. In this system, Glu89 was free to populate the inner and outer conformations, overcoming an energy barrier of ∼4.5 kcal/mol, calculated from metadynamics simulations. In addition, we computed the energetics for the full release of the leaving group in the presence or absence of MgC using confined well-tempered metadynamic simulations. (48) The energetic barrier for AMP departure was ∼16.5 kcal/mol in the presence of MgC and ∼35 kcal/mol in its absence.
The Gibbs free energy (ΔG‡) for the overall catalytic process of hExo1 is 19.6 kcal/mol, computed using the experimental kcat for hExo1 (see SI for further information). (31) This energy value corresponds fairly well to our estimation of the free-energy barrier for the unbinding process of the leaving group in the presence of the third ion, i.e., ∼16.5 kcal/mol. The leaving group departure may therefore be rate limiting for the exonuclease catalytic process in hExo1, as already proposed for other metallonucleases (e.g., FENs, APE1, PvuII, MunI, NaeI, SfiI, EcoRI, EcoRV). (52−59)
These results suggest a mechanism where Glu89 recruits a third metal ion in the reactant state. Clearly, quantum calculations are needed to evaluate the mechanistic implications of this additional ion for the chemical step of DNA hydrolysis. (60−63) However, from these classical MD simulations, it emerges that the third ion promotes leaving group departure, acting as a shuttle for the exit of the nucleotide monophosphate product from the catalytic site. Notably, this result is in line with evidence of a third-ion-mediated leaving mechanism for pyrophosphate departure in polymerase enzymes. (47)
To further test this mechanistic hypothesis and investigate whether this enzymatic strategy is shared by other nucleases, we performed sequence alignments via the Needleman–Wunsch algorithm (64) using 10 different eukaryotic species of Exo1 (see SI for more information). We found that the Glu89 is fully conserved among these enzymes, as for those residues forming the reaction center, and second-shell residues like Lys85 and Arg92, and the guide residues Tyr32 and His36 (Figure S11). This suggests that Glu89 is an integral part of the enzymatic machinery for efficient catalysis in Exo1.
We also performed structural comparisons using recent crystallographic structures of additional nucleases and identified a shared spatial localization in these enzymes of an acidic residue (Glu/Asp), in analogy to Glu89 in hExo1. In such enzymes, in fact, we always identified the presence of a Glu/Asp residue located in a second-shell sphere cantered on the two-metal-ion active site. This acidic residue is always situated in a solvent-accessible position (thus able to recruit ions from the bulk), being strategically located on the side of the expected exit path for leaving group departure, with respect to the catalytic center. For example, human ExoG, (65,66) which is 5′ metallo-exonuclease enzyme cocrystallized in complex with the DNA substrate, has a glutamate (Glu317) residue located near the terminal 5′ phosphate. Here, Glu317 resides in a solvent-exposed area. Notably, Glu317 can assume different orientations in the available crystals (see PDB ID 5T5C vs PDB ID 5T40), (65) which suggests that this glutamate may act as a recruiter of metal ions in the same way as for Glu89 in hExo1 (Figure 7A). Another case is the human λ-Exonuclease, (67,68) where Glu36 is located close to the 5′ phosphate of the DNA substrate (Figure 7B). Here, too, it has been hypothesized that a third metal ion may transiently bind close to the two-metal-ion site, likely aiding the leaving group departure. (49) A further example is RecJ nuclease, (69) where Asp158 is solvent exposed and close to the active site in a similar position as Glu89 in hExo1 (Figure 7C).

Figure 7

Figure 7. Close views of the active site of 5′ metallonuclease members that possess an analogous acid residue (light green) close to the two-metal-ion center (MA, MB, in orange), the active site (in cyan), and the leaving group (indicated by a dashed line). (A) Human ExoG, in which Glu317 is pointing in the inner (PDB ID 5T5C) and (A′) outer conformations (PDB ID 5T40, merged with the DNA substrate from PDB ID 5T5C). (B) Escherichia phage T5Fen (PDB ID 5HNK). (C) Human λ-Exonuclease X (PDB ID 3SM4). (D) D. radiodurans RecJ (PDB ID 5F55). (E) Sequence alignment of M. smegmatis FenA and E. phage T5Fen. Conserved acid residue (Glu/Asp) is indicated in orange.

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Then we looked at the hExo1 family member bacteriophage T5 flap endonuclease (T5 Fen, PDB ID 5HNK). (16) We identified Glu83, which is located along the mobile arch, in analogy to Glu89 in hExo1. Finally, we considered the recent high-resolution X-ray structure of M. smegmatis FenA, (17) which is a 5′ structure-specific nuclease and close homologue of phage T5Fen, and its sequence alignment with T5Fen. It suggests that Glu83 in T5Fen may correspond to Asp85 in FenA (Figure 7D and 7E). Notably, holo forms of T5Fen and FenA were crystallized in complex with three metal ions in the active site. (16,17) This further corroborates the idea that multimetal-ion catalytic sites may be necessary for nucleic-acid processing in these enzymes. Indeed, while the presence of a third metal ion, located in the vicinity of the two-metal-ion active site, is a novel aspect in polymerases and nucleases, the exact position of such additional metal ion with respect to the catalytic center can vary. (4) For example, T5FEN and FenA enzymes were solved with a third ion in a different relative position, although always in close proximity of the reactive two-metal-ion center. It is thus plausible that the third transient ion may play different roles during catalysis, according to its specific location at the catalytic center.
Another intriguing aspect is the recurring presence of second-shell positively charged residues that surround the metal-aided catalytic site in nucleic-acid-processing enzyme. (3) During our extended MD simulations, we observed the interaction of Arg93, along the helical arch, with the terminal phosphate of the substrate. We structurally aligned hExo1 (PDB ID 5V0E) (23) with hFEN1 (PDB ID 5KSE), (34) and we noted that the terminal guanidine, amide, and amine groups of Arg93, Asn124 (in hExo1), and Lys132 (in hFEN1) residues were all within a sphere of ∼3 Å and close to the 5′ phosphate in the leaving group (Figure S12). This result further suggests that Arg93, together with Arg96, may have a crucial role in 5′ phosphate steering. (34) In this respect, Arg93 is an important point of control in Exo1 poly(ADP-ribose) binding, as proved by in vitro and in vivo assays on natural Arg93Gly mutation. (70) Moreover, a common feature of different nucleic-acid-processing enzymes is a solvent-exposed and positively charged residue that interacts with the negatively charged moiety in the leaving group (e.g., Arg96–5′ phosphate). (71) Interestingly, this Arg96–5′ phosphate interaction comes in addition to an already complex architecture characterized by a number of positively charged residues surrounding the active site. Indeed, hExo1 is one of a large set of nucleic-acid-processing enzymes characterized by the recurring presence of positively charged elements in the vicinity of the reactive site. (3) These elements in hExo1 comprise the catalytic Lys85 and Arg92. These positively charged residues are thought to play a role in the phosphate steering during the threading mechanism. These residues therefore seem crucial for substrate recognition, binding, catalysis, translocation, and initial product release. However, the Glu89-mediated recruitment and binding of a third ion from the bulk also seems necessary for the full departure of the leaving group from the enzyme’s catalytic site in hExo1 and likely in several other nuclease enzymes.

Conclusions

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Our results provide new insights into the functional role of a third metal ion, which was recently found transiently located at the catalytic site of nuclease enzymes during catalysis. Using molecular dynamics and free-energy simulations applied to multiple systems, we considered the conformational switch of the side chain of a specific residue, Glu89, which is located near the active site in hExo1. We noted that this conformational switch favors the recruitment of a third metal ion from the bulk. The third metal ion is thus promptly positioned near the catalytic center, in accordance with the structural evidence. Our simulations also indicate that this ion serves as an exit shuttle for the leaving group departure from the catalytic site after DNA hydrolysis. The exit mechanism is also favored by the initial involvement of positively charged residues, which are located in an extended and highly structured second-shell area at the two-metal-ion active site. (3,72,73) Finally, our structural analyses of nuclease enzymes show that such a negatively charged residue (Glu/Asp) is persistently found in a similar, structurally conserved, and strategic position in several other 5′ structure-specific nucleases, which seem to share this enzymatic mechanism to promote DNA hydrolysis. These findings may have an implication for de novo enzyme engineering and structure-based drug design. (74,75)

Methods

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Structural Models

We used six different model systems: (i) the wild-type (wt) reactant state (RS3M), based on the recent time-resolved X-ray structure of the complex of the hExo1(PDB ID 5V06), which includes the 5′ recessed-end DNA substrate (in this structure, the third metal ion is located close to the catalytic site); (ii) the same wt reactant state with the third metal ion manually removed (RS2M); (iii) the mutated Glu89Ala reactant state (RSGlu89Ala), modeled on the same X-ray structure (PDB ID 5V06); (iv) the product state (PS2M), based on the recent time-resolved X-ray structure of the ternary complex of hExo1(PDB ID 5V0A), characterized by the newly formed 5′ recessed-end DNA substrate and the leaving adenosine monophosphate (AMP); (v) the product state with the third metal ion close to the 5′ phosphate of the AMP (PS3M) (this system is modeled on the X-ray structure of the prereactive complex, in which we manually cleaved the scissile phosphate); (vi) the mutated Glu89Ala product state (PSGlu89Ala), modeled on the same X-ray structure (PDB ID 5V0A). (23) Protein coordinates and relevant trajectory files are available from the authors upon request.

Classical Molecular Dynamics Simulations

To investigate the functional dynamics of the hExo1/DNA complex, we used extensive force-field-based MD simulations, which are highly informative for complex enzyme/nucleic acid assemblies. (76−80) Here, the AMBER/ff14SB (81) and OL15 (82,83) force fields were used to treat the hExo1 enzyme and the DNA, respectively. The terminal 5′ thymine monophosphate, the 5′ adenosine monophosphate, and the terminal 5′ guanidine monophosphate were treated with the general Amber force field (GAFF). (84) The atomic charges were derived by fitting the electrostatic potential according to the Merz–Singh–Kollman scheme, (85) the RESP fitting procedure. (86) The length of all bonds involving hydrogen atoms was constrained to the equilibrium using the P-LINCS algorithm, (87) and a time integration step of 2 fs was used. All simulations were performed using GROMACS 5.1 code. (88) Long-range electrostatic interactions were calculated with the particle mesh Ewald method with a Fourier grid spacing of 1.6 Å. (89,90) Periodic boundary conditions in the three directions of Cartesian space were applied. The magnesium ions were treated with a nonbonded approach based on the “atoms in molecules” theory partitioning scheme. (91,92) The systems were solvated with TIP3P water molecules (93) and neutralized adding Mg2+, Na+, K+, and Cl– ions, as indicated in the crystallization procedure. (23) The total number of atoms was ∼60 000 for each system (see SI for more information). We followed a two-step procedure for the MD simulations: first, the equilibration phase in which we followed different procedures depending on the system (see SI). Then the production phase was carried out in an NPT ensemble, a constant temperature of 310 K imposed using the velocity-rescaling thermostat, (94) and a constant pressure of 1 bar maintained with a Parrinello–Rahman barostat. (95) We collected MD simulations of ∼0.7 μs for RS3M and ∼1 μs for each of RS2M, RSGlu89Ala, PS2M, PS3M, and PSGlu89Ala for a total of ∼6 μs of MD.

Free-Energy Calculations

We used well-tempered metadynamics (96) to characterize and estimate the free-energy landscape of Glu89 conformational flexibility. We selected a collective variable (CV) that distinguished between the inner, the outer, and the intermediate conformations adopted by Glu89 during the MD simulations. Thus, the selected CV was the pseudodihedral angle ϕ defined by the N, Cα, Cδ, and Cγ atoms on Glu89 (see SI). In particular, based on our MD simulations, inner conformations are adopted at ∼70° < ϕ < ∼100°, intermediate conformations at ∼−40° < ϕ < ∼−10°, and outer conformations at ∼−150° < ϕ < ∼−100°. We performed well-tempered metadynamics by biasing the CV using an initial hill height of 0.02 kcal mol–1, a hill width of 0.35 rad, a fictitious CV temperature of 1550 K, and a deposition rate of 1 ps–1. The simulations were conducted until convergence (see SI for more information).
Well-tempered metadynamics was also used to evaluate possible pathways and the semiquantitative energetics for the release of the leaving group (i.e., adenosine 5′-monophosphate, AMP) from the active site. We used a confined metadynamics approach, (48) which excludes regions of the conformational space that are not relevant to the chemical event under investigation. The selected CV was the distance between the center of mass (COM) of the heavy atoms of AMP and the COM of the Cα of the aspartates (Asp152, Asp171, Asp173) in the first coordination shell of the two catalytic metal ions (see SI). This CV indicates the degree of departure of AMP from the active site in our MD simulations. We used an initial hill height of 0.29 kcal mol–1, a hill width of 0.6 Å, a fictitious CV temperature of 3720 K, and a deposition rate of 1 ps–1. The simulations were conducted until convergence (see SI for more information).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.9b10656.

  • Setup protocols of the force-field-based MD simulations; details on enhanced sampling free energy calculations and additional analyses; PDB analyses, structure, and sequence alignments (PDF)

  • Illustrative movie of the overall catalytic process of hExo1, created by merging PDB structures with fragments of trajectories of equilibrium and metadynamics simulations (MP4)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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M.D.V. thanks the Italian Association for Cancer Research (AIRC) for financial support (IG 23679). V.G. thanks the European Molecular Biology Organization (EMBO) for financial support (ALTF 103-2018). We thank Giuseppina La Sala for helpful discussions. We thank Grace Fox for her proofreading and copyediting.

References

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    Zheng, L.; Jia, J.; Finger, L. D.; Guo, Z.; Zer, C.; Shen, B. Functional Regulation of FEN1 Nuclease and Its Link to Cancer. Nucleic Acids Res. 2011, 39 (3), 781– 794,  DOI: 10.1093/nar/gkq884
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    11
    Functional regulation of FEN1 nuclease and its link to cancer
    Zheng, Li; Jia, Jia; Finger, L. David; Guo, Zhigang; Zer, Cindy; Shen, Binghui
    Nucleic Acids Research (2011), 39 (3), 781-794CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    A review. Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5'-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochem. and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be crit. to regulate protein-protein interactions and cellular localizations of FEN1. Lastly, we also review the biol. significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models.
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  12. 12
    Peltomäki, P. Role of DNA Mismatch Repair Defects in the Pathogenesis of Human Cancer. J. Clin. Oncol. 2003, 21 (6), 1174– 1179,  DOI: 10.1200/JCO.2003.04.060
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    12
    Role of DNA mismatch repair defects in the pathogenesis of human cancer
    Peltomaki, Paivi
    Journal of Clinical Oncology (2003), 21 (6), 1174-1179CODEN: JCONDN; ISSN:0732-183X. (American Society of Clinical Oncology)
    A review. The DNA mismatch repair (MMR) system is necessary for the maintenance of genomic stability. In a broad sense, all main functions of the MMR system, including the correction of biosynthetic errors, DNA damage surveillance, and prevention of recombination between nonidentical sequences serve this important purpose. Failure to accomplish these functions may lead to cancer. It is therefore not surprising that inherited defects in the MMR system underlie one of the most prevalent cancer syndromes in humans, hereditary nonpolyposis colon cancer (HNPCC). In addn., acquired defects of the same system may account for 15% to 25%, or even a higher percentage, of sporadic cancers of different organs of the "HNPCC spectrum," including the colon and rectum, uterine endometrium, stomach, and ovaries. Recent studies indicate that the MMR genes may be involved in the pathogenesis of even a broader spectrum of tumors in one way or another. An updated review of the different features of the human MMR system will be provided, with the emphasis on their implications in cancer development.
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  13. 13
    Dai, Y.; Tang, Z.; Yang, Z.; Zhang, L.; Deng, Q.; Zhang, X.; Yu, Y.; Liu, X.; Zhu, J. EXO1 Overexpression Is Associated with Poor Prognosis of Hepatocellular Carcinoma Patients. Cell Cycle 2018, 17 (19–20), 2386– 2397,  DOI: 10.1080/15384101.2018.1534511
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    13
    EXO1 overexpression is associated with poor prognosis of hepatocellular carcinoma patients
    Dai, Yaoyao; Tang, Zuxiong; Yang, Zongguo; Zhang, Lan; Deng, Qing; Zhang, Xiaofeng; Yu, Yongchun; Liu, Xing; Zhu, Junfeng
    Cell Cycle (2018), 17 (19-20), 2386-2397CODEN: CCEYAS; ISSN:1551-4005. (Taylor & Francis Ltd.)
    The roles of exonuclease 1 (EXO1) in hepatocellular carcinoma (HCC) tumorigenesis and progression remain unclear. This study aimed to assess the prognostic value and therapeutic potential of EXO1 in HCC. Exo1 gene copy nos. were obtained from three Oncomine microarray datasets (n = 447). EXO1 mRNA expression was validated by semi-quant. PCR and QuantiGene 2.0 assays. Cell growth curve and colony formation were performed to asses the cell proliferation. Clonogenic assay, flow cytometry, and immunofluorescence were adopted to acess the effects of EXO1 knockdown and radiation on cell survival, cell cycle distribution and DNA repair. Western blots were performed to reveal the related mechanism. A significant copy no. variation (CNV) of the Exo1 gene was found in HCC specimens in three sep. sets of published microarray data. In the 143 cases treated by our team, EXO1 expression levels were elevated (86.71%, 124/143). In addn., EXO1 overexpression was correlated with larger tumor size (P = 0.002), increased lymph node metastasis (P=0.033) and lower Edmondson grade (P = 0.018). High EXO1 expression unfavorably affected overall survival (OS) (P = 0.009). Both univariate and multivariate Cox regression analyses identified EXO1 as an independent predictor of OS (univariate, P = 0.012; multivariate, P = 0.039). Silencing of EXO1 in vitro reduced cell proliferation. EXO1 knockdown further suppressed clonogenic cell survival, abrogated radiation-induced G2/M phase arrest, and enhanced γ-H2AX foci after exposure to irradn. The accumulation of ataxiatelangiectasia mutated (ATM) might partially regulate the EXO1 related radiosensitivity. In summary, EXO1 could be a promising prognostic marker, with a potential therapeutic value in HCC.
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  14. 14
    Ivanov, I.; Tainer, J. A.; McCammon, J. A. Unraveling the Three-Metal-Ion Catalytic Mechanism of the DNA Repair Enzyme Endonuclease IV. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (5), 1465– 1470,  DOI: 10.1073/pnas.0603468104
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    14
    Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV
    Ivanov, Ivaylo; Tainer, John A.; McCammon, J. Andrew
    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (5), 1465-1470, S1465/1-S1465/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Endonuclease IV belongs to a class of important apurinic/apyrimidinic endonucleases involved in DNA repair. Although a structure-based mechanistic hypothesis has been put forth for this enzyme, the detailed catalytic mechanism has remained unknown. Using thermodn. integration in the context of ab initio quantum mechanics/mol. mechanics mol. dynamics, we examd. certain aspects of the phosphodiester cleavage step in the mechanism. We found the reaction proceeded through a synchronous bimol. (ANDN) mechanism with reaction free energy and barrier of -3.5 and 20.6 kcal/mol, in agreement with exptl. ests. In the course of the reaction the trinuclear active site of endonuclease IV underwent dramatic local conformational changes: shifts in the mode of coordination of both substrate and first-shell ligands. This qual. finding supports the notion that structural rearrangements in the active sites of multinuclear enzymes are integral to biol. function.
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  15. 15
    Prieto, J.; Redondo, P.; Merino, N.; Villate, M.; Montoya, G.; Blanco, F. J.; Molina, R. Structure of the I-SceI Nuclease Complexed with Its DsDNA Target and Three Catalytic Metal Ions. Acta Crystallogr., Sect. F: Struct. Biol. Commun. 2016, 72 (6), 473– 479,  DOI: 10.1107/S2053230X16007512
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    15
    Structure of the I-SceI nuclease complexed with its dsDNA target and three catalytic metal ions
    Prieto, Jesus; Redondo, Pilar; Merino, Nekane; Villate, Maider; Montoya, Guillermo; Blanco, Francisco J.; Molina, Rafael
    Acta Crystallographica, Section F: Structural Biology Communications (2016), 72 (6), 473-479CODEN: ACSFEN; ISSN:2053-230X. (International Union of Crystallography)
    Homing endonucleases (HEs) are highly specific DNA-cleaving enzymes that recognize and cleave long stretches of DNA. The engineering of these enzymes provides instruments for genome modification in a wide range of fields, including gene targeting. The homing endonuclease I-SceI from the yeast Saccharomyces cerevisiae has been purified after overexpression in Escherichia coli and its crystal structure has been detd. in complex with its target DNA. In order to evaluate the no. of ions that are involved in the cleavage process, thus detg. the catalytic mechanism, crystn. expts. were performed in the presence of Mn2+, yielding crystals that were suitable for X-ray diffraction anal. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 80.11, b = 80.57, c = 130.87 Å, α = β = γ = 90°. The self-rotation function and the Matthews coeff. suggested the presence of two protein-DNA complexes in the asym. unit. The crystals diffracted to a resoln. limit of 2.9 Å using synchrotron radiation. From the anomalous data, it was detd. that three cations are involved in catalysis and it was confirmed that I-SceI follows a two-metal-ion DNA-strand cleavage mechanism.
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  16. 16
    AlMalki, F. A.; Flemming, C. S.; Zhang, J.; Feng, M.; Sedelnikova, S. E.; Ceska, T.; Rafferty, J. B.; Sayers, J. R.; Artymiuk, P. J. Direct Observation of DNA Threading in Flap Endonuclease Complexes. Nat. Struct. Mol. Biol. 2016, 23 (7), 640– 646,  DOI: 10.1038/nsmb.3241
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    16
    Direct observation of DNA threading in flap endonuclease complexes
    AlMalki Faizah A; Flemming Claudia S; Sedelnikova Svetlana E; Rafferty John B; Artymiuk Peter J; Zhang Jing; Feng Min; Sayers Jon R; Ceska Tom; Rafferty John B; Sayers Jon R; Sayers Jon R
    Nature structural & molecular biology (2016), 23 (7), 640-6 ISSN:.
    Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 ÅA. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.
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  17. 17
    Uson, M. L.; Carl, A.; Goldgur, Y.; Shuman, S. Crystal Structure and Mutational Analysis of Mycobacterium Smegmatis FenA Highlight Active Site Amino Acids and Three Metal Ions Essential for Flap Endonuclease and 5 Exonuclease Activities. Nucleic Acids Res. 2018, 46 (8), 4164– 4175,  DOI: 10.1093/nar/gky238
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    17
    Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities
    Uson, Maria Loressa; Carl, Ayala; Goldgur, Yehuda; Shuman, Stewart
    Nucleic Acids Research (2018), 46 (8), 4164-4175CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).
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  18. 18
    Yang, W. Nucleases: Diversity of Structure, Function and Mechanism. Q. Rev. Biophys. 2011, 44 (1), 1– 93,  DOI: 10.1017/S0033583510000181
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    18
    Nucleases: diversity of structure, function and mechanism
    Yang, Wei
    Quarterly Reviews of Biophysics (2011), 44 (1), 1-93CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
    A review. Nucleases cleave the phosphodiester bonds of nucleic acids and may be endo or exo, DNase or RNase, topoisomerases, recombinases, ribozymes, or RNA splicing enzymes. In this review, I survey nuclease activities with known structures and catalytic machinery and classify them by reaction mechanism and metal-ion dependence and by their biol. function ranging from DNA replication, recombination, repair, RNA maturation, processing, interference, to defense, nutrient regeneration or cell death. Several general principles emerge from this anal. There is little correlation between catalytic mechanism and biol. function. A single catalytic mechanism can be adapted in a variety of reactions and biol. pathways. Conversely, a single biol. process can often be accomplished by multiple tertiary and quaternary folds and by more than one catalytic mechanism. Two-metal-ion-dependent nucleases comprise the largest no. of different tertiary folds and mediate the most diverse set of biol. functions. Metal-ion-dependent cleavage is exclusively assocd. with exonucleases producing mononucleotides and endonucleases that cleave double- or single-stranded substrates in helical and base-stacked conformations. All metal-ion-independent RNases generate 2'.3'-cyclic phosphate products, and all metal-ion-independent DNases form phospho-protein intermediates. I also find several previously unnoted relationships between different nucleases and shared catalytic configurations.
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  19. 19
    Palermo, G.; Cavalli, A.; Klein, M. L.; Alfonso-Prieto, M.; Dal Peraro, M.; De Vivo, M. Catalytic Metal Ions and Enzymatic Processing of DNA and RNA. Acc. Chem. Res. 2015, 48 (2), 220– 228,  DOI: 10.1021/ar500314j
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    19
    Catalytic metal ions and enzymatic processing of DNA and RNA
    Palermo, Giulia; Cavalli, Andrea; Klein, Michael L.; Alfonso-Prieto, Mercedes; Dal Peraro, Matteo; De Vivo, Marco
    Accounts of Chemical Research (2015), 48 (2), 220-228CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion (2M) mechanism. Several high-resoln. x-ray structures portraying the two-metal-aided catalytic site, together with mutagenesis and kinetics studies, have demonstrated a functional role of the ions for catalysis in numerous metallonucleases. Overall, the exptl. data confirm the general mechanistic hypothesis for 2M-aided phosphoryl transfer originally reported by T. A. Steitz and J. A. Steitz (1993). This seminal paper proposed that one metal ion favors the formation of the nucleophile, while the nearby 2nd metal ion facilitates leaving group departure during RNA hydrolysis. Both metals were suggested to stabilize the enzymic transition state. Nevertheless, static x-ray structures alone cannot exhaustively unravel how the two ions execute their functional role along the enzymic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states. Here, the authors discuss the role of multiscale mol. simulations in further disclosing mechanistic insights of 2M-aided catalysis for two prototypical enzymic targets for drug discovery, namely, RNase H (RNase H) and type II topoisomerase (topoII). In both examples, first-principles mol. simulations, integrated with structural data, emphasize a cooperative motion of the bimetal motif during catalysis. The coordinated motion of both ions is crucial for maintaining a flexible metal-centered structural architecture exquisitely tailored to accommodate the DNA or RNA sugar-phosphate backbone during phosphodiester bond cleavage. Furthermore, the anal. of RNase H and the N-terminal domain (PAN) of influenza polymerase shows that classical mol. dynamics simulations coupled with enhanced sampling techniques have contributed to describe the modulatory effect of metal ion concn. and metal uptake on the 2M mechanism and efficiency. These aspects all point to the emerging and intriguing role of addnl. adjacent ions potentially involved in the modulation of phosphoryl transfer reactions and enzymic turnover in 2M-catalysis, as recently obsd. exptl. in polymerase η and homing endonuclease I DmoI. These computational results, integrated with exptl. findings, describe and reinforce the nascent concept of a functional and cooperative dynamics of the catalytic metal ions during the 2M-dependent enzymic processing of DNA and RNA. Encouraged by the insights provided by computational approaches, the authors foresee further expts. that will feature the functional and joint dynamics of the catalytic metal ions for nucleic acid processing. This could impact the de novo design of artificial metallonucleases and the rational design of potent metal-chelating inhibitors of pharmaceutically relevant enzymes.
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  20. 20
    Palermo, G.; Stenta, M.; Cavalli, A.; Dal Peraro, M.; De Vivo, M. Molecular Simulations Highlight the Role of Metals in Catalysis and Inhibition of Type II Topoisomerase. J. Chem. Theory Comput. 2013, 9 (2), 857– 862,  DOI: 10.1021/ct300691u
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    20
    Molecular Simulations Highlight the Role of Metals in Catalysis and Inhibition of Type II Topoisomerase
    Palermo, Giulia; Stenta, Marco; Cavalli, Andrea; Dal Peraro, Matteo; De Vivo, Marco
    Journal of Chemical Theory and Computation (2013), 9 (2), 857-862CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    Type II topoisomerase (topoII) is a metalloenzyme targeted by clin. antibiotics and anticancer agents. Here, we integrate existing structural data with mol. simulation and propose a model for the yet uncharacterized structure of the reactant state of topoII. This model describes a canonical two-metal-ion mechanism and suggests how the metals could rearrange at the catalytic pocket during enzymic turnover, explaining also exptl. evidence for topoII inhibition. These results call for further exptl. validation.
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  21. 21
    Schmidt, B. H.; Burgin, A. B.; Deweese, J. E.; Osheroff, N.; Berger, J. M. A Novel and Unified Two-Metal Mechanism for DNA Cleavage by Type II and IA Topoisomerases. Nature 2010, 465 (7298), 641– 644,  DOI: 10.1038/nature08974
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    21
    A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases
    Schmidt, Bryan H.; Burgin, Alex B.; Deweese, Joseph E.; Osheroff, Neil; Berger, James M.
    Nature (London, United Kingdom) (2010), 465 (7298), 641-644CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Type II topoisomerases are required for the management of DNA tangles and supercoils, and are targets of clin. antibiotics and anti-cancer agents. These enzymes catalyze the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or gates'. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyze G-segment cleavage. Recent efforts have proposed that strand scission relies on a "two-metal mechanism", a ubiquitous biochem. strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topoisomerase II covalently linked to DNA through its active-site tyrosine at 2.5 Å resoln., revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking expts. indicate that cleavage is catalyzed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a mol. "trapdoor" to prevent subunit dissocn. during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.
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  22. 22
    Perera, L.; Freudenthal, B. D.; Beard, W. A.; Shock, D. D.; Pedersen, L. G.; Wilson, S. H. Requirement for Transient Metal Ions Revealed through Computational Analysis for DNA Polymerase Going in Reverse. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (38), E5228– E5236,  DOI: 10.1073/pnas.1511207112
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    22
    Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse
    Perera, Lalith; Freudenthal, Bret D.; Beard, William A.; Shock, David D.; Pedersen, Lee G.; Wilson, Samuel H.
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (38), E5228-E5236CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chem. equil. by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3' of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallog. studies of DNA polymerases have identified an addnl. metal ion (product metal) assocd. with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mech./mol. mech. calcns. of the reverse reaction in the confines of the DNA polymerase β active site. Addnl., site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallog. structures. The transition barrier for pyrophosphorolysis was estd. to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the resp. reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chem. equil. of a reaction that is central to genome stability.
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  23. 23
    Shi, Y.; Hellinga, H. W.; Beese, L. S. Interplay of Catalysis, Fidelity, Threading, and Processivity in the Exo- and Endonucleolytic Reactions of Human Exonuclease I. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (23), 6010– 6015,  DOI: 10.1073/pnas.1704845114
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    23
    Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I
    Shi, Yuqian; Hellinga, Homme W.; Beese, Lorena S.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (23), 6010-6015CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends were accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guided the 2 substrate types into a shared reaction mechanism that catalyzed their cleavage by an elaborated variant of the 2-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppressed inappropriate or premature cleavage, enhancing processing fidelity. The striking redn. in flap conformational entropy was catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the obsd. reaction sequence, hExo1 reset without relinquishing DNA binding, suggesting a structural basis for its processivity.
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  24. 24
    Jimeno, S.; Herrera-Moyano, E.; Ortega, P.; Aguilera, A. Differential Effect of the Overexpression of Rad2/XPG Family Endonucleases on Genome Integrity in Yeast and Human Cells. DNA Repair 2017, 57, 66– 75,  DOI: 10.1016/j.dnarep.2017.06.030
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    24
    Differential effect of the overexpression of Rad2/XPG family endonucleases on genome integrity in yeast and human cells
    Jimeno, Sonia; Herrera-Moyano, Emilia; Ortega, Pedro; Aguilera, Andres
    DNA Repair (2017), 57 (), 66-75CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)
    Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metab. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are crit. to preserve genome integrity. In addn. to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.
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  25. 25
    Emmert, S.; Schneider, T. D.; Khan, S. G.; Kraemer, K. H. The Human XPG Gene: Gene Architecture, Alternative Splicing and Single Nucleotide Polymorphisms. Nucleic Acids Res. 2001, 29 (7), 1443– 1452,  DOI: 10.1093/nar/29.7.1443
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    25
    The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms
    Emmert, Steffen; Schneider, Thomas D.; Khan, Sikandar G.; Kraemer, Kenneth H.
    Nucleic Acids Research (2001), 29 (7), 1443-1452CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, detn. of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Anal. of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amt. of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in assocn. with multiple alternatively spliced isoforms of XPG mRNA.
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  26. 26
    Qiu, J.; Qian, Y.; Chen, V.; Guan, M.-X.; Shen, B. Human Exonuclease 1 Functionally Complements Its Yeast Homologues in DNA Recombination, RNA Primer Removal, and Mutation Avoidance. J. Biol. Chem. 1999, 274 (25), 17893– 17900,  DOI: 10.1074/jbc.274.25.17893
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    26
    Human exonuclease 1 functionally complements its yeast homologues in DNA recombination, RNA primer removal, and mutation avoidance
    Qiu, Junzhuan; Qian, Ying; Chen, Victoria; Guan, Min-Xin; Shen, Binghui
    Journal of Biological Chemistry (1999), 274 (25), 17893-17900CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Yeast exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, the authors show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Δ mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5'-3' double strand DNA exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.
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  27. 27
    Genschel, J.; Bazemore, L. R.; Modrich, P. Human Exonuclease I Is Required for 5′ and 3′ Mismatch Repair. J. Biol. Chem. 2002, 277 (15), 13302– 13311,  DOI: 10.1074/jbc.M111854200
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    Human exonuclease I is required for 5' and 3' mismatch repair
    Genschel, Jochen; Bazemore, Laura R.; Modrich, Paul
    Journal of Biological Chemistry (2002), 277 (15), 13302-13311CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The authors have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear ext. fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted exts. requires both hMutSα and hMutLα. However, excision directed by a 5' strand break requires hMutSα but can occur in absence of hMutLα. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSα in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.
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  28. 28
    Wei, K.; Clark, A. B.; Wong, E.; Kane, M. F.; Mazur, D. J.; Parris, T.; Kolas, N. K.; Russell, R.; Hou, H.; Kneitz, B.; Yang, G.; Kunkel, T. A.; Kolodner, R. D.; Cohen, P. E.; Edelmann, W. Inactivation of Exonuclease I in Mice Results in DNA Mismatch Repair Defects, Increased Cancer Susceptibility, and Male and Female Sterility. Genes Dev. 2003, 17 (5), 603– 614,  DOI: 10.1101/gad.1060603
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    28
    Inactivation of exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility
    Wei, Kaichun; Clark, Alan B.; Wong, Edmund; Kane, Michael F.; Mazur, Dan J.; Parris, Tchaiko; Kolas, Nadine K.; Russell, Robert; Hou, Harry, Jr.; Kneitz, Burkhard; Yang, Guohze; Kunkel, Thomas A.; Kolodner, Richard D.; Cohen, Paula E.; Edelmann, Winfried
    Genes & Development (2003), 17 (5), 603-614CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
    Exonuclease 1 (Exo1) is a 5'-3' exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Anal. of Exo1-/- cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5' and 3' nick-directed repair. The repair defect in Exo1-/- cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1-/- animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addn., Exo1-/- male and female mice were sterile because of a meiotic defect. Meiosis in Exo1-/- animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis.
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  29. 29
    Zhu, Z.; Chung, W. H.; Shim, E. Y.; Lee, S. E.; Ira, G. Sgs1 Helicase and Two Nucleases Dna2 and Exo1 Resect DNA Double-Strand Break Ends. Cell 2008, 134 (6), 981– 994,  DOI: 10.1016/j.cell.2008.08.037
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    29
    Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends
    Zhu, Zhu; Chung, Woo-Hyun; Shim, Eun Yong; Lee, Sang Eun; Ira, Grzegorz
    Cell (Cambridge, MA, United States) (2008), 134 (6), 981-994CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. The authors monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. The Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degrdn., whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Δ sgs1Δ double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.
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  30. 30
    Vallur, A. C.; Maizels, N. Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats. J. Biol. Chem. 2010, 285 (37), 28514– 28519,  DOI: 10.1074/jbc.M110.132738
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    Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats
    Vallur, Aarthy C.; Maizels, Nancy
    Journal of Biological Chemistry (2010), 285 (37), 28514-28519CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Trinucleotide repeats can form stable secondary structures that promote genomic instability. To det. how such structures are resolved, we have defined biochem. activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flap-bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. HEXO1 did not exhibit endonuclease activity on 5'-flap-bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by DNA quadruplex (G4) structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity vs. hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, resp. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.
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    Lee, B. I.; Wilson, D. M. The RAD2 Domain of Human Exonuclease 1 Exhibits 5′ to 3′ Exonuclease and Flap Structure-Specific Endonuclease Activities. J. Biol. Chem. 1999, 274 (53), 37763– 37769,  DOI: 10.1074/jbc.274.53.37763
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    The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities
    Lee, Byung-In; Wilson, David M., III
    Journal of Biological Chemistry (1999), 274 (53), 37763-37769CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg2+ being the preferred metal ion. HEX1-N2 is ∼3-fold less active in Mn2+-contg. buffers and exhibits <5% activity in the presence of Co2+, Zn2+, or Ca2+. The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (kcat) and site of incision, and is as efficient (kcat/Km) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.
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    Keijzers, G.; Bohr, V. A.; Rasmussen, L. J. Human Exonuclease 1 (EXO1) Activity Characterization and Its Function on FLAP Structures. Biosci. Rep. 2015, 35 (3), e00206  DOI: 10.1042/BSR20150058
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    Orans, J.; McSweeney, E. A.; Iyer, R. R.; Hast, M. A.; Hellinga, H. W.; Modrich, P.; Beese, L. S. Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family. Cell 2011, 145 (2), 212– 223,  DOI: 10.1016/j.cell.2011.03.005
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    Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family
    Orans, Jillian; McSweeney, Elizabeth A.; Iyer, Ravi R.; Hast, Michael A.; Hellinga, Homme W.; Modrich, Paul; Beese, Lorena S.
    Cell (Cambridge, MA, United States) (2011), 145 (2), 212-223CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. HExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant soln. to a complex geometrical puzzle of substrate recognition and processing.
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  34. 34
    Tsutakawa, S. E.; Thompson, M. J.; Arvai, A. S.; Neil, A. J.; Shaw, S. J.; Algasaier, S. I.; Kim, J. C.; Finger, L. D.; Jardine, E.; Gotham, V. J. B.; Sarker, A. H.; Her, M. Z.; Rashid, F.; Hamdan, S. M.; Mirkin, S. M.; Grasby, J. A.; Tainer, J. A. Phosphate Steering by Flap Endonuclease 1 Promotes 5′-Flap Specificity and Incision to Prevent Genome Instability. Nat. Commun. 2017, 8, 15855,  DOI: 10.1038/ncomms15855
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    Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability
    Tsutakawa, Susan E.; Thompson, Mark J.; Arvai, Andrew S.; Neil, Alexander J.; Shaw, Steven J.; Algasaier, Sana I.; Kim, Jane C.; Finger, L. David; Jardine, Emma; Gotham, Victoria J. B.; Sarker, Altaf H.; Her, Mai Z.; Rashid, Fahad; Hamdan, Samir M.; Mirkin, Sergei M.; Grasby, Jane A.; Tainer, John A.
    Nature Communications (2017), 8 (), 15855CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallog., biochem. and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via 'phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold redn. in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.
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    Steitz, T. A.; Steitz, J. A. A General Two-Metal-Ion Mechanism for Catalytic RNA. Proc. Natl. Acad. Sci. U. S. A. 1993, 90 (14), 6498– 6502,  DOI: 10.1073/pnas.90.14.6498
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    A general two-metal-ion mechanism for catalytic RNA
    Steitz, Thomas A.; Steitz, Joan A.
    Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (14), 6498-502CODEN: PNASA6; ISSN:0027-8424.
    A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chem. catalysis is facilitated by 2 divalent metal ions 3.9 Å apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar OH group, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a 2-metal ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the 2 catalytic metal ions and properly orient the substrates via 3 specific binding sites.
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    Yang, W.; Lee, J. Y.; Nowotny, M. Making and Breaking Nucleic Acids: Two-Mg2+-Ion Catalysis and Substrate Specificity. Mol. Cell 2006, 22 (1), 5– 13,  DOI: 10.1016/j.molcel.2006.03.013
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    Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity
    Yang, Wei; Lee, Jae Young; Nowotny, Marcin
    Molecular Cell (2006), 22 (1), 5-13CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
    A review. DNA and a large proportion of RNA are antiparallel duplexes composed of an unvarying phosphosugar backbone surrounding uniformly stacked and highly similar base pairs. How do the myriad of enzymes (including ribozymes) that perform catalysis on nucleic acids achieve exquisite structure or sequence specificity. In all DNA and RNA polymerases and many nucleases and transposases, two Mg2+ ions are jointly coordinated by the nucleic acid substrate and catalytic residues of the enzyme. Based on the exquisite sensitivity of Mg2+ ions to the ligand geometry and electrostatic environment, we propose that two-metal-ion catalysis greatly enhances substrate recognition and catalytic specificity.
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    Dupureur, C. M. One Is Enough: Insights into the Two-Metal Ion Nuclease Mechanism from Global Analysis and Computational Studies. Metallomics. 2010, 2 (9), 609– 620,  DOI: 10.1039/c0mt00013b
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    One is enough: insights into the two-metal ion nuclease mechanism from global analysis and computational studies
    Dupureur, Cynthia M.
    Metallomics (2010), 2 (9), 609-620CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
    A review. The mechanistic details of metallonuclease reactions, typically supported by Mg(II), have a long and contentious history. Two-metal ion mechanisms have enjoyed much favor, based largely in the multitude of X-ray crystal structures of these enzymes with more than one metal ion per active site. Most recently, this mechanism has come under challenge. Reviewed herein are the applications of different exptl. strategies that collectively support a mechanism in which only one metal ion is necessary for nucleic acid hydrolysis. Based on global kinetic anal., anal. of reactions in which the nonsupportive Ca(II) is added, and a no. of computational approaches, secondary sites are proposed to either be occupied by activity-modulating metal ions or occupied in turn by a single metal that changes position during the course of the reaction.
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    Beese, L. S.; Steitz, T. A. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase 1: a two metal ion mechanism. EMBO J. 1991, 10 (1), 25– 33,  DOI: 10.1002/j.1460-2075.1991.tb07917.x
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    Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism
    Beese, Lorena S.; Steitz, Thomas A.
    EMBO Journal (1991), 10 (1), 25-33CODEN: EMJODG; ISSN:0261-4189.
    The refined crystal structures of the large proteolytic fragment (Klenow fragment) of E. coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5'exonuclease active site. The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6- and 3.1-Å resoln. resp. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carred out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 Å from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.
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    De Vivo, M.; Dal Peraro, M.; Klein, M. L. Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism. J. Am. Chem. Soc. 2008, 130 (33), 10955– 10962,  DOI: 10.1021/ja8005786
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    Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism
    De Vivo, Marco; Dal Peraro, Matteo; Klein, Michael L.
    Journal of the American Chemical Society (2008), 130 (33), 10955-10962CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    RNase H belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA•DNA hybrids. This enzyme is implicated in replication initiation and DNA topol. restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resoln. crystal structures of the complex RNase H/RNA•DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/mol. mechanics calcns. for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water vs. hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg2+ metals also support the formation of a meta-stable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated β-phosphoglucomutase crystal (Lahiri, S. D.; et al. Science 2003, 299 (5615), 2067-2071). The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water mols. Due to the highly conserved nature of the enzymic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily.
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  40. 40
    Genna, V.; Vidossich, P.; Ippoliti, E.; Carloni, P.; De Vivo, M. A Self-Activated Mechanism for Nucleic Acid Polymerization Catalyzed by DNA/RNA Polymerases. J. Am. Chem. Soc. 2016, 138 (44), 14592– 14598,  DOI: 10.1021/jacs.6b05475
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    40
    A Self-Activated Mechanism for Nucleic Acid Polymerization Catalyzed by DNA/RNA Polymerases
    Genna, Vito; Vidossich, Pietro; Ippoliti, Emiliano; Carloni, Paolo; Vivo, Marco De
    Journal of the American Chemical Society (2016), 138 (44), 14592-14598CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The enzymic polymn. of DNA and RNA is at the basis of genetic inheritance for all living organisms. It is catalyzed by the DNA/RNA polymerase (Pol) superfamily. Here, bioinformatics anal. revealed that the incoming nucleotide substrate always forms an H-bond between its 3'-OH and β-phosphate moieties upon formation of the Michaelis complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addn. and, importantly, nucleic acid translocation. Thus, SAM allows an elegant and efficient closed-loop sequence of chem. and phys. steps for Pol catalysis. This is markedly different from previous mechanistic hypotheses. This proposed mechanism was corroborated via ab initio QM/MM simulations on a specific Pol, human DNA polymerase-η, an enzyme involved in repairing damaged DNA. The structural conservation of DNA and RNA Pols supports the possible extension of SAM to Pol enzymes from the 3 domains of life.
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  41. 41
    Shaw, S. J.; Finger, L. D.; Grasby, J. A. Human Exonuclease 1 Threads 5′-Flap Substrates through Its Helical Arch. Biochemistry 2017, 56 (29), 3704– 3707,  DOI: 10.1021/acs.biochem.7b00507
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    Human Exonuclease 1 Threads 5'-Flap Substrates through Its Helical Arch
    Shaw, Steven J.; Finger, L. David; Grasby, Jane A.
    Biochemistry (2017), 56 (29), 3704-3707CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Human exonuclease 1 (hEXO1) is a member of the 5'-nuclease superfamily and plays important roles in DNA repair. Along with acting as a 5'-exonuclease on blunt, gapped, nicked, and 3'-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5'-single-stranded flaps from duplex ends. How hEXO1 and related 5'-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5'-flaps has been controversial. Here we report the first functional data that imply that hEXO1 threads the 5'-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single strands. Conjugation of bulky 5'-streptavidin that would "block" threading through the arch drastically slowed the hEXO1 reaction. In contrast, addn. of streptavidin to a preformed hEXO1 5'-biotin flap DNA complex trapped a portion of the substrate in a highly reactive threaded conformation. However, another fraction behaves as if it were "blocked" and decayed very slowly, implying there were both threaded and unthreaded forms of the substrate present. The reaction of an unmodified hEXO1-flap DNA complex did not exhibit marked biphasic kinetics, suggesting a fast re-equilibration occurs that produces more threaded substrate when some decays. The finding that a threading mechanism like that used by hFEN1 is also used by hEXO1 unifies the mode of operation for members of the 5'-nuclease superfamily that act on discontinuous substrates. As with hFEN1, intrinsic disorder of the arch region of the protein may explain how flaps can be threaded without a need for a coupled energy source.
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  42. 42
    Tomlinson, C. G.; Atack, J. M.; Chapados, B.; Tainer, J. A.; Grasby, J. A. Substrate Recognition and Catalysis by Flap Endonucleases and Related Enzymes. Biochem. Soc. Trans. 2010, 38 (2), 433– 437,  DOI: 10.1042/BST0380433
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    Substrate recognition and catalysis by flap endonucleases and related enzymes
    Tomlinson, Christopher G.; Atack, John M.; Chapados, Brian; Tainer, John A.; Grasby, Jane A.
    Biochemical Society Transactions (2010), 38 (2), 433-437CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
    A review. Flap endonucleases (FENs) and related FEN-like enzymes [exonuclease-1, gap endonuclease 1 (GEN-1), and xeroderma pigmentosum complementation group G] are a family of divalent metal ion-dependent nucleases that catalyze structure-specific hydrolysis of DNA duplex-contg. nucleic acid structures during DNA replication, repair, and recombination. In the case of FENs, the ability to catalyze reactions on a variety of substrates has been rationalized as a result of combined functional and structural studies. Analyses of FENs also exemplify controversies regarding the 2-metal-ion mechanism. However, kinetic studies of phage T5 FEN reveal that a 2-metal-ion-like mechanism for chem. catalysis is plausible. Consideration of the metallobiochem. and the positioning of substrate in metal-free structures has led to the proposal that the duplex termini of substrates are unpaired in the catalytically active form and that FENs and related enzymes may recognize breathing duplex termini within more complex structures. An outstanding issue in FEN catalysis is the role played by the intermediate (I) domain arch or clamp. It has been proposed that FENs thread the 5'-portion of their substrates through this arch, which is wide enough to accommodate single-stranded, but not double-stranded, DNA. However, FENs exhibit gap endonuclease activity acting upon substrates that have a region of 5'-duplex. Moreover, the action of other FEN family members such as GEN-1, proposed to target Holliday junctions without termini, appears incompatible with a threading mechanism. An alterative is that the I domain is used as a clamp. A future challenge is to clarify the role of this domain in FENs and related enzymes.
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  43. 43
    Bennet, I. A; Finger, L D.; Baxter, N. J; Ambrose, B.; Hounslow, A. M; Thompson, M. J; Exell, J. C; Shahari, N. N. B M.; Craggs, T. D; Waltho, J. P; Grasby, J. A Regional Conformational Flexibility Couples Substrate Specificity and Scissile Phosphate Diester Selectivity in Human Flap Endonuclease 1. Nucleic Acids Res. 2018, 46 (11), 5618– 5633,  DOI: 10.1093/nar/gky293
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    Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1
    Bennet, Ian A.; Finger, L. David; Baxter, Nicola J.; Ambrose, Benjamin; Hounslow, Andrea M.; Thompson, Mark J.; Exell, Jack C.; Shahari, Nur Nazihah B. Md.; Craggs, Timothy D.; Waltho, Jonathan P.; Grasby, Jane A.
    Nucleic Acids Research (2018), 46 (11), 5618-5633CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the soln. conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-mol. FRET data show a shift in the conformational ensemble in the arch and loop region upon addn. of DNA. Furthermore, the addn. of divalent metal ions to the active site of the hFEN1- DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.
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  44. 44
    Raper, A. T.; Reed, A. J.; Suo, Z. Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion. Chem. Rev. 2018, 118 (2), 6000– 6025,  DOI: 10.1021/acs.chemrev.7b00685
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    Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion
    Raper, Austin T.; Reed, Andrew J.; Suo, Zucai
    Chemical Reviews (Washington, DC, United States) (2018), 118 (12), 6000-6025CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases, has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyl transfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyl transfer (i.e. chem.), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallog. studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidences for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.
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  45. 45
    Black, C. B.; Huang, H. W.; Cowan, J. A. Biological Coordination Chemistry of Magnesium, Sodium, and Potassium Ions. Protein and Nucleotide Binding Sites. Coord. Chem. Rev. 1994, 135–136 (C), 165– 202,  DOI: 10.1016/0010-8545(94)80068-5
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    Biological coordination chemistry of magnesium, sodium, and potassium ions. Protein and nucleotide binding sites
    Black, C. B.; Huang, H.-W.; Cowan, J. A.
    Coordination Chemistry Reviews (1994), 135/136 (), 165-202CODEN: CCHRAM; ISSN:0010-8545. (Elsevier)
    This review with 78 refs. explores Mg2+ binding to proteins and enzymes, K+ activated enzymes, metal ions and membranes, and metal-nucleotide binding domains.
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  46. 46
    Ho, M.-H.; De Vivo, M.; Dal Peraro, M.; Klein, M. L. Understanding the Effect of Magnesium Ion Concentration on the Catalytic Activity of Ribonuclease H through Computation: Does a Third Metal Binding Site Modulate Endonuclease Activity?. J. Am. Chem. Soc. 2010, 132 (39), 13702– 13712,  DOI: 10.1021/ja102933y
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    46
    Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: Does a third metal binding site modulate endonuclease activity?
    Ho, Ming-Hsun; De Vivo, Marco; Dal Peraro, Matteo; Klein, Michael L.
    Journal of the American Chemical Society (2010), 132 (39), 13702-13712CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    RNase H belongs to the nucleotidyltransferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallog. data have located 2 ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, expts. have shown a dependency of the catalytic activity on the Mg2+ concn. Moreover, in RNase H, Glu-188 has been shown to be essential for full enzymic activation, regardless of Mg2+ concn. The catalytic center is known to contain 3 Mg2+ ions, and Glu-188 is not one of the primary metal ligands. Here, classical mol. dynamics (MD) simulations were employed to study the metal-ligand coordination in Bacillus halodurans RNase H at different concns. of Mg2+. Importantly, the presence of a 3rd Mg2+ ion, bound to 2nd-shell ligand Glu-188, was a persistent feature of the MD simulations. Free energy calcns. identified 2 distinct conformations, depending on the concn. of Mg2+. At std. concn., a 3rd Mg2+ was found in the catalytic pocket, but it did not perturb the optimal RNase H active conformation. However, at higher concns., the 3rd Mg2+ ion heavily perturbed the nucleophilic water and thereby influenced the catalytic efficiency of RNase H. In addn., the E188A mutant showed no ability to engage addnl. Mg2+ ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of mutant E188A and also supports the key role of Glu-188 in localizing the 3rd Mg2+ ion at the active site. Glu residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calcns. support the hypothesis that RNase H can accommodate 3 divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.
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    Genna, V.; Gaspari, R.; Dal Peraro, M.; De Vivo, M. Cooperative Motion of a Key Positively Charged Residue and Metal Ions for DNA Replication Catalyzed by Human DNA Polymerase-η. Nucleic Acids Res. 2016, 44 (6), 2827– 2836,  DOI: 10.1093/nar/gkw128
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    Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase-η
    Genna Vito; Gaspari Roberto; Dal Peraro Matteo; De Vivo Marco
    Nucleic acids research (2016), 44 (6), 2827-36 ISSN:.
    Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases.
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  48. 48
    La Sala, G.; Riccardi, L.; Gaspari, R.; Cavalli, A.; Hantschel, O.; De Vivo, M. HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase. J. Chem. Theory Comput. 2016, 12 (11), 5563– 5574,  DOI: 10.1021/acs.jctc.6b00600
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    HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase
    La Sala, Giuseppina; Riccardi, Laura; Gaspari, Roberto; Cavalli, Andrea; Hantschel, Oliver; De Vivo, Marco
    Journal of Chemical Theory and Computation (2016), 12 (11), 5563-5574CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    A no. of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Tyr-412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, the binding of the SH2 domain to the N-lobe of the Abl kinase domain was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, the authors report on microsecond-long mol. dynamics simulations coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain, and with the DFG motif flipped in 2 ways (in or out conformation). Through comparative anal., the simulations augment the interpretation of the existing Abl exptl. data, revealing a dynamical network of interactions that interconnect SH2 domain-binding with A-loop plasticity and Tyr-412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely the EPK-ELK H-bond network and the HRD motif. These results show that signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.
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    Hwang, W.; Yoo, J.; Lee, Y.; Park, S.; Hoang, P. L.; Cho, H.; Yu, J.; Hoa Vo, T. M.; Shin, M.; Jin, M. S.; Park, D.; Hyeon, C.; Lee, G. Dynamic Coordination of Two-Metal-Ions Orchestrates λ-Exonuclease Catalysis. Nat. Commun. 2018, 9 (1), 4404,  DOI: 10.1038/s41467-018-06750-9
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    Dynamic coordination of two-metal-ions orchestrates λ-exonuclease catalysis
    Hwang Wonseok; Lee Yuno; Hyeon Changbong; Hwang Wonseok; Yoo Jungmin; Park Suyeon; Hoang Phuong Lien; Cho HyeokJin; Yu Jeongmin; Hoa Vo Thi Minh; Jin Mi Sun; Park Daeho; Lee Gwangrog; Lee Yuno; Shin Minsang
    Nature communications (2018), 9 (1), 4404 ISSN:.
    Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg(2+) binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while MgA(2+) and MgB(2+) have similar binding constants, the dissociation rate of MgA(2+) is two order of magnitude lower than that of MgB(2+) due to a kinetic-barrier-difference. At physiological Mg(2+) concentration, the MgB(2+) ion near the 5'-terminal side of the scissile phosphate dissociates each-round of degradation, facilitating a series of DNA cleavages via fast product-release concomitant with enzyme-translocation. At a low magnesium concentration, occasional dissociation and slow re-coordination of MgA(2+) result in pauses during processive degradation. Our study highlights the importance of metal-ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis.
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    Samara, N. L.; Yang, W. Cation Trafficking Propels RNA Hydrolysis. Nat. Struct. Mol. Biol. 2018, 25 (8), 715– 721,  DOI: 10.1038/s41594-018-0099-4
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    Cation trafficking propels RNA hydrolysis
    Samara, Nadine L.; Yang, Wei
    Nature Structural & Molecular Biology (2018), 25 (8), 715-721CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
    Catalysis by members of the RNase H superfamily of enzymes is generally believed to require only two Mg2+ ions that are coordinated by active-site carboxylates. By examg. the catalytic process of Bacillus halodurans RNase H1 in crystallo, however, we found that the two canonical Mg2+ ions and an addnl. K+ failed to align the nucleophilic water for RNA cleavage. Substrate alignment and product formation required a second K+ and a third Mg2+, which replaced the first K+ and departed immediately after cleavage. A third transient Mg2+ has also been obsd. for DNA synthesis, but in that case it coordinates the leaving group instead of the nucleophile as in the case of the RNase H1 hydrolysis reaction. These transient cations have no contact with the enzymes. Other DNA and RNA enzymes that catalyze consecutive cleavage and strand-transfer reactions in a single active site may similarly require cation trafficking coordinated by the substrate.
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    Molina, R.; Stella, S.; Redondo, P.; Gomez, H.; Marcaida, M. J.; Orozco, M.; Prieto, J.; Montoya, G. Visualizing Phosphodiester-Bond Hydrolysis by an Endonuclease. Nat. Struct. Mol. Biol. 2015, 22 (1), 65– 72,  DOI: 10.1038/nsmb.2932
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    Visualizing phosphodiester-bond hydrolysis by an endonuclease
    Molina, Rafael; Stella, Stefano; Redondo, Pilar; Gomez, Hansel; Marcaida, Maria Jose; Orozco, Modesto; Prieto, Jesus; Montoya, Guillermo
    Nature Structural & Molecular Biology (2015), 22 (1), 65-72CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    The enzymic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chem. reaction has not yet been obsd. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We obsd. the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.
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    Sasnauskas, G.; Jeltsch, A.; Pingoud, A.; Siksnys, V. Plasmid DNA Cleavage by MunI Restriction Enzyme: Single-Turnover and Steady-State Kinetic Analysis. Biochemistry 1999, 38 (13), 4028– 4036,  DOI: 10.1021/bi982456n
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    Plasmid DNA cleavage by MunI restriction enzyme: single-turnover and steady-state kinetic analysis
    Sasnauskas, Giedrius; Jeltsch, Albert; Pingoud, Alfred; Siksnys, Virginijus
    Biochemistry (1999), 38 (13), 4028-4036CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Mutational anal. has previously indicated that D83 and E98 residues are essential for DNA cleavage activity and presumably chelate a Mg2+ ion at the active site of MunI restriction enzyme. In the absence of metal ions, protonation of an ionizable residue with a pKa > 7.0, most likely one of the active site carboxylates, controls the DNA binding specificity of MunI [Lagunavicius, A., Grazulis, S., Balciunaite, E., Vainius, D., and Siksnys, V. (1997) Biochem. 36, 11093-11099.]. Thus, competition between H+ and Mg2+ binding at the active site of MunI presumably plays an important role in catalysis/binding. In the present study we have identified elementary steps and intermediates in the reaction pathway of plasmid DNA cleavage by MunI and elucidated the effect of pH and Mg2+ ions on the individual steps of the DNA cleavage reaction. The kinetic anal. indicated that the multiple-turnover rate of plasmid cleavage by MunI is limited by product release throughout the pH range 6.0-9.3. Quenched-flow expts. revealed that open circle DNA is an obligatory intermediate in the reaction pathway. Under optimal reaction conditions, open circle DNA remains bound to the MunI; however it is released into the soln. at low [MgCl2]. Rate consts. for the phosphodiester bond hydrolysis of the first (k1) and second (k2) strand of plasmid DNA at pH 7.0 and 10 mM MgCl2 more than 100-fold exceed the kcat value which is limited by product dissocn. The anal. of the pH and [Mg2+] dependences of k1 and k2 revealed that both H+ and Mg2+ ions compete for the binding to the same residue at the active site of MunI. Thus, the decreased rate of phosphodiester hydrolysis by MunI at pH < 7.0 may be due to the redn. of affinity for the Mg2+ binding at the active site. Kinetic anal. of DNA cleavage by MunI yielded ests. for the assocn.-dissocn. rate consts. of enzyme-substrate complex and demonstrated the decreased stability of the MunI-DNA complex at pH values above 8.0.
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    Xie, F.; Dupureur, C. M. Kinetic Analysis of Product Release and Metal Ions in a Metallonuclease. Arch. Biochem. Biophys. 2009, 483 (1), 1– 9,  DOI: 10.1016/j.abb.2009.01.001
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    Kinetic analysis of product release and metal ions in a metallonuclease
    Xie, Fuqian; Dupureur, Cynthia M.
    Archives of Biochemistry and Biophysics (2009), 483 (1), 1-9CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)
    Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equil. and kinetic expts. are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equil. fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre-steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre-steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, anal. of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no pos. cooperativity between the two metal ions binding each active site.
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  54. 54
    Sengerová, B.; Tomlinson, C.; Atack, J. M.; Williams, R.; Sayers, J. R.; Williams, N. H.; Grasby, J. A. Brønsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substrates. Biochemistry 2010, 49 (37), 8085– 8093,  DOI: 10.1021/bi100895j
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    Bronsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substrates
    Sengerova, Blanka; Tomlinson, Christopher; Atack, John M.; Williams, Ryan; Sayers, Jon R.; Williams, Nicholas H.; Grasby, Jane A.
    Biochemistry (2010), 49 (37), 8085-8093CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    During replication and repair flap endonucleases (FENs) catalyze endonucleolytic and exonucleolytic (EXO) DNA hydrolyzes. Altering the leaving group pKa, by replacing the departing nucleoside with analogs, had minimal effect on kcat/Km in a T5FEN-catalyzed EXO reaction, producing a very low Bronsted coeff., βlg. Investigation of the viscosity dependence of kcat/Km revealed that reactions of EXO substrates are rate limited by diffusional encounter of enzyme and substrate, explaining the small βlg. However, the maximal single turnover rate of the FEN EXO reaction also yields a near zero βlg. A low βlg was also obsd. when evaluating kcat/Km for D201I/D204S FEN-catalyzed reactions, even though these reactions were not affected by added viscogen. But an active site K83A mutant produced a βlg = -1.2±0.10, closer to the value obsd. for soln. hydrolysis of phosphate diesters. The pH-maximal rate profiles of the WT and K83A FEN reactions both reach a max. at high pH and do not support an explanation of the data that involves catalysis of leaving group departure by Lys 83 functioning as a general acid. Instead, a rate-limiting phys. step, such as substrate unpairing or helical arch ordering, that occurs after substrate assocn. must kinetically hide an inherent large βlg. It is suggested that K83 acts as an electrostatic catalyst that stabilizes the transition state for phosphate diester hydrolysis. When K83 is removed from the active site, chem. becomes rate limiting and the leaving group sensitivity of the FEN-catalyzed reaction is revealed.
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    Taylor, J. D.; Halford, S. E. Discrimination between DNA Sequences by the EcoRV Restriction Endonuclease. Biochemistry 1989, 28 (15), 6198– 6207,  DOI: 10.1021/bi00441a011
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    Discrimination between DNA sequences by the EcoRV restriction endonuclease
    Taylor, John D.; Halford, Stephen E.
    Biochemistry (1989), 28 (15), 6198-207CODEN: BICHAW; ISSN:0006-2960.
    Restrictive endonuclease EcoRV cleaves not only its recognition sequence on DNA (GATAC), but also, at vastly reduced rates, a no. of alternative DNA sequences. Plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by 1 base pair. EcoRV nuclease showed a marked preference for one particular site from among these alternatives. This noncognate site was located at sequence GTTATC, and the mechanism of action of EcRV at this site was analyzed. The mechanism differed from that at the cognate site in 3 respects. First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this could not not account for the specificity of EcoRV as measured from kcat/Km values. Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appeared to be a key factor in limiting the rates of DNA cleavage at alternative sites. Third, the reaction pathway at the noncognate site differed from that at the cognate site. At the former, EcoRV cleaved 1st one strand of the DNA and then the other, whereas at the latter, both strands were cut in a single concerted reaction. The difference in reaction pathway allowed DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site. The addn. of DNA ligase to reactions with EcoRV made no differences to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.
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    Yang, C. C.; Baxter, B. K.; Topal, M. D. DNA Cleavage by NaeI: Protein Purification, Rate-Limiting Step, and Accuracy. Biochemistry 1994, 33 (49), 14918– 14925,  DOI: 10.1021/bi00253a031
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    DNA Cleavage by NaeI: Protein Purification, Rate-Limiting Step, and Accuracy
    Yang, Charles C.; Baxter, Bonnie K.; Topal, Michael D.
    Biochemistry (1994), 33 (49), 14918-25CODEN: BICHAW; ISSN:0006-2960.
    NaeI endonuclease must bind two DNA sites for cleavage to occur. NaeI was purified to apparent homogeneity and used to det. the rate-limiting step for DNA cleavage and to measure NaeI's specificity for its cognate recognition site. Steady-state cleavage by NaeI in the presence of effector DNA (activated) gave values of 0.045 s-1 and 10 nM for kcat and KM for M13 DNA substrate, resp., but values of 0.4 s-1 and 170 nM, resp., for an M13 DNA fragment substrate. Single-turnover cleavage of M13 DNA demonstrated that DNA strand scission is not rate-limiting for turnover of NaeI. Transient kinetic anal. of M13 DNA cleavage by NaeI showed an initial burst of substrate cleavage that was proportional to NaeI concn., implying that product release is rate-limiting for turnover of NaeI. The NaeI effector and substrate binding sites were found to prefer cognate over noncognate sequences by 103-fold and at least 40-500-fold, resp. Kcat for noncognate recognition sequence was at least 106-fold lower than that for cognate. The specificity of activated NaeI, as measured by kcat/KM, for noncognate recognition sequence was 108-fold lower than that for cognate, and over 1011-fold lower when the decreased affinity for noncognate sequence at the effector binding site was taken into account. This specificity is approx. 104-fold larger than for any other restriction enzyme measured.
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    Williams, R.; Sengerova, B.; Osborne, S.; Syson, K.; Ault, S.; Kilgour, A.; Chapados, B. R.; Tainer, J. A.; Sayers, J. R.; Grasby, J. A. Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease. J. Mol. Biol. 2007, 371 (1), 34– 48,  DOI: 10.1016/j.jmb.2007.04.063
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    Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease
    Williams, Ryan; Sengerova, Blanka; Osborne, Sadie; Syson, Karl; Ault, Sophie; Kilgour, Anna; Chapados, Brian R.; Tainer, John A.; Sayers, Jon R.; Grasby, Jane A.
    Journal of Molecular Biology (2007), 371 (1), 34-48CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Flap endonucleases (FENs) catalyze the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalyzed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterized. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs contg. two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover nos. for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilize the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concns. below satn. with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.
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    Finger, D. L.; Blanchard, M. S.; Theimer, C. A.; Sengerová, B.; Singh, P. S.; Chavez, V.; Liu, F.; Grasby, J. A.; Shen, B. The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis. J. Biol. Chem. 2009, 284 (33), 22184– 22194,  DOI: 10.1074/jbc.M109.015065
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    The 3'-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis
    Finger, L. David; Blanchard, M. Suzette; Theimer, Carla A.; Sengerova, Blanka; Singh, Purnima; Chavez, Valerie; Liu, Fei; Grasby, Jane A.; Shen, Binghui
    Journal of Biological Chemistry (2009), 284 (33), 22184-22194CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest esp. in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that although they share similar folds, the FEN1s of higher organisms contain a 3'-extrahelical nucleotide (3'-flap) binding pocket. When presented with 5'-flap substrates having a 3'-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well-defined DNA substrates was conducted. The presence of a 3'-flap on substrates reduced Km and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiol. salt concns. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3'-flap, and the absence of a 3'-flap from a 5'-flap substrate was more detrimental to hFEN1 activity than removal of the 5'-flap or introduction of a hairpin into the 5'-flap structure. HFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3'-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5'-phosphorylated product. Together the results indicate that the presence of a 3'-flap is the crit. feature for efficient hFEN1 substrate recognition and catalysis.
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    Kuznetsova, A. A.; Fedorova, O. S.; Kuznetsov, N. A. Kinetic Features of 3′-5′ Exonuclease Activity of Human AP-Endonuclease APE1. Molecules 2018, 23 (9), 2101,  DOI: 10.3390/molecules23092101
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    Kinetic features of 3'-5' exonuclease activity of human AP-endonuclease APE1
    Kuznetsova, Alexandra A.; Fedorova, Olga S.; Kuznetsov, Nikita A.
    Molecules (2018), 23 (9), 2101/1-2101/14CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5' to the damage. In addn. to the AP-endonuclease activity, APE1 possesses 3'-5' exonuclease activity, which presumably is responsible for cleaning up nonconventional 3' ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3'-end nucleotide removal in the 3'-5' exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5' dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3' end of the short oligonucleotide. The impact of the 3'-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3'-5' exonuclease activity was detd. Kinetic data revealed that the rate-limiting step of 3' nucleotide removal by APE1 in the 3'-5' exonuclease process is the release of the detached nucleotide from the enzyme's active site.
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    Warshel, A.; Levitt, M. Theoretical Studies of Enzymic Reactions: Dielectric, Electrostatic and Steric Stabilization of the Carbonium Ion in the Reaction of Lysozyme. J. Mol. Biol. 1976, 103 (2), 227– 249,  DOI: 10.1016/0022-2836(76)90311-9
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    Theoretical studies of enzymic reactions: dielectric, electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme
    Warshel, A.; Levitt, M.
    Journal of Molecular Biology (1976), 103 (2), 227-49CODEN: JMOBAK; ISSN:0022-2836.
    A general method for detailed study of enzymic reactions is presented. The method considers the complete enzyme-substrate complex together with the surrounding solvent and evaluates all the different quantum mech. and classical energy factors that can affect the reaction pathway. These factors include the quantum mech. energies assocd. with bond cleavage and charge redistribution of the substrate and the classical energies of steric and electrostatic interactions between the substrate and the enzyme. The electrostatic polarization of the enzyme atoms and the orientation of the dipoles of the surrounding H2O mols. is simulated by a microscopic dielec. model. The solvation energy resulting from this polarization is considerable and must be included in any realistic calcn. of chem. reactions involving anything more than an isolated mol. in vacuo. Without it, acidic groups can never become ionized and the charge distribution on the substrate will not be reasonable. The same dielec. model can also be used to study the reaction of the substrate in soln. In this way the reaction in soln. can be compared with the enzymic reaction. The stability of the carbonium ion intermediate formed in the cleavage of a glycosidic bond by lysozyme was studied. Electrostatic stabilization is an important factor in increasing the rate of the reaction step that leads to the formation of the carbonium ion intermediate. Steric factors, such as the strain of the substrate on binding to lysozyme, do not seem to contribute significantly.
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    Brunk, E.; Rothlisberger, U. Mixed Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulations of Biological Systems in Ground and Electronically Excited States. Chem. Rev. 2015, 115 (12), 6217– 6263,  DOI: 10.1021/cr500628b
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    Mixed Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulations of Biological Systems in Ground and Electronically Excited States
    Brunk, Elizabeth; Rothlisberger, Ursula
    Chemical Reviews (Washington, DC, United States) (2015), 115 (12), 6217-6263CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The quantum nature of electrons and nuclei is manifested in countless biol. events, including the rearrangements of electrons in biochem. reactions, electron and proton tunneling, coupled proton-electron transfers, photoexcitations, and long-lived quantum coherences and quantum entanglement. Quantum mech. (QM) phenomena are thus at the core of fundamental biol. processes. The introduction of mixed quantum mech./mol. mech. (QM/MM) methods that allow one to treat electronic quantum phenomena in complex classical environments represented a seminal step toward the quantum mech. treatment of realistic biol. systems. Subsequent extension of the QM/MM approach from adiabatic simulations in the electronic ground state to nonadiabatic dynamics in electronically excited states added an addnl. layer of complexity by making it necessary to account for the quantum nature of nuclear degrees of freedom. The present review examines the current state of the art of QM/MM mol. dynamics approaches in ground and electronically excited states and their applications to biol. problems.
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    Stevens, D. R.; Hammes-Schiffer, S. Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations. J. Am. Chem. Soc. 2018, 140 (28), 8965– 8969,  DOI: 10.1021/jacs.8b05177
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    Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations
    Stevens, David R.; Hammes-Schiffer, Sharon
    Journal of the American Chemical Society (2018), 140 (28), 8965-8969CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The enzyme human DNA polymerase η (Pol η) is crit. for bypassing lesions during DNA replication. In addn. to the two Mg2+ ions aligning the active site, expts. suggest that a third Mg2+ ion could play an essential catalytic role. Herein the role of this third metal ion is investigated with quantum mech./mol. mech. (QM/MM) free energy simulations of the phosphoryl transfer reaction and a proposed self-activating proton transfer from the incoming nucleotide to the pyrophosphate leaving group. The simulations with only two metal ions in the active site support a sequential mechanism, with phosphoryl transfer followed by relatively fast proton transfer. The simulations with three metal ions in the active site suggest that the third metal ion may play a catalytic role through electrostatic interactions with the leaving group. These electrostatic interactions stabilize the product, making the phosphoryl transfer reaction more thermodynamically favorable with a lower free energy barrier relative to the activated state corresponding to the deprotonated 3'OH nucleophile, and also inhibit the subsequent proton transfer.
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    Field, M. J.; Bash, P. A.; Karplus, M. A Combined Quantum Mechanical and Molecular Mechanical Potential for Molecular Dynamics Simulations. J. Comput. Chem. 1990, 11 (6), 700– 733,  DOI: 10.1002/jcc.540110605
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    A combined quantum mechanical and molecular mechanical potential for molecular dynamics simulations
    Field, Martin J.; Bash, Paul A.; Karplus, Martin
    Journal of Computational Chemistry (1990), 11 (6), 700-33CODEN: JCCHDD; ISSN:0192-8651.
    A combined quantum mech. (QM) and mol. mech. (MM) potential has been developed for the study of reactions in condensed phases. For the quantum mech. calcns. semiempirical methods of the MNDO and AM1 type are used, while the mol. mechanics part is treated with the HARMM force field. Specific prescriptions are given for the interactions between the QM and MM portions of the system; cases in which the QM and MM methodol. is applied to parts of the same mol. or to different mols. are considered. The details of the method and a range of test calcns., including comparisons with ab initio and exptl. results, are given. In many cases satisfactory results are obtained. However, there are limitations to this type of approach, some of which arise from the AM1 or MNDO methods themselves and others from the present QM/MM implementation. This suggests that it is important to test the applicability of the method to each particular case prior to its use. Possible areas of improvement in the methodol. are discussed.
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    Needleman, S. B.; Wunsch, C. D. A General Method Applicable to Search for Similarities in Amino Acid Sequence of Two Proteins. J. Mol. Biol. 1970, 48 (3), 443– 453,  DOI: 10.1016/0022-2836(70)90057-4
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    General method applicable to the search for similarities in the amino acid sequence of two proteins
    Needleman, Saul B.; Wunsch, Christian D.
    Journal of Molecular Biology (1970), 48 (3), 443-53CODEN: JMOBAK; ISSN:0022-2836.
    A computer adaptable method for finding similarities in the amino acid sequences of two proteins has been developed, making it possible to det. whether significant homology exists between the proteins. This information is used to trace their possible evolutionary development. The max. match is a no. dependent upon the similarity of the sequences. One of its definitions is the largest no. of amino acids of one protein that can be matched with those of a second protein allowing for all possible interruptions in either of the sequences. While the interruptions give rise to a very large no. of comparisons, the method efficiently excludes from consideration those comparisons that cannot contribute to the max. match. Comparisons are made from the smallest unit of significance, a pair of amino acids, one from each protein.
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    Szymanski, M. R.; Yu, W.; Gmyrek, A. M.; White, M. A.; Molineux, I. J.; Lee, J. C.; Yin, Y. W. A Domain in Human EXOG Converts Apoptotic Endonuclease to DNA-Repair Exonuclease. Nat. Commun. 2017, 8, 14959,  DOI: 10.1038/ncomms14959
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    A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease
    Szymanski Michal R; Yu Wangsheng; Yin Y Whitney; Szymanski Michal R; Yu Wangsheng; White Mark A; Lee J Ching; Yin Y Whitney; Gmyrek Aleksandra M; White Mark A; Lee J Ching; Molineux Ian J
    Nature communications (2017), 8 (), 14959 ISSN:.
    Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 ÅA resolution, respectively. A Wing domain, absent in other ββα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic 'tape-measure'. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase γ.
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    Wu, C.-C.; Lin, J. L. J.; Yang-Yen, H.-F.; Yuan, H. S. A Unique Exonuclease ExoG Cleaves between RNA and DNA in Mitochondrial DNA Replication. Nucleic Acids Res. 2019, 47 (10), 5405– 5419,  DOI: 10.1093/nar/gkz241
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    A unique exonuclease ExoG cleaves between RNA and DNA in mitochondrial DNA replication
    Wu, Chyuan-Chuan; Lin, Jason L. J.; Yang-Yen, Hsin-Fang; Yuan, Hanna S.
    Nucleic Acids Research (2019), 47 (10), 5405-5419CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5' end of nascent DNA. A subsequent 5'-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5'-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA-DNA junctions to remove the 5'-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound resp. with DNA, RNA/DNA hybrid and RNA-DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA-DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the mol. basis of ExoG functioning as a unique 5'-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.
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  67. 67
    Zhang, J.; Mccabe, K. A.; Bell, C. E. Crystal Structures of λ Exonuclease in Complex with DNA Suggest an Electrostatic Ratchet Mechanism for Processivity. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (29), 11872– 11877,  DOI: 10.1073/pnas.1103467108
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    67
    Crystal structures of λ exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity
    Zhang, Jinjin; McCabe, Kimberly A.; Bell, Charles E.
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (29), 11872-11877, S11872/1-S11872/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5'-ended strand to form 5' mononucleotides and a long 3' overhang. The crystal structure of λ exonuclease revealed a toroidal homo-trimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topol. linkage of the trimer to the DNA was proposed. Here, we have detd. the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resoln. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5'-ended strand insert into the active site of one subunit of the trimer, while the 3'-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5' phosphate of the DNA binds to a pos. charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg2+ ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5' phosphate to the pos. charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.
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  68. 68
    Zhang, J.; Pan, X.; Bell, C. E. Crystal Structure of λ Exonuclease in Complex with DNA and Ca2+. Biochemistry 2014, 53 (47), 7415– 7425,  DOI: 10.1021/bi501155q
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    68
    Crystal Structure of λ Exonuclease in Complex with DNA and Ca2+
    Zhang, Jinjin; Pan, Xinlei; Bell, Charles E.
    Biochemistry (2014), 53 (47), 7415-7425CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Bacteriophage λ exonuclease (λexo) is a ring-shaped homotrimer that resects double-stranded DNA ends in the 5'-3' direction to generate a long 3'-overhang that is a substrate for recombination. λexo is a member of the type II restriction endonuclease-like superfamily of proteins that use a Mg2+-dependent mechanism for nucleotide cleavage. A previous structure of λexo in complex with DNA and Mg2+ was detd. using a nuclease-defective K131A variant to trap a stable complex. This structure revealed the detailed coordination of the two active site Mg2+ ions but did not show the interactions involving the side chain of the conserved active site Lys-131 residue. Here, we have detd. the crystal structure of wild-type (WT) λexo in complex with the same DNA substrate, but in the presence of Ca2+ instead of Mg2+. Surprisingly, there is only one Ca2+ bound in the active site, near the position of MgA in the structure with Mg2+. The scissile phosphate is displaced by 2.2 Å relative to its position in the structure with Mg2+, and the network of interactions involving the attacking water mol. is broken. Thus, the structure does not represent a catalytic configuration. However, the crystal structure does show clear electron d. for the side chain of Lys-131, which is held in place by interactions with Gln-157 and Glu-129. By combining the K131A-Mg2+ and WT-Ca2+ structures, we constructed a composite model to show the likely interactions of Lys-131 during catalysis. The implications with regard to the catalytic mechanism are discussed.
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  69. 69
    Cheng, K.; Xu, H.; Chen, X.; Wang, L.; Tian, B.; Zhao, Y.; Hua, Y. Structural Basis for DNA 5′-End Resection by RecJ. eLife 2016, 5, e14294  DOI: 10.7554/eLife.14294
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    69
    Structural basis for DNA 5'-end resection by RecJ
    Cheng, Kaiying; Xu, Hong; Chen, Xuanyi; Wang, Liangyan; Tian, Bing; Zhao, Ye; Hua, Yuejin
    eLife (2016), 5 (), e14294/1-e14294/21CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    The resection of DNA strand with a 5' end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5' nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5'-phosphate-binding pocket above the active site dets. the 5'-3' polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180 ° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination.
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  70. 70
    Zhang, F.; Shi, J.; Chen, S. H.; Bian, C.; Yu, X. The PIN Domain of EXO1 Recognizes Poly(ADP-Ribose) in DNA Damage Response. Nucleic Acids Res. 2015, 43 (22), 10782– 10794,  DOI: 10.1093/nar/gkv939
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    70
    The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response
    Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun
    Nucleic Acids Research (2015), 43 (22), 10782-10794CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Following DNA double-strand breaks, poly(ADPribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a no. of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We obsd. that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response.
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    Genna, V.; Carloni, P.; De Vivo, M. A Strategically Located Arg/Lys Residue Promotes Correct Base Paring during Nucleic Acid Biosynthesis in Polymerases. J. Am. Chem. Soc. 2018, 140 (9), 3312– 3321,  DOI: 10.1021/jacs.7b12446
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    71
    A Strategically Located Arg/Lys Residue Promotes Correct Base Paring During Nucleic Acid Biosynthesis in Polymerases
    Genna, Vito; Carloni, Paolo; De Vivo, Marco
    Journal of the American Chemical Society (2018), 140 (9), 3312-3321CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Polymerases (Pols) synthesize the double-stranded nucleic acids in the Watson-Crick (W-C) conformation, which is crit. for DNA and RNA functioning. Yet, the mol. basis to catalyze the W-C base pairing during Pol-mediated nucleic acids biosynthesis remains unclear. Here, through bioinformatics analyses on a large data set of Pol/DNA structures, we first describe the conserved presence of one pos. charged residue (Lys or Arg), which is similarly located near the enzymic two-metal active site, always interacting directly with the incoming substrate (d)NTP. Incidentally, we noted that some Pol/DNA structures showing the alternative Hoogsteen base pairing were often solved with this specific residue either mutated, displaced, or missing. We then used quantum and classical simulations coupled to free-energy calcns. to illustrate how, in human DNA Pol-η, the conserved Arg61 favors W-C base pairing through defined interactions with the incoming nucleotide. Taken together, these structural observations and computational results suggest a structural framework in which this specific residue is crit. for stabilizing the incoming (d)NTP nucleotide and base pairing during Pol-mediated nucleic acid biosynthesis. These results may benefit enzyme engineering for nucleic acid processing and encourage new drug discovery strategies to modulate Pols function.
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    Palermo, G.; Miao, Y.; Walker, R. C.; Jinek, M.; McCammon, J. A. CRISPR-Cas9 Conformational Activation as Elucidated from Enhanced Molecular Simulations. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (28), 7260– 7265,  DOI: 10.1073/pnas.1707645114
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    72
    CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations
    Palermo, Giulia; Miao, Yinglong; Walker, Ross C.; Jinek, Martin; McCammon, J. Andrew
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (28), 7260-7265CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    CRISPR-Cas9 has become a facile genome editing technol., yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive mol. simulations in an enhanced sampling regime, using a Gaussian-accelerated mol. dynamics (GaMD) methodol., which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a pos. charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose exptl. characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage. These results provide at.-level information on the mol. mechanism of CRISPR-Cas9 that will inspire future exptl. investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.
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    Mulholland, A. J.; Roitberg, A. E.; Tuñón, I. Enzyme Dynamics and Catalysis in the Mechanism of DNA Polymerase. Theor. Chem. Acc. 2012, 131, 1286,  DOI: 10.1007/s00214-012-1286-8
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    Riccardi, L.; Genna, V.; De Vivo, M. Metal–Ligand Interactions in Drug Design. Nat. Rev. Chem. 2018, 2 (7), 100– 112,  DOI: 10.1038/s41570-018-0018-6
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    Metal-ligand interactions in drug design
    Riccardi, Laura; Genna, Vito; De Vivo, Marco
    Nature Reviews Chemistry (2018), 2 (7), 100-112CODEN: NRCAF7; ISSN:2397-3358. (Nature Research)
    The fast-growing body of exptl. data on metalloenzymes and organometallic compds. is fostering the exploitation of metal-ligand interactions for the design of new drugs. Atomistic understanding of the metal-ligand interactions will help us identify potent metalloenzyme inhibitors and metallodrugs. Static docking calcns. have proved effective in identifying hit compds. that target metalloproteins. However, the flexibility, dynamics and electronic structure of metal-centered complexes pose difficult challenges for shaping metal-ligand interactions in structure-based drug design. In this respect, once-prohibitive quantum mechanics-based strategies and extensive mol. simulations are rapidly becoming practical approaches for fast-paced drug discovery. These methods account for ligand exchange and structural flexibility at metal-centered complexes and provide good ests. of the thermodn. and kinetics of metal-aided drug binding. This Perspective examines the successes, limitations and new avenues for modeling metalloenzyme inhibitors and metallodrugs to further explore and expand the unconventional chem. space of these distinctive drugs.
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    De Vivo, M. Bridging Quantum Mechanics and Structure-Based Drug Design. Front. Biosci., Landmark Ed. 2011, 16, 1619– 1633,  DOI: 10.2741/3809
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    Bridging quantum mechanics and structure-based drug design
    De Vivo, Marco
    Frontiers in Bioscience, Landmark Edition (2011), 16 (5), 1619-1633CODEN: FRBIF6; ISSN:1093-4715. (Frontiers in Bioscience)
    A review. The last decade has seen great advances in the use of quantum mechanics (QM) to solve biol. problems of pharmaceutical relevance. For instance, enzymic catalysis is often investigated by means of the so-called QM/MM approach, which uses QM and mol. mechanics (MM) methods to det. the (free) energy landscape of the enzymic reaction mechanism. Here, I will discuss a few representative examples of QM and QM/MM studies of important metalloenzymes of pharmaceutical interest (i.e. metallophosphatases and metallo-beta-lactamases). This review article aims to show how QM-based methods can be used to elucidate ligand-receptor interactions. The challenge is then to exploit this knowledge for the structure-based design of new and potent inhibitors, such as transition state (TS) analogs that resemble the structure and physicochem. properties of the enzymic TS. Given the results and potential expressed to date by QM-based methods in studying biol. problems, the application of QM in structure-based drug design will likely increase, making of these once-prohibitive computations a routinely used tool for drug design.
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    Genna, V.; Marcia, M.; De Vivo, M. A Transient and Flexible Cation−π Interaction Promotes Hydrolysis of Nucleic Acids in DNA and RNA Nucleases. J. Am. Chem. Soc. 2019, 141 (27), 10770– 10776,  DOI: 10.1021/jacs.9b03663
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    76
    A Transient and Flexible Cation-π Interaction Promotes Hydrolysis of Nucleic Acids in DNA and RNA Nucleases
    Genna, Vito; Marcia, Marco; Vivo, Marco De
    Journal of the American Chemical Society (2019), 141 (27), 10770-10776CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Metal-dependent DNA and RNA nucleases are enzymes that cleave nucleic acids with great efficiency and precision. These enzyme-mediated hydrolytic reactions are fundamental for the replication, repair, and storage of genetic information within the cell. Here, extensive classical and quantum-based free-energy mol. simulations show that a cation-π interaction is transiently formed in situ at the metal core of Bacteriophage-λ Exonuclease (Exo-λ), during catalysis. This noncovalent interaction (Lys131-Tyr154) triggers nucleophile activation for nucleotide excision. Then, our simulations also show the oscillatory dynamics and swinging of the newly formed cation-π dyad, whose conformational change may favor proton release from the cationic Lys131 to the bulk soln., thus restoring the precatalytic protonation state in Exo-λ. Altogether, we report on the novel mechanistic character of cation-π interactions for catalysis. Structural and bioinformatic analyses support that flexible orientation and transient formation of mobile cation-π interactions may represent a common catalytic strategy to promote nucleic acid hydrolysis in DNA and RNA nucleases.
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    Yan, C.; Dodd, T.; He, Y.; Tainer, J. A.; Tsutakawa, S. E.; Ivanov, I. Transcription Preinitiation Complex Structure and Dynamics Provide Insight into Genetic Diseases. Nat. Struct. Mol. Biol. 2019, 26 (6), 397– 406,  DOI: 10.1038/s41594-019-0220-3
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    Transcription preinitiation complex structure and dynamics provide insight into genetic diseases
    Yan, Chunli; Dodd, Thomas; He, Yuan; Tainer, John A.; Tsutakawa, Susan E.; Ivanov, Ivaylo
    Nature Structural & Molecular Biology (2019), 26 (6), 397-406CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
    Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly's global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE-p62 interactions that link core-PIC to TFIIH.p62 rigging interlaces p34, p44 and XPD while capping the DNA-binding and ATP-binding sites of XPD. PIC kinks and locks substrate DNA, creating neg.supercoiling within the Pol II cleft to facilitate promoter opening. Mapping disease mutations assocd. with xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome onto defined communities reveals clustering into three mechanistic classes that affect TFIIH helicase functions, protein interactions and interface dynamics.
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  78. 78
    Casalino, L.; Palermo, G.; Spinello, A.; Rothlisberger, U.; Magistrato, A. All-Atom Simulations Disentangle the Functional Dynamics Underlying Gene Maturation in the Intron Lariat Spliceosome. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (26), 6584– 6589,  DOI: 10.1073/pnas.1802963115
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    78
    All-atom simulations disentangle the functional dynamics underlying gene maturation in the intron lariat spliceosome
    Casalino, Lorenzo; Palermo, Giulia; Spinello, Angelo; Rothlisberger, Ursula; Magistrato, Alessandra
    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (26), 6584-6589CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The spliceosome (SPL) is a majestic macromol. machinery composed of five small nuclear RNAs and hundreds of proteins. SPL removes noncoding introns from precursor mRNAs (pre-mRNAs) and ligates coding exons, giving rise to functional mRNAs. Building on the first SPL structure solved at near-at.-level resoln., here we elucidate the functional dynamics of the intron lariat spliceosome (ILS) complex through multi-microsecond-long mol.-dynamics simulations of ∼1,000,000 atoms models. The ILS essential dynamics unveils (i) the leading role of the Spp42 protein, which heads the gene maturation by tuning the motions of distinct SPL components, and (ii) the crit. participation of the Cwf19 protein in displacing the intron lariat/U2 branch helix. These findings provide unprecedented details on the SPL functional dynamics, thus contributing to move a step forward toward a thorough understanding of eukaryotic pre-mRNA splicing.
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    Orellana, L.; Thorne, A. H.; Lema, R.; Gustavsson, J.; Parisian, A. D.; Hospital, A.; Cordeiro, T. N.; Bernadó, P.; Scott, A. M.; Brun-Heath, I.; Lindahl, E.; Cavenee, W. K.; Furnari, F. B.; Orozco, M. Oncogenic Mutations at the EGFR Ectodomain Structurally Converge to Remove a Steric Hindrance on a Kinase-Coupled Cryptic Epitope. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (20), 10009– 10018,  DOI: 10.1073/pnas.1821442116
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    79
    Oncogenic mutations at the EGFR ectodomain structurally converge to remove a steric hindrance on a kinase-coupled cryptic epitope
    Orellana, Laura; Thorne, Amy H.; Lema, Rafael; Gustavsson, Johan; Parisian, Alison D.; Hospital, Adam; Cordeiro, Tiago N.; Bernado, Pau; Scott, Andrew M.; Brun-Heath, Isabelle; Lindahl, Erik; Cavenee, Webster K.; Furnari, Frank B.; Orozco, Modesto
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (20), 10009-10018CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Epidermal growth factor receptor (EGFR) signaling is initiated by a large ligand-favored conformational change of the extracellular domain (ECD) from a closed, self-inhibited tethered monomer, to an open untethered state, which exposes a loop required for strong dimerization and activation. In glioblastomas (GBMs), structurally heterogeneous missense and deletion mutations conc. at the ECD for unclear reasons. We explore the conformational impact of GBM missense mutations, combining elastic network models (ENMs) with multiple mol. dynamics (MD) trajectories. Our simulations reveal that the main missense class, located at the I-II interface away from the self-inhibitory tether, can unexpectedly favor spontaneous untethering to a compact intermediate state, here validated by small-angle X-ray scattering (SAXS). Significantly, such intermediate is characterized by the rotation of a large ECD fragment (N-TR1), deleted in the most common GBM mutation, EGFRvIII, and that makes accessible a cryptic epitope characteristic of cancer cells. This observation suggested potential structural equivalence of missense and deletion ECD changes in GBMs. Corroborating this hypothesis, our FACS, in vitro, and in vivo data demonstrate that entirely different ECD variants all converge to remove N-TR1 steric hindrance from the 806-epitope, which we show is allosterically coupled to an intermediate kinase and hallmarks increased oncogenicity. Finally, the detected extraintracellular coupling allows for synergistic cotargeting of the intermediate with mAb806 and inhibitors, which is proved herein.
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    Palermo, G.; Casalino, L.; Magistrato, A.; McCammon, A. J. Understanding the Mechanistic Basis of Non-Coding RNA through Molecular Dynamics Simulations. J. Struct. Biol. 2019, 206 (3), 267– 279,  DOI: 10.1016/j.jsb.2019.03.004
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    Understanding the mechanistic basis of non-coding RNA through molecular dynamics simulations
    Palermo, Giulia; Casalino, Lorenzo; Magistrato, Alessandra; Andrew McCammon, J.
    Journal of Structural Biology (2019), 206 (3), 267-279CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)
    A review. Noncoding RNA (ncRNA) has a key role in regulating gene expression, mediating fundamental processes and diseases via a variety of yet unknown mechanisms. Here, we review recent applications of conventional and enhanced Mol. Dynamics (MD) simulations methods to address the mechanistic function of large biomol. systems that are tightly involved in the ncRNA function and that are of key importance in life sciences. This compendium focuses of three biomol. systems, namely the CRISPR-Cas9 genome editing machinery, group II intron ribozyme and the ribonucleoprotein complex of the spliceosome, which edit and process ncRNA. We show how the application of a novel accelerated MD simulations method has been key in disclosing the conformational transitions underlying RNA binding in the CRISPR-Cas9 complex, suggesting a mechanism for RNA recruitment and clarifying the conformational changes required for attaining genome editing. As well, we discuss the use of mixed quantum-classical MD simulations in deciphering the catalytic mechanism of RNA splicing as operated by group II intron ribozyme, one of the largest ncRNA structures crystd. so far. Finally, we debate the future challenges and opportunities in the field, discussing the recent application of MD simulations for unraveling the functional biophysics of the spliceosome, a multi-mega Dalton complex of proteins and small nuclear RNAs that performs RNA splicing in humans. This showcase of applications highlights the current talent of MD simulations to dissect at.-level details of complex biomol. systems instrumental for the design of finely engineered genome editing machines. As well, this review aims at inspiring future investigations of several other ncRNA regulatory systems, such as micro and small interfering RNAs, which achieve their function and specificity using RNA-based recognition and targeting strategies.
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    Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
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    81
    ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
    Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
    Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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    Galindo-Murillo, R.; Robertson, J. C.; Zgarbová, M.; Šponer, J.; Otyepka, M.; Jurečka, P.; Cheatham, T. E. Assessing the Current State of Amber Force Field Modifications for DNA. J. Chem. Theory Comput. 2016, 12 (8), 4114– 4127,  DOI: 10.1021/acs.jctc.6b00186
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    Assessing the Current State of Amber Force Field Modifications for DNA
    Galindo-Murillo, Rodrigo; Robertson, James C.; Zgarbova, Marie; Sponer, Jiri; Otyepka, Michal; Jurecka, Petr; Cheatham, Thomas E.
    Journal of Chemical Theory and Computation (2016), 12 (8), 4114-4127CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    The utility of mol. dynamics (MD) simulations to model biomol. structure, dynamics, and interactions has witnessed enormous advances in recent years due to the availability of optimized MD software and access to significant computational power, including GPU multicore computing engines and other specialized hardware. This has led researchers to routinely extend conformational sampling times to the microsecond level and beyond. The extended sampling time has allowed the community not only to converge conformational ensembles through complete sampling but also to discover deficiencies and overcome problems with the force fields. Accuracy of the force fields is a key component, along with sampling, toward being able to generate accurate and stable structures of biopolymers. The Amber force field for nucleic acids has been used extensively since the 1990s, and multiple artifacts have been discovered, cor., and reassessed by different research groups. We present a direct comparison of two of the most recent and state-of-the-art Amber force field modifications, bsc1 and OL15, that focus on accurate modeling of double-stranded DNA. After extensive MD simulations with five test cases and two different water models, we conclude that both modifications are a remarkable improvement over the previous bsc0 force field. Both force field modifications show better agreement when compared to exptl. structures. To ensure convergence, the Drew-Dickerson dodecamer (DDD) system was simulated using 100 independent MD simulations, each extended to at least 10 μs, and the independent MD simulations were concatenated into a single 1 ms long trajectory for each combination of force field and water model. This is significantly beyond the time scale needed to converge the conformational ensemble of the internal portions of a DNA helix absent internal base pair opening. Considering all of the simulations discussed in the current work, the MD simulations performed to assess and validate the current force fields and water models aggregate over 14 ms of simulation time. The results suggest that both the bsc1 and OL15 force fields render av. structures that deviate significantly less than 1 Å from the av. exptl. structures. This can be compared to similar but less exhaustive simulations with the CHARMM 36 force field that aggregate to the ∼90 μs time scale and also perform well but do not produce structures as close to the DDD NMR av. structures (with root-mean-square deviations of 1.3 Å) as the newer Amber force fields. On the basis of these analyses, any future research involving double-stranded DNA simulations using the Amber force fields should employ the bsc1 or OL15 modification.
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    How accurate are accurate force-fields for B-DNA?
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    Nucleic Acids Research (2017), 45 (7), 4217-4230CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Last generation of force-fields are raising expectations on the quality of mol. dynamics (MD) simulations of DNA, as well as to the belief that theor. models can substitute exptl. ones in several cases. However these claims are based on limited benchmarks, where MD simulations have shown the ability to reproduce already existing 'exptl. models', which in turn, have an unclear accuracy to represent DNA conformation in soln. In this work we explore the ability of different force-fields to predict the structure of two new B-DNA dodecamers, detd. herein by means of 1H NMR. The study allowed us to check directly for exptl. NMR observables on duplexes previously not solved, and also to assess the reliability of 'exptl. structures'. We obsd. that tech. details in the annealing procedures can induce non-negligible local changes in the final structures. We also found that while not all theor. simulations are equally reliable, those obtained using last generation of AMBER force-fields (BSC1 and BSC0OL15) show predictive power in the multi-microsecond timescale and can be safely used to reproduce global structure of DNA duplexes and fine sequence-dependent details.
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    Development and testing of a general Amber force field
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  2. Rajwinder Kaur, Dylan J. Nikkel, Stacey D. Wetmore. Mechanism of Nucleic Acid Phosphodiester Bond Cleavage by Human Endonuclease V: MD and QM/MM Calculations Reveal a Versatile Metal Dependence. The Journal of Physical Chemistry B 2024, 128 (39) , 9455-9469. https://doi.org/10.1021/acs.jpcb.4c05846
  3. Dylan J. Nikkel, Stacey D. Wetmore. Distinctive Formation of a DNA–Protein Cross-Link during the Repair of DNA Oxidative Damage: Insights into Human Disease from MD Simulations and QM/MM Calculations. Journal of the American Chemical Society 2023, 145 (24) , 13114-13125. https://doi.org/10.1021/jacs.3c01773
  4. Elisa Donati, Pietro Vidossich, Marco De Vivo. Molecular Mechanism of Phosphate Steering for DNA Binding, Cleavage Localization, and Substrate Release in Nucleases. ACS Catalysis 2021, 11 (21) , 13244-13254. https://doi.org/10.1021/acscatal.1c03346
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  6. Marco Marcia, Jacopo Manigrasso, Marco De Vivo. Finding the Ion in the RNA-Stack: Can Computational Models Accurately Predict Key Functional Elements in Large Macromolecular Complexes?. Journal of Chemical Information and Modeling 2021, 61 (6) , 2511-2515. https://doi.org/10.1021/acs.jcim.1c00572
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Cite this: J. Am. Chem. Soc. 2020, 142, 6, 2823–2834
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  • Abstract

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    Figure 1

    Figure 1. Catalytic domain of hExo1 in complex with DNA substrate and the two catalytic metal ions (PDB ID 5V06). (Left) hExo1 in cartoon and with colors for different structural motifs. (Right) Closer view of the active site. Three metal ions (MgA, MgB, MgC) are in orange, nucleophilic water molecule (Nu) is in red, two guide residues (Tyr32, His36) in yellow, and residues of the catalytic pocket (Gly2, Asp30, Asp78, Asp152, Asp171, Asp173) are in cyan. Scissile phosphate is correctly positioned for the nucleophilic attack, and MgC is coordinated by the 5′ terminal phosphate.

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    Scheme 1

    Scheme 1. Schematic Representation of the Motion of the Transient Third Metal Ion, Which We Found to Be Intermittently Recruited/Released by Glu89 (switching its inner/outer conformations) during Exonuclease Catalysisa
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    aThis structural evidence suggests that the transient third ion may be crucial for substrate hydrolysis and/or leaving group departure.

    Figure 2

    Figure 2. Radial distribution function, g(r), calculated for ions around 10 Å from the center of mass of the 5′ phosphate group. Plot shows the presence of ions ∼3 Å from the 5′ phosphate group for the RS2M system. In this system, a K+ ion approached the negatively charged group. For the RSGlu89Ala and PSGlu89Ala systems, there are no ions within ∼5.5 Å of the 5′ phosphate, as indicated by the g(r) values of ∼0. In the upper right corner, the 5′ phosphate group and Glu89Ala residues are shown in licorice (taken from the PSGlu89Ala simulations).

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    Figure 3

    Figure 3. (A) Distance (dMG in yellow) between the third Mg2+ ion, from the bulk (Mgbulk), and the phosphorus of the 5′ phosphate group of AMP. Inset, representation (snapshot from the PS2M simulations) of Mgbulk approaching the terminal 5′ phosphate as well as the pseudo-dihedral angle ϕ of Glu89 side chain (defined by the N–Cα–Cδ−-Cγ atoms). (B) Probability density of the pseudo-dihedral angle ϕ in Glu89 during the simulation: (blue) probability density for dMG values > 4 Å shows the outer conformation as the most populated; (red) probability density for dMG values < 4 Å shows the inner conformation is the most populated.

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    Figure 4

    Figure 4. (A) Graphic representation of d1 and d2 distances (PDB ID 5V0A). (B) Probability density of the distances d1 and d2 calculated during simulations of the systems PS2M (in red), PS3M (in orange), and PSGlu89Ala (in green).

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    Figure 5

    Figure 5. (Bottom) Free energy surface obtained through well-tempered metadynamics simulations for RS3M (red), RS2M (blue), PS2M (green), and PS3M (light purple) systems. Results show three conformations (outer, intermediate, inner). (Top) Graphic representations, taken from PS3M simulaitons, of the three conformations are shown in licorice.

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    Figure 6

    Figure 6. Free energy surface obtained through confined well-tempered metadynamics simulations for PS2M (blue) and PS3M (yellow) systems. (Top) Schematic representation of the CV1, exemplified using a snapshot from PS3M simulations. It represents the distance between the center of mass (COM) of the heavy atoms of the nucleotide leaving group and the COM of the Cα of the aspartates (Asp152, Asp171, Asp173) in the first coordination shell of MgA, MgB. Two different minima, at 8.8 and 12.4 Å, agree with the MD results, in which a partial exit of the leaving group (CV1 ≈ 12.4 Å) was seen only in the presence of MgC (Figure S8).

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    Figure 7

    Figure 7. Close views of the active site of 5′ metallonuclease members that possess an analogous acid residue (light green) close to the two-metal-ion center (MA, MB, in orange), the active site (in cyan), and the leaving group (indicated by a dashed line). (A) Human ExoG, in which Glu317 is pointing in the inner (PDB ID 5T5C) and (A′) outer conformations (PDB ID 5T40, merged with the DNA substrate from PDB ID 5T5C). (B) Escherichia phage T5Fen (PDB ID 5HNK). (C) Human λ-Exonuclease X (PDB ID 3SM4). (D) D. radiodurans RecJ (PDB ID 5F55). (E) Sequence alignment of M. smegmatis FenA and E. phage T5Fen. Conserved acid residue (Glu/Asp) is indicated in orange.

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      A review. The human genome encodes at least 14 DNA-dependent DNA polymerases - a surprisingly large no. These include the more abundant, high-fidelity enzymes that replicate the bulk of genomic DNA, together with eight or more specialized DNA polymerases that have been discovered in the past decade. Although the roles of the newly recognized polymerases are still being defined, one of their crucial functions is to allow synthesis past DNA damage that blocks replication-fork progression. We explore the reasons that might justify the need for so many DNA polymerases, describe their function and mode of regulation, and finally consider links between mutations in DNA polymerases and human disease.
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      Functional regulation of FEN1 nuclease and its link to cancer
      Zheng, Li; Jia, Jia; Finger, L. David; Guo, Zhigang; Zer, Cindy; Shen, Binghui
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      A review. Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5'-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochem. and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be crit. to regulate protein-protein interactions and cellular localizations of FEN1. Lastly, we also review the biol. significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models.
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      Dai, Yaoyao; Tang, Zuxiong; Yang, Zongguo; Zhang, Lan; Deng, Qing; Zhang, Xiaofeng; Yu, Yongchun; Liu, Xing; Zhu, Junfeng
      Cell Cycle (2018), 17 (19-20), 2386-2397CODEN: CCEYAS; ISSN:1551-4005. (Taylor & Francis Ltd.)
      The roles of exonuclease 1 (EXO1) in hepatocellular carcinoma (HCC) tumorigenesis and progression remain unclear. This study aimed to assess the prognostic value and therapeutic potential of EXO1 in HCC. Exo1 gene copy nos. were obtained from three Oncomine microarray datasets (n = 447). EXO1 mRNA expression was validated by semi-quant. PCR and QuantiGene 2.0 assays. Cell growth curve and colony formation were performed to asses the cell proliferation. Clonogenic assay, flow cytometry, and immunofluorescence were adopted to acess the effects of EXO1 knockdown and radiation on cell survival, cell cycle distribution and DNA repair. Western blots were performed to reveal the related mechanism. A significant copy no. variation (CNV) of the Exo1 gene was found in HCC specimens in three sep. sets of published microarray data. In the 143 cases treated by our team, EXO1 expression levels were elevated (86.71%, 124/143). In addn., EXO1 overexpression was correlated with larger tumor size (P = 0.002), increased lymph node metastasis (P=0.033) and lower Edmondson grade (P = 0.018). High EXO1 expression unfavorably affected overall survival (OS) (P = 0.009). Both univariate and multivariate Cox regression analyses identified EXO1 as an independent predictor of OS (univariate, P = 0.012; multivariate, P = 0.039). Silencing of EXO1 in vitro reduced cell proliferation. EXO1 knockdown further suppressed clonogenic cell survival, abrogated radiation-induced G2/M phase arrest, and enhanced γ-H2AX foci after exposure to irradn. The accumulation of ataxiatelangiectasia mutated (ATM) might partially regulate the EXO1 related radiosensitivity. In summary, EXO1 could be a promising prognostic marker, with a potential therapeutic value in HCC.
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    14. 14
      Ivanov, I.; Tainer, J. A.; McCammon, J. A. Unraveling the Three-Metal-Ion Catalytic Mechanism of the DNA Repair Enzyme Endonuclease IV. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (5), 1465– 1470,  DOI: 10.1073/pnas.0603468104
      14
      Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV
      Ivanov, Ivaylo; Tainer, John A.; McCammon, J. Andrew
      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (5), 1465-1470, S1465/1-S1465/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Endonuclease IV belongs to a class of important apurinic/apyrimidinic endonucleases involved in DNA repair. Although a structure-based mechanistic hypothesis has been put forth for this enzyme, the detailed catalytic mechanism has remained unknown. Using thermodn. integration in the context of ab initio quantum mechanics/mol. mechanics mol. dynamics, we examd. certain aspects of the phosphodiester cleavage step in the mechanism. We found the reaction proceeded through a synchronous bimol. (ANDN) mechanism with reaction free energy and barrier of -3.5 and 20.6 kcal/mol, in agreement with exptl. ests. In the course of the reaction the trinuclear active site of endonuclease IV underwent dramatic local conformational changes: shifts in the mode of coordination of both substrate and first-shell ligands. This qual. finding supports the notion that structural rearrangements in the active sites of multinuclear enzymes are integral to biol. function.
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    15. 15
      Prieto, J.; Redondo, P.; Merino, N.; Villate, M.; Montoya, G.; Blanco, F. J.; Molina, R. Structure of the I-SceI Nuclease Complexed with Its DsDNA Target and Three Catalytic Metal Ions. Acta Crystallogr., Sect. F: Struct. Biol. Commun. 2016, 72 (6), 473– 479,  DOI: 10.1107/S2053230X16007512
      15
      Structure of the I-SceI nuclease complexed with its dsDNA target and three catalytic metal ions
      Prieto, Jesus; Redondo, Pilar; Merino, Nekane; Villate, Maider; Montoya, Guillermo; Blanco, Francisco J.; Molina, Rafael
      Acta Crystallographica, Section F: Structural Biology Communications (2016), 72 (6), 473-479CODEN: ACSFEN; ISSN:2053-230X. (International Union of Crystallography)
      Homing endonucleases (HEs) are highly specific DNA-cleaving enzymes that recognize and cleave long stretches of DNA. The engineering of these enzymes provides instruments for genome modification in a wide range of fields, including gene targeting. The homing endonuclease I-SceI from the yeast Saccharomyces cerevisiae has been purified after overexpression in Escherichia coli and its crystal structure has been detd. in complex with its target DNA. In order to evaluate the no. of ions that are involved in the cleavage process, thus detg. the catalytic mechanism, crystn. expts. were performed in the presence of Mn2+, yielding crystals that were suitable for X-ray diffraction anal. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 80.11, b = 80.57, c = 130.87 Å, α = β = γ = 90°. The self-rotation function and the Matthews coeff. suggested the presence of two protein-DNA complexes in the asym. unit. The crystals diffracted to a resoln. limit of 2.9 Å using synchrotron radiation. From the anomalous data, it was detd. that three cations are involved in catalysis and it was confirmed that I-SceI follows a two-metal-ion DNA-strand cleavage mechanism.
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    16. 16
      AlMalki, F. A.; Flemming, C. S.; Zhang, J.; Feng, M.; Sedelnikova, S. E.; Ceska, T.; Rafferty, J. B.; Sayers, J. R.; Artymiuk, P. J. Direct Observation of DNA Threading in Flap Endonuclease Complexes. Nat. Struct. Mol. Biol. 2016, 23 (7), 640– 646,  DOI: 10.1038/nsmb.3241
      16
      Direct observation of DNA threading in flap endonuclease complexes
      AlMalki Faizah A; Flemming Claudia S; Sedelnikova Svetlana E; Rafferty John B; Artymiuk Peter J; Zhang Jing; Feng Min; Sayers Jon R; Ceska Tom; Rafferty John B; Sayers Jon R; Sayers Jon R
      Nature structural & molecular biology (2016), 23 (7), 640-6 ISSN:.
      Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 ÅA. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.
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    17. 17
      Uson, M. L.; Carl, A.; Goldgur, Y.; Shuman, S. Crystal Structure and Mutational Analysis of Mycobacterium Smegmatis FenA Highlight Active Site Amino Acids and Three Metal Ions Essential for Flap Endonuclease and 5 Exonuclease Activities. Nucleic Acids Res. 2018, 46 (8), 4164– 4175,  DOI: 10.1093/nar/gky238
      17
      Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities
      Uson, Maria Loressa; Carl, Ayala; Goldgur, Yehuda; Shuman, Stewart
      Nucleic Acids Research (2018), 46 (8), 4164-4175CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).
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    18. 18
      Yang, W. Nucleases: Diversity of Structure, Function and Mechanism. Q. Rev. Biophys. 2011, 44 (1), 1– 93,  DOI: 10.1017/S0033583510000181
      18
      Nucleases: diversity of structure, function and mechanism
      Yang, Wei
      Quarterly Reviews of Biophysics (2011), 44 (1), 1-93CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
      A review. Nucleases cleave the phosphodiester bonds of nucleic acids and may be endo or exo, DNase or RNase, topoisomerases, recombinases, ribozymes, or RNA splicing enzymes. In this review, I survey nuclease activities with known structures and catalytic machinery and classify them by reaction mechanism and metal-ion dependence and by their biol. function ranging from DNA replication, recombination, repair, RNA maturation, processing, interference, to defense, nutrient regeneration or cell death. Several general principles emerge from this anal. There is little correlation between catalytic mechanism and biol. function. A single catalytic mechanism can be adapted in a variety of reactions and biol. pathways. Conversely, a single biol. process can often be accomplished by multiple tertiary and quaternary folds and by more than one catalytic mechanism. Two-metal-ion-dependent nucleases comprise the largest no. of different tertiary folds and mediate the most diverse set of biol. functions. Metal-ion-dependent cleavage is exclusively assocd. with exonucleases producing mononucleotides and endonucleases that cleave double- or single-stranded substrates in helical and base-stacked conformations. All metal-ion-independent RNases generate 2'.3'-cyclic phosphate products, and all metal-ion-independent DNases form phospho-protein intermediates. I also find several previously unnoted relationships between different nucleases and shared catalytic configurations.
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    19. 19
      Palermo, G.; Cavalli, A.; Klein, M. L.; Alfonso-Prieto, M.; Dal Peraro, M.; De Vivo, M. Catalytic Metal Ions and Enzymatic Processing of DNA and RNA. Acc. Chem. Res. 2015, 48 (2), 220– 228,  DOI: 10.1021/ar500314j
      19
      Catalytic metal ions and enzymatic processing of DNA and RNA
      Palermo, Giulia; Cavalli, Andrea; Klein, Michael L.; Alfonso-Prieto, Mercedes; Dal Peraro, Matteo; De Vivo, Marco
      Accounts of Chemical Research (2015), 48 (2), 220-228CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion (2M) mechanism. Several high-resoln. x-ray structures portraying the two-metal-aided catalytic site, together with mutagenesis and kinetics studies, have demonstrated a functional role of the ions for catalysis in numerous metallonucleases. Overall, the exptl. data confirm the general mechanistic hypothesis for 2M-aided phosphoryl transfer originally reported by T. A. Steitz and J. A. Steitz (1993). This seminal paper proposed that one metal ion favors the formation of the nucleophile, while the nearby 2nd metal ion facilitates leaving group departure during RNA hydrolysis. Both metals were suggested to stabilize the enzymic transition state. Nevertheless, static x-ray structures alone cannot exhaustively unravel how the two ions execute their functional role along the enzymic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states. Here, the authors discuss the role of multiscale mol. simulations in further disclosing mechanistic insights of 2M-aided catalysis for two prototypical enzymic targets for drug discovery, namely, RNase H (RNase H) and type II topoisomerase (topoII). In both examples, first-principles mol. simulations, integrated with structural data, emphasize a cooperative motion of the bimetal motif during catalysis. The coordinated motion of both ions is crucial for maintaining a flexible metal-centered structural architecture exquisitely tailored to accommodate the DNA or RNA sugar-phosphate backbone during phosphodiester bond cleavage. Furthermore, the anal. of RNase H and the N-terminal domain (PAN) of influenza polymerase shows that classical mol. dynamics simulations coupled with enhanced sampling techniques have contributed to describe the modulatory effect of metal ion concn. and metal uptake on the 2M mechanism and efficiency. These aspects all point to the emerging and intriguing role of addnl. adjacent ions potentially involved in the modulation of phosphoryl transfer reactions and enzymic turnover in 2M-catalysis, as recently obsd. exptl. in polymerase η and homing endonuclease I DmoI. These computational results, integrated with exptl. findings, describe and reinforce the nascent concept of a functional and cooperative dynamics of the catalytic metal ions during the 2M-dependent enzymic processing of DNA and RNA. Encouraged by the insights provided by computational approaches, the authors foresee further expts. that will feature the functional and joint dynamics of the catalytic metal ions for nucleic acid processing. This could impact the de novo design of artificial metallonucleases and the rational design of potent metal-chelating inhibitors of pharmaceutically relevant enzymes.
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    20. 20
      Palermo, G.; Stenta, M.; Cavalli, A.; Dal Peraro, M.; De Vivo, M. Molecular Simulations Highlight the Role of Metals in Catalysis and Inhibition of Type II Topoisomerase. J. Chem. Theory Comput. 2013, 9 (2), 857– 862,  DOI: 10.1021/ct300691u
      20
      Molecular Simulations Highlight the Role of Metals in Catalysis and Inhibition of Type II Topoisomerase
      Palermo, Giulia; Stenta, Marco; Cavalli, Andrea; Dal Peraro, Matteo; De Vivo, Marco
      Journal of Chemical Theory and Computation (2013), 9 (2), 857-862CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Type II topoisomerase (topoII) is a metalloenzyme targeted by clin. antibiotics and anticancer agents. Here, we integrate existing structural data with mol. simulation and propose a model for the yet uncharacterized structure of the reactant state of topoII. This model describes a canonical two-metal-ion mechanism and suggests how the metals could rearrange at the catalytic pocket during enzymic turnover, explaining also exptl. evidence for topoII inhibition. These results call for further exptl. validation.
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    21. 21
      Schmidt, B. H.; Burgin, A. B.; Deweese, J. E.; Osheroff, N.; Berger, J. M. A Novel and Unified Two-Metal Mechanism for DNA Cleavage by Type II and IA Topoisomerases. Nature 2010, 465 (7298), 641– 644,  DOI: 10.1038/nature08974
      21
      A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases
      Schmidt, Bryan H.; Burgin, Alex B.; Deweese, Joseph E.; Osheroff, Neil; Berger, James M.
      Nature (London, United Kingdom) (2010), 465 (7298), 641-644CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Type II topoisomerases are required for the management of DNA tangles and supercoils, and are targets of clin. antibiotics and anti-cancer agents. These enzymes catalyze the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or gates'. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyze G-segment cleavage. Recent efforts have proposed that strand scission relies on a "two-metal mechanism", a ubiquitous biochem. strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topoisomerase II covalently linked to DNA through its active-site tyrosine at 2.5 Å resoln., revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking expts. indicate that cleavage is catalyzed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a mol. "trapdoor" to prevent subunit dissocn. during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.
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    22. 22
      Perera, L.; Freudenthal, B. D.; Beard, W. A.; Shock, D. D.; Pedersen, L. G.; Wilson, S. H. Requirement for Transient Metal Ions Revealed through Computational Analysis for DNA Polymerase Going in Reverse. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (38), E5228– E5236,  DOI: 10.1073/pnas.1511207112
      22
      Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse
      Perera, Lalith; Freudenthal, Bret D.; Beard, William A.; Shock, David D.; Pedersen, Lee G.; Wilson, Samuel H.
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (38), E5228-E5236CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chem. equil. by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3' of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallog. studies of DNA polymerases have identified an addnl. metal ion (product metal) assocd. with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mech./mol. mech. calcns. of the reverse reaction in the confines of the DNA polymerase β active site. Addnl., site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallog. structures. The transition barrier for pyrophosphorolysis was estd. to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the resp. reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chem. equil. of a reaction that is central to genome stability.
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    23. 23
      Shi, Y.; Hellinga, H. W.; Beese, L. S. Interplay of Catalysis, Fidelity, Threading, and Processivity in the Exo- and Endonucleolytic Reactions of Human Exonuclease I. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (23), 6010– 6015,  DOI: 10.1073/pnas.1704845114
      23
      Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I
      Shi, Yuqian; Hellinga, Homme W.; Beese, Lorena S.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (23), 6010-6015CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends were accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guided the 2 substrate types into a shared reaction mechanism that catalyzed their cleavage by an elaborated variant of the 2-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppressed inappropriate or premature cleavage, enhancing processing fidelity. The striking redn. in flap conformational entropy was catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the obsd. reaction sequence, hExo1 reset without relinquishing DNA binding, suggesting a structural basis for its processivity.
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    24. 24
      Jimeno, S.; Herrera-Moyano, E.; Ortega, P.; Aguilera, A. Differential Effect of the Overexpression of Rad2/XPG Family Endonucleases on Genome Integrity in Yeast and Human Cells. DNA Repair 2017, 57, 66– 75,  DOI: 10.1016/j.dnarep.2017.06.030
      24
      Differential effect of the overexpression of Rad2/XPG family endonucleases on genome integrity in yeast and human cells
      Jimeno, Sonia; Herrera-Moyano, Emilia; Ortega, Pedro; Aguilera, Andres
      DNA Repair (2017), 57 (), 66-75CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)
      Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metab. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are crit. to preserve genome integrity. In addn. to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.
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    25. 25
      Emmert, S.; Schneider, T. D.; Khan, S. G.; Kraemer, K. H. The Human XPG Gene: Gene Architecture, Alternative Splicing and Single Nucleotide Polymorphisms. Nucleic Acids Res. 2001, 29 (7), 1443– 1452,  DOI: 10.1093/nar/29.7.1443
      25
      The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms
      Emmert, Steffen; Schneider, Thomas D.; Khan, Sikandar G.; Kraemer, Kenneth H.
      Nucleic Acids Research (2001), 29 (7), 1443-1452CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, detn. of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Anal. of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amt. of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in assocn. with multiple alternatively spliced isoforms of XPG mRNA.
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    26. 26
      Qiu, J.; Qian, Y.; Chen, V.; Guan, M.-X.; Shen, B. Human Exonuclease 1 Functionally Complements Its Yeast Homologues in DNA Recombination, RNA Primer Removal, and Mutation Avoidance. J. Biol. Chem. 1999, 274 (25), 17893– 17900,  DOI: 10.1074/jbc.274.25.17893
      26
      Human exonuclease 1 functionally complements its yeast homologues in DNA recombination, RNA primer removal, and mutation avoidance
      Qiu, Junzhuan; Qian, Ying; Chen, Victoria; Guan, Min-Xin; Shen, Binghui
      Journal of Biological Chemistry (1999), 274 (25), 17893-17900CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Yeast exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, the authors show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Δ mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5'-3' double strand DNA exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.
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    27. 27
      Genschel, J.; Bazemore, L. R.; Modrich, P. Human Exonuclease I Is Required for 5′ and 3′ Mismatch Repair. J. Biol. Chem. 2002, 277 (15), 13302– 13311,  DOI: 10.1074/jbc.M111854200
      27
      Human exonuclease I is required for 5' and 3' mismatch repair
      Genschel, Jochen; Bazemore, Laura R.; Modrich, Paul
      Journal of Biological Chemistry (2002), 277 (15), 13302-13311CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The authors have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear ext. fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted exts. requires both hMutSα and hMutLα. However, excision directed by a 5' strand break requires hMutSα but can occur in absence of hMutLα. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSα in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.
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    28. 28
      Wei, K.; Clark, A. B.; Wong, E.; Kane, M. F.; Mazur, D. J.; Parris, T.; Kolas, N. K.; Russell, R.; Hou, H.; Kneitz, B.; Yang, G.; Kunkel, T. A.; Kolodner, R. D.; Cohen, P. E.; Edelmann, W. Inactivation of Exonuclease I in Mice Results in DNA Mismatch Repair Defects, Increased Cancer Susceptibility, and Male and Female Sterility. Genes Dev. 2003, 17 (5), 603– 614,  DOI: 10.1101/gad.1060603
      28
      Inactivation of exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility
      Wei, Kaichun; Clark, Alan B.; Wong, Edmund; Kane, Michael F.; Mazur, Dan J.; Parris, Tchaiko; Kolas, Nadine K.; Russell, Robert; Hou, Harry, Jr.; Kneitz, Burkhard; Yang, Guohze; Kunkel, Thomas A.; Kolodner, Richard D.; Cohen, Paula E.; Edelmann, Winfried
      Genes & Development (2003), 17 (5), 603-614CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
      Exonuclease 1 (Exo1) is a 5'-3' exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Anal. of Exo1-/- cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5' and 3' nick-directed repair. The repair defect in Exo1-/- cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1-/- animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addn., Exo1-/- male and female mice were sterile because of a meiotic defect. Meiosis in Exo1-/- animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis.
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    29. 29
      Zhu, Z.; Chung, W. H.; Shim, E. Y.; Lee, S. E.; Ira, G. Sgs1 Helicase and Two Nucleases Dna2 and Exo1 Resect DNA Double-Strand Break Ends. Cell 2008, 134 (6), 981– 994,  DOI: 10.1016/j.cell.2008.08.037
      29
      Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends
      Zhu, Zhu; Chung, Woo-Hyun; Shim, Eun Yong; Lee, Sang Eun; Ira, Grzegorz
      Cell (Cambridge, MA, United States) (2008), 134 (6), 981-994CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. The authors monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. The Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degrdn., whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Δ sgs1Δ double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.
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    30. 30
      Vallur, A. C.; Maizels, N. Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats. J. Biol. Chem. 2010, 285 (37), 28514– 28519,  DOI: 10.1074/jbc.M110.132738
      30
      Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats
      Vallur, Aarthy C.; Maizels, Nancy
      Journal of Biological Chemistry (2010), 285 (37), 28514-28519CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Trinucleotide repeats can form stable secondary structures that promote genomic instability. To det. how such structures are resolved, we have defined biochem. activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flap-bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. HEXO1 did not exhibit endonuclease activity on 5'-flap-bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by DNA quadruplex (G4) structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity vs. hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, resp. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.
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    31. 31
      Lee, B. I.; Wilson, D. M. The RAD2 Domain of Human Exonuclease 1 Exhibits 5′ to 3′ Exonuclease and Flap Structure-Specific Endonuclease Activities. J. Biol. Chem. 1999, 274 (53), 37763– 37769,  DOI: 10.1074/jbc.274.53.37763
      31
      The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities
      Lee, Byung-In; Wilson, David M., III
      Journal of Biological Chemistry (1999), 274 (53), 37763-37769CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg2+ being the preferred metal ion. HEX1-N2 is ∼3-fold less active in Mn2+-contg. buffers and exhibits <5% activity in the presence of Co2+, Zn2+, or Ca2+. The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (kcat) and site of incision, and is as efficient (kcat/Km) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.
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    32. 32
      Keijzers, G.; Bohr, V. A.; Rasmussen, L. J. Human Exonuclease 1 (EXO1) Activity Characterization and Its Function on FLAP Structures. Biosci. Rep. 2015, 35 (3), e00206  DOI: 10.1042/BSR20150058
      There is no corresponding record for this reference.
    33. 33
      Orans, J.; McSweeney, E. A.; Iyer, R. R.; Hast, M. A.; Hellinga, H. W.; Modrich, P.; Beese, L. S. Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family. Cell 2011, 145 (2), 212– 223,  DOI: 10.1016/j.cell.2011.03.005
      33
      Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family
      Orans, Jillian; McSweeney, Elizabeth A.; Iyer, Ravi R.; Hast, Michael A.; Hellinga, Homme W.; Modrich, Paul; Beese, Lorena S.
      Cell (Cambridge, MA, United States) (2011), 145 (2), 212-223CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. HExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant soln. to a complex geometrical puzzle of substrate recognition and processing.
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    34. 34
      Tsutakawa, S. E.; Thompson, M. J.; Arvai, A. S.; Neil, A. J.; Shaw, S. J.; Algasaier, S. I.; Kim, J. C.; Finger, L. D.; Jardine, E.; Gotham, V. J. B.; Sarker, A. H.; Her, M. Z.; Rashid, F.; Hamdan, S. M.; Mirkin, S. M.; Grasby, J. A.; Tainer, J. A. Phosphate Steering by Flap Endonuclease 1 Promotes 5′-Flap Specificity and Incision to Prevent Genome Instability. Nat. Commun. 2017, 8, 15855,  DOI: 10.1038/ncomms15855
      34
      Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability
      Tsutakawa, Susan E.; Thompson, Mark J.; Arvai, Andrew S.; Neil, Alexander J.; Shaw, Steven J.; Algasaier, Sana I.; Kim, Jane C.; Finger, L. David; Jardine, Emma; Gotham, Victoria J. B.; Sarker, Altaf H.; Her, Mai Z.; Rashid, Fahad; Hamdan, Samir M.; Mirkin, Sergei M.; Grasby, Jane A.; Tainer, John A.
      Nature Communications (2017), 8 (), 15855CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallog., biochem. and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via 'phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold redn. in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.
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    35. 35
      Steitz, T. A.; Steitz, J. A. A General Two-Metal-Ion Mechanism for Catalytic RNA. Proc. Natl. Acad. Sci. U. S. A. 1993, 90 (14), 6498– 6502,  DOI: 10.1073/pnas.90.14.6498
      35
      A general two-metal-ion mechanism for catalytic RNA
      Steitz, Thomas A.; Steitz, Joan A.
      Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (14), 6498-502CODEN: PNASA6; ISSN:0027-8424.
      A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chem. catalysis is facilitated by 2 divalent metal ions 3.9 Å apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar OH group, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a 2-metal ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the 2 catalytic metal ions and properly orient the substrates via 3 specific binding sites.
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    36. 36
      Yang, W.; Lee, J. Y.; Nowotny, M. Making and Breaking Nucleic Acids: Two-Mg2+-Ion Catalysis and Substrate Specificity. Mol. Cell 2006, 22 (1), 5– 13,  DOI: 10.1016/j.molcel.2006.03.013
      36
      Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity
      Yang, Wei; Lee, Jae Young; Nowotny, Marcin
      Molecular Cell (2006), 22 (1), 5-13CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
      A review. DNA and a large proportion of RNA are antiparallel duplexes composed of an unvarying phosphosugar backbone surrounding uniformly stacked and highly similar base pairs. How do the myriad of enzymes (including ribozymes) that perform catalysis on nucleic acids achieve exquisite structure or sequence specificity. In all DNA and RNA polymerases and many nucleases and transposases, two Mg2+ ions are jointly coordinated by the nucleic acid substrate and catalytic residues of the enzyme. Based on the exquisite sensitivity of Mg2+ ions to the ligand geometry and electrostatic environment, we propose that two-metal-ion catalysis greatly enhances substrate recognition and catalytic specificity.
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    37. 37
      Dupureur, C. M. One Is Enough: Insights into the Two-Metal Ion Nuclease Mechanism from Global Analysis and Computational Studies. Metallomics. 2010, 2 (9), 609– 620,  DOI: 10.1039/c0mt00013b
      37
      One is enough: insights into the two-metal ion nuclease mechanism from global analysis and computational studies
      Dupureur, Cynthia M.
      Metallomics (2010), 2 (9), 609-620CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
      A review. The mechanistic details of metallonuclease reactions, typically supported by Mg(II), have a long and contentious history. Two-metal ion mechanisms have enjoyed much favor, based largely in the multitude of X-ray crystal structures of these enzymes with more than one metal ion per active site. Most recently, this mechanism has come under challenge. Reviewed herein are the applications of different exptl. strategies that collectively support a mechanism in which only one metal ion is necessary for nucleic acid hydrolysis. Based on global kinetic anal., anal. of reactions in which the nonsupportive Ca(II) is added, and a no. of computational approaches, secondary sites are proposed to either be occupied by activity-modulating metal ions or occupied in turn by a single metal that changes position during the course of the reaction.
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    38. 38
      Beese, L. S.; Steitz, T. A. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase 1: a two metal ion mechanism. EMBO J. 1991, 10 (1), 25– 33,  DOI: 10.1002/j.1460-2075.1991.tb07917.x
      38
      Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism
      Beese, Lorena S.; Steitz, Thomas A.
      EMBO Journal (1991), 10 (1), 25-33CODEN: EMJODG; ISSN:0261-4189.
      The refined crystal structures of the large proteolytic fragment (Klenow fragment) of E. coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5'exonuclease active site. The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6- and 3.1-Å resoln. resp. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carred out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 Å from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.
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    39. 39
      De Vivo, M.; Dal Peraro, M.; Klein, M. L. Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism. J. Am. Chem. Soc. 2008, 130 (33), 10955– 10962,  DOI: 10.1021/ja8005786
      39
      Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism
      De Vivo, Marco; Dal Peraro, Matteo; Klein, Michael L.
      Journal of the American Chemical Society (2008), 130 (33), 10955-10962CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      RNase H belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA•DNA hybrids. This enzyme is implicated in replication initiation and DNA topol. restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resoln. crystal structures of the complex RNase H/RNA•DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/mol. mechanics calcns. for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water vs. hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg2+ metals also support the formation of a meta-stable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated β-phosphoglucomutase crystal (Lahiri, S. D.; et al. Science 2003, 299 (5615), 2067-2071). The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water mols. Due to the highly conserved nature of the enzymic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily.
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    40. 40
      Genna, V.; Vidossich, P.; Ippoliti, E.; Carloni, P.; De Vivo, M. A Self-Activated Mechanism for Nucleic Acid Polymerization Catalyzed by DNA/RNA Polymerases. J. Am. Chem. Soc. 2016, 138 (44), 14592– 14598,  DOI: 10.1021/jacs.6b05475
      40
      A Self-Activated Mechanism for Nucleic Acid Polymerization Catalyzed by DNA/RNA Polymerases
      Genna, Vito; Vidossich, Pietro; Ippoliti, Emiliano; Carloni, Paolo; Vivo, Marco De
      Journal of the American Chemical Society (2016), 138 (44), 14592-14598CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The enzymic polymn. of DNA and RNA is at the basis of genetic inheritance for all living organisms. It is catalyzed by the DNA/RNA polymerase (Pol) superfamily. Here, bioinformatics anal. revealed that the incoming nucleotide substrate always forms an H-bond between its 3'-OH and β-phosphate moieties upon formation of the Michaelis complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addn. and, importantly, nucleic acid translocation. Thus, SAM allows an elegant and efficient closed-loop sequence of chem. and phys. steps for Pol catalysis. This is markedly different from previous mechanistic hypotheses. This proposed mechanism was corroborated via ab initio QM/MM simulations on a specific Pol, human DNA polymerase-η, an enzyme involved in repairing damaged DNA. The structural conservation of DNA and RNA Pols supports the possible extension of SAM to Pol enzymes from the 3 domains of life.
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    41. 41
      Shaw, S. J.; Finger, L. D.; Grasby, J. A. Human Exonuclease 1 Threads 5′-Flap Substrates through Its Helical Arch. Biochemistry 2017, 56 (29), 3704– 3707,  DOI: 10.1021/acs.biochem.7b00507
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      Human Exonuclease 1 Threads 5'-Flap Substrates through Its Helical Arch
      Shaw, Steven J.; Finger, L. David; Grasby, Jane A.
      Biochemistry (2017), 56 (29), 3704-3707CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Human exonuclease 1 (hEXO1) is a member of the 5'-nuclease superfamily and plays important roles in DNA repair. Along with acting as a 5'-exonuclease on blunt, gapped, nicked, and 3'-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5'-single-stranded flaps from duplex ends. How hEXO1 and related 5'-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5'-flaps has been controversial. Here we report the first functional data that imply that hEXO1 threads the 5'-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single strands. Conjugation of bulky 5'-streptavidin that would "block" threading through the arch drastically slowed the hEXO1 reaction. In contrast, addn. of streptavidin to a preformed hEXO1 5'-biotin flap DNA complex trapped a portion of the substrate in a highly reactive threaded conformation. However, another fraction behaves as if it were "blocked" and decayed very slowly, implying there were both threaded and unthreaded forms of the substrate present. The reaction of an unmodified hEXO1-flap DNA complex did not exhibit marked biphasic kinetics, suggesting a fast re-equilibration occurs that produces more threaded substrate when some decays. The finding that a threading mechanism like that used by hFEN1 is also used by hEXO1 unifies the mode of operation for members of the 5'-nuclease superfamily that act on discontinuous substrates. As with hFEN1, intrinsic disorder of the arch region of the protein may explain how flaps can be threaded without a need for a coupled energy source.
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    42. 42
      Tomlinson, C. G.; Atack, J. M.; Chapados, B.; Tainer, J. A.; Grasby, J. A. Substrate Recognition and Catalysis by Flap Endonucleases and Related Enzymes. Biochem. Soc. Trans. 2010, 38 (2), 433– 437,  DOI: 10.1042/BST0380433
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      Substrate recognition and catalysis by flap endonucleases and related enzymes
      Tomlinson, Christopher G.; Atack, John M.; Chapados, Brian; Tainer, John A.; Grasby, Jane A.
      Biochemical Society Transactions (2010), 38 (2), 433-437CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
      A review. Flap endonucleases (FENs) and related FEN-like enzymes [exonuclease-1, gap endonuclease 1 (GEN-1), and xeroderma pigmentosum complementation group G] are a family of divalent metal ion-dependent nucleases that catalyze structure-specific hydrolysis of DNA duplex-contg. nucleic acid structures during DNA replication, repair, and recombination. In the case of FENs, the ability to catalyze reactions on a variety of substrates has been rationalized as a result of combined functional and structural studies. Analyses of FENs also exemplify controversies regarding the 2-metal-ion mechanism. However, kinetic studies of phage T5 FEN reveal that a 2-metal-ion-like mechanism for chem. catalysis is plausible. Consideration of the metallobiochem. and the positioning of substrate in metal-free structures has led to the proposal that the duplex termini of substrates are unpaired in the catalytically active form and that FENs and related enzymes may recognize breathing duplex termini within more complex structures. An outstanding issue in FEN catalysis is the role played by the intermediate (I) domain arch or clamp. It has been proposed that FENs thread the 5'-portion of their substrates through this arch, which is wide enough to accommodate single-stranded, but not double-stranded, DNA. However, FENs exhibit gap endonuclease activity acting upon substrates that have a region of 5'-duplex. Moreover, the action of other FEN family members such as GEN-1, proposed to target Holliday junctions without termini, appears incompatible with a threading mechanism. An alterative is that the I domain is used as a clamp. A future challenge is to clarify the role of this domain in FENs and related enzymes.
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    43. 43
      Bennet, I. A; Finger, L D.; Baxter, N. J; Ambrose, B.; Hounslow, A. M; Thompson, M. J; Exell, J. C; Shahari, N. N. B M.; Craggs, T. D; Waltho, J. P; Grasby, J. A Regional Conformational Flexibility Couples Substrate Specificity and Scissile Phosphate Diester Selectivity in Human Flap Endonuclease 1. Nucleic Acids Res. 2018, 46 (11), 5618– 5633,  DOI: 10.1093/nar/gky293
      43
      Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1
      Bennet, Ian A.; Finger, L. David; Baxter, Nicola J.; Ambrose, Benjamin; Hounslow, Andrea M.; Thompson, Mark J.; Exell, Jack C.; Shahari, Nur Nazihah B. Md.; Craggs, Timothy D.; Waltho, Jonathan P.; Grasby, Jane A.
      Nucleic Acids Research (2018), 46 (11), 5618-5633CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the soln. conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-mol. FRET data show a shift in the conformational ensemble in the arch and loop region upon addn. of DNA. Furthermore, the addn. of divalent metal ions to the active site of the hFEN1- DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.
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    44. 44
      Raper, A. T.; Reed, A. J.; Suo, Z. Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion. Chem. Rev. 2018, 118 (2), 6000– 6025,  DOI: 10.1021/acs.chemrev.7b00685
      44
      Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion
      Raper, Austin T.; Reed, Andrew J.; Suo, Zucai
      Chemical Reviews (Washington, DC, United States) (2018), 118 (12), 6000-6025CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases, has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyl transfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyl transfer (i.e. chem.), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallog. studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidences for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.
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    45. 45
      Black, C. B.; Huang, H. W.; Cowan, J. A. Biological Coordination Chemistry of Magnesium, Sodium, and Potassium Ions. Protein and Nucleotide Binding Sites. Coord. Chem. Rev. 1994, 135–136 (C), 165– 202,  DOI: 10.1016/0010-8545(94)80068-5
      45
      Biological coordination chemistry of magnesium, sodium, and potassium ions. Protein and nucleotide binding sites
      Black, C. B.; Huang, H.-W.; Cowan, J. A.
      Coordination Chemistry Reviews (1994), 135/136 (), 165-202CODEN: CCHRAM; ISSN:0010-8545. (Elsevier)
      This review with 78 refs. explores Mg2+ binding to proteins and enzymes, K+ activated enzymes, metal ions and membranes, and metal-nucleotide binding domains.
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    46. 46
      Ho, M.-H.; De Vivo, M.; Dal Peraro, M.; Klein, M. L. Understanding the Effect of Magnesium Ion Concentration on the Catalytic Activity of Ribonuclease H through Computation: Does a Third Metal Binding Site Modulate Endonuclease Activity?. J. Am. Chem. Soc. 2010, 132 (39), 13702– 13712,  DOI: 10.1021/ja102933y
      46
      Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: Does a third metal binding site modulate endonuclease activity?
      Ho, Ming-Hsun; De Vivo, Marco; Dal Peraro, Matteo; Klein, Michael L.
      Journal of the American Chemical Society (2010), 132 (39), 13702-13712CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      RNase H belongs to the nucleotidyltransferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallog. data have located 2 ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, expts. have shown a dependency of the catalytic activity on the Mg2+ concn. Moreover, in RNase H, Glu-188 has been shown to be essential for full enzymic activation, regardless of Mg2+ concn. The catalytic center is known to contain 3 Mg2+ ions, and Glu-188 is not one of the primary metal ligands. Here, classical mol. dynamics (MD) simulations were employed to study the metal-ligand coordination in Bacillus halodurans RNase H at different concns. of Mg2+. Importantly, the presence of a 3rd Mg2+ ion, bound to 2nd-shell ligand Glu-188, was a persistent feature of the MD simulations. Free energy calcns. identified 2 distinct conformations, depending on the concn. of Mg2+. At std. concn., a 3rd Mg2+ was found in the catalytic pocket, but it did not perturb the optimal RNase H active conformation. However, at higher concns., the 3rd Mg2+ ion heavily perturbed the nucleophilic water and thereby influenced the catalytic efficiency of RNase H. In addn., the E188A mutant showed no ability to engage addnl. Mg2+ ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of mutant E188A and also supports the key role of Glu-188 in localizing the 3rd Mg2+ ion at the active site. Glu residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calcns. support the hypothesis that RNase H can accommodate 3 divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.
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    47. 47
      Genna, V.; Gaspari, R.; Dal Peraro, M.; De Vivo, M. Cooperative Motion of a Key Positively Charged Residue and Metal Ions for DNA Replication Catalyzed by Human DNA Polymerase-η. Nucleic Acids Res. 2016, 44 (6), 2827– 2836,  DOI: 10.1093/nar/gkw128
      47
      Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase-η
      Genna Vito; Gaspari Roberto; Dal Peraro Matteo; De Vivo Marco
      Nucleic acids research (2016), 44 (6), 2827-36 ISSN:.
      Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases.
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    48. 48
      La Sala, G.; Riccardi, L.; Gaspari, R.; Cavalli, A.; Hantschel, O.; De Vivo, M. HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase. J. Chem. Theory Comput. 2016, 12 (11), 5563– 5574,  DOI: 10.1021/acs.jctc.6b00600
      48
      HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase
      La Sala, Giuseppina; Riccardi, Laura; Gaspari, Roberto; Cavalli, Andrea; Hantschel, Oliver; De Vivo, Marco
      Journal of Chemical Theory and Computation (2016), 12 (11), 5563-5574CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      A no. of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Tyr-412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, the binding of the SH2 domain to the N-lobe of the Abl kinase domain was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, the authors report on microsecond-long mol. dynamics simulations coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain, and with the DFG motif flipped in 2 ways (in or out conformation). Through comparative anal., the simulations augment the interpretation of the existing Abl exptl. data, revealing a dynamical network of interactions that interconnect SH2 domain-binding with A-loop plasticity and Tyr-412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely the EPK-ELK H-bond network and the HRD motif. These results show that signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.
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    49. 49
      Hwang, W.; Yoo, J.; Lee, Y.; Park, S.; Hoang, P. L.; Cho, H.; Yu, J.; Hoa Vo, T. M.; Shin, M.; Jin, M. S.; Park, D.; Hyeon, C.; Lee, G. Dynamic Coordination of Two-Metal-Ions Orchestrates λ-Exonuclease Catalysis. Nat. Commun. 2018, 9 (1), 4404,  DOI: 10.1038/s41467-018-06750-9
      49
      Dynamic coordination of two-metal-ions orchestrates λ-exonuclease catalysis
      Hwang Wonseok; Lee Yuno; Hyeon Changbong; Hwang Wonseok; Yoo Jungmin; Park Suyeon; Hoang Phuong Lien; Cho HyeokJin; Yu Jeongmin; Hoa Vo Thi Minh; Jin Mi Sun; Park Daeho; Lee Gwangrog; Lee Yuno; Shin Minsang
      Nature communications (2018), 9 (1), 4404 ISSN:.
      Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg(2+) binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while MgA(2+) and MgB(2+) have similar binding constants, the dissociation rate of MgA(2+) is two order of magnitude lower than that of MgB(2+) due to a kinetic-barrier-difference. At physiological Mg(2+) concentration, the MgB(2+) ion near the 5'-terminal side of the scissile phosphate dissociates each-round of degradation, facilitating a series of DNA cleavages via fast product-release concomitant with enzyme-translocation. At a low magnesium concentration, occasional dissociation and slow re-coordination of MgA(2+) result in pauses during processive degradation. Our study highlights the importance of metal-ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis.
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    50. 50
      Samara, N. L.; Yang, W. Cation Trafficking Propels RNA Hydrolysis. Nat. Struct. Mol. Biol. 2018, 25 (8), 715– 721,  DOI: 10.1038/s41594-018-0099-4
      50
      Cation trafficking propels RNA hydrolysis
      Samara, Nadine L.; Yang, Wei
      Nature Structural & Molecular Biology (2018), 25 (8), 715-721CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
      Catalysis by members of the RNase H superfamily of enzymes is generally believed to require only two Mg2+ ions that are coordinated by active-site carboxylates. By examg. the catalytic process of Bacillus halodurans RNase H1 in crystallo, however, we found that the two canonical Mg2+ ions and an addnl. K+ failed to align the nucleophilic water for RNA cleavage. Substrate alignment and product formation required a second K+ and a third Mg2+, which replaced the first K+ and departed immediately after cleavage. A third transient Mg2+ has also been obsd. for DNA synthesis, but in that case it coordinates the leaving group instead of the nucleophile as in the case of the RNase H1 hydrolysis reaction. These transient cations have no contact with the enzymes. Other DNA and RNA enzymes that catalyze consecutive cleavage and strand-transfer reactions in a single active site may similarly require cation trafficking coordinated by the substrate.
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    51. 51
      Molina, R.; Stella, S.; Redondo, P.; Gomez, H.; Marcaida, M. J.; Orozco, M.; Prieto, J.; Montoya, G. Visualizing Phosphodiester-Bond Hydrolysis by an Endonuclease. Nat. Struct. Mol. Biol. 2015, 22 (1), 65– 72,  DOI: 10.1038/nsmb.2932
      51
      Visualizing phosphodiester-bond hydrolysis by an endonuclease
      Molina, Rafael; Stella, Stefano; Redondo, Pilar; Gomez, Hansel; Marcaida, Maria Jose; Orozco, Modesto; Prieto, Jesus; Montoya, Guillermo
      Nature Structural & Molecular Biology (2015), 22 (1), 65-72CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      The enzymic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chem. reaction has not yet been obsd. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We obsd. the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.
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    52. 52
      Sasnauskas, G.; Jeltsch, A.; Pingoud, A.; Siksnys, V. Plasmid DNA Cleavage by MunI Restriction Enzyme: Single-Turnover and Steady-State Kinetic Analysis. Biochemistry 1999, 38 (13), 4028– 4036,  DOI: 10.1021/bi982456n
      52
      Plasmid DNA cleavage by MunI restriction enzyme: single-turnover and steady-state kinetic analysis
      Sasnauskas, Giedrius; Jeltsch, Albert; Pingoud, Alfred; Siksnys, Virginijus
      Biochemistry (1999), 38 (13), 4028-4036CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Mutational anal. has previously indicated that D83 and E98 residues are essential for DNA cleavage activity and presumably chelate a Mg2+ ion at the active site of MunI restriction enzyme. In the absence of metal ions, protonation of an ionizable residue with a pKa > 7.0, most likely one of the active site carboxylates, controls the DNA binding specificity of MunI [Lagunavicius, A., Grazulis, S., Balciunaite, E., Vainius, D., and Siksnys, V. (1997) Biochem. 36, 11093-11099.]. Thus, competition between H+ and Mg2+ binding at the active site of MunI presumably plays an important role in catalysis/binding. In the present study we have identified elementary steps and intermediates in the reaction pathway of plasmid DNA cleavage by MunI and elucidated the effect of pH and Mg2+ ions on the individual steps of the DNA cleavage reaction. The kinetic anal. indicated that the multiple-turnover rate of plasmid cleavage by MunI is limited by product release throughout the pH range 6.0-9.3. Quenched-flow expts. revealed that open circle DNA is an obligatory intermediate in the reaction pathway. Under optimal reaction conditions, open circle DNA remains bound to the MunI; however it is released into the soln. at low [MgCl2]. Rate consts. for the phosphodiester bond hydrolysis of the first (k1) and second (k2) strand of plasmid DNA at pH 7.0 and 10 mM MgCl2 more than 100-fold exceed the kcat value which is limited by product dissocn. The anal. of the pH and [Mg2+] dependences of k1 and k2 revealed that both H+ and Mg2+ ions compete for the binding to the same residue at the active site of MunI. Thus, the decreased rate of phosphodiester hydrolysis by MunI at pH < 7.0 may be due to the redn. of affinity for the Mg2+ binding at the active site. Kinetic anal. of DNA cleavage by MunI yielded ests. for the assocn.-dissocn. rate consts. of enzyme-substrate complex and demonstrated the decreased stability of the MunI-DNA complex at pH values above 8.0.
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    53. 53
      Xie, F.; Dupureur, C. M. Kinetic Analysis of Product Release and Metal Ions in a Metallonuclease. Arch. Biochem. Biophys. 2009, 483 (1), 1– 9,  DOI: 10.1016/j.abb.2009.01.001
      53
      Kinetic analysis of product release and metal ions in a metallonuclease
      Xie, Fuqian; Dupureur, Cynthia M.
      Archives of Biochemistry and Biophysics (2009), 483 (1), 1-9CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)
      Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equil. and kinetic expts. are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equil. fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre-steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre-steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, anal. of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no pos. cooperativity between the two metal ions binding each active site.
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    54. 54
      Sengerová, B.; Tomlinson, C.; Atack, J. M.; Williams, R.; Sayers, J. R.; Williams, N. H.; Grasby, J. A. Brønsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substrates. Biochemistry 2010, 49 (37), 8085– 8093,  DOI: 10.1021/bi100895j
      54
      Bronsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substrates
      Sengerova, Blanka; Tomlinson, Christopher; Atack, John M.; Williams, Ryan; Sayers, Jon R.; Williams, Nicholas H.; Grasby, Jane A.
      Biochemistry (2010), 49 (37), 8085-8093CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      During replication and repair flap endonucleases (FENs) catalyze endonucleolytic and exonucleolytic (EXO) DNA hydrolyzes. Altering the leaving group pKa, by replacing the departing nucleoside with analogs, had minimal effect on kcat/Km in a T5FEN-catalyzed EXO reaction, producing a very low Bronsted coeff., βlg. Investigation of the viscosity dependence of kcat/Km revealed that reactions of EXO substrates are rate limited by diffusional encounter of enzyme and substrate, explaining the small βlg. However, the maximal single turnover rate of the FEN EXO reaction also yields a near zero βlg. A low βlg was also obsd. when evaluating kcat/Km for D201I/D204S FEN-catalyzed reactions, even though these reactions were not affected by added viscogen. But an active site K83A mutant produced a βlg = -1.2±0.10, closer to the value obsd. for soln. hydrolysis of phosphate diesters. The pH-maximal rate profiles of the WT and K83A FEN reactions both reach a max. at high pH and do not support an explanation of the data that involves catalysis of leaving group departure by Lys 83 functioning as a general acid. Instead, a rate-limiting phys. step, such as substrate unpairing or helical arch ordering, that occurs after substrate assocn. must kinetically hide an inherent large βlg. It is suggested that K83 acts as an electrostatic catalyst that stabilizes the transition state for phosphate diester hydrolysis. When K83 is removed from the active site, chem. becomes rate limiting and the leaving group sensitivity of the FEN-catalyzed reaction is revealed.
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    55. 55
      Taylor, J. D.; Halford, S. E. Discrimination between DNA Sequences by the EcoRV Restriction Endonuclease. Biochemistry 1989, 28 (15), 6198– 6207,  DOI: 10.1021/bi00441a011
      55
      Discrimination between DNA sequences by the EcoRV restriction endonuclease
      Taylor, John D.; Halford, Stephen E.
      Biochemistry (1989), 28 (15), 6198-207CODEN: BICHAW; ISSN:0006-2960.
      Restrictive endonuclease EcoRV cleaves not only its recognition sequence on DNA (GATAC), but also, at vastly reduced rates, a no. of alternative DNA sequences. Plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by 1 base pair. EcoRV nuclease showed a marked preference for one particular site from among these alternatives. This noncognate site was located at sequence GTTATC, and the mechanism of action of EcRV at this site was analyzed. The mechanism differed from that at the cognate site in 3 respects. First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this could not not account for the specificity of EcoRV as measured from kcat/Km values. Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appeared to be a key factor in limiting the rates of DNA cleavage at alternative sites. Third, the reaction pathway at the noncognate site differed from that at the cognate site. At the former, EcoRV cleaved 1st one strand of the DNA and then the other, whereas at the latter, both strands were cut in a single concerted reaction. The difference in reaction pathway allowed DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site. The addn. of DNA ligase to reactions with EcoRV made no differences to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXksFejt7o%253D&md5=024551c9fc3222ef4672096b59ce1357
    56. 56
      Yang, C. C.; Baxter, B. K.; Topal, M. D. DNA Cleavage by NaeI: Protein Purification, Rate-Limiting Step, and Accuracy. Biochemistry 1994, 33 (49), 14918– 14925,  DOI: 10.1021/bi00253a031
      56
      DNA Cleavage by NaeI: Protein Purification, Rate-Limiting Step, and Accuracy
      Yang, Charles C.; Baxter, Bonnie K.; Topal, Michael D.
      Biochemistry (1994), 33 (49), 14918-25CODEN: BICHAW; ISSN:0006-2960.
      NaeI endonuclease must bind two DNA sites for cleavage to occur. NaeI was purified to apparent homogeneity and used to det. the rate-limiting step for DNA cleavage and to measure NaeI's specificity for its cognate recognition site. Steady-state cleavage by NaeI in the presence of effector DNA (activated) gave values of 0.045 s-1 and 10 nM for kcat and KM for M13 DNA substrate, resp., but values of 0.4 s-1 and 170 nM, resp., for an M13 DNA fragment substrate. Single-turnover cleavage of M13 DNA demonstrated that DNA strand scission is not rate-limiting for turnover of NaeI. Transient kinetic anal. of M13 DNA cleavage by NaeI showed an initial burst of substrate cleavage that was proportional to NaeI concn., implying that product release is rate-limiting for turnover of NaeI. The NaeI effector and substrate binding sites were found to prefer cognate over noncognate sequences by 103-fold and at least 40-500-fold, resp. Kcat for noncognate recognition sequence was at least 106-fold lower than that for cognate. The specificity of activated NaeI, as measured by kcat/KM, for noncognate recognition sequence was 108-fold lower than that for cognate, and over 1011-fold lower when the decreased affinity for noncognate sequence at the effector binding site was taken into account. This specificity is approx. 104-fold larger than for any other restriction enzyme measured.
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    57. 57
      Williams, R.; Sengerova, B.; Osborne, S.; Syson, K.; Ault, S.; Kilgour, A.; Chapados, B. R.; Tainer, J. A.; Sayers, J. R.; Grasby, J. A. Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease. J. Mol. Biol. 2007, 371 (1), 34– 48,  DOI: 10.1016/j.jmb.2007.04.063
      57
      Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease
      Williams, Ryan; Sengerova, Blanka; Osborne, Sadie; Syson, Karl; Ault, Sophie; Kilgour, Anna; Chapados, Brian R.; Tainer, John A.; Sayers, Jon R.; Grasby, Jane A.
      Journal of Molecular Biology (2007), 371 (1), 34-48CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Flap endonucleases (FENs) catalyze the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalyzed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterized. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs contg. two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover nos. for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilize the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concns. below satn. with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.
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    58. 58
      Finger, D. L.; Blanchard, M. S.; Theimer, C. A.; Sengerová, B.; Singh, P. S.; Chavez, V.; Liu, F.; Grasby, J. A.; Shen, B. The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis. J. Biol. Chem. 2009, 284 (33), 22184– 22194,  DOI: 10.1074/jbc.M109.015065
      58
      The 3'-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis
      Finger, L. David; Blanchard, M. Suzette; Theimer, Carla A.; Sengerova, Blanka; Singh, Purnima; Chavez, Valerie; Liu, Fei; Grasby, Jane A.; Shen, Binghui
      Journal of Biological Chemistry (2009), 284 (33), 22184-22194CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest esp. in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that although they share similar folds, the FEN1s of higher organisms contain a 3'-extrahelical nucleotide (3'-flap) binding pocket. When presented with 5'-flap substrates having a 3'-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well-defined DNA substrates was conducted. The presence of a 3'-flap on substrates reduced Km and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiol. salt concns. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3'-flap, and the absence of a 3'-flap from a 5'-flap substrate was more detrimental to hFEN1 activity than removal of the 5'-flap or introduction of a hairpin into the 5'-flap structure. HFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3'-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5'-phosphorylated product. Together the results indicate that the presence of a 3'-flap is the crit. feature for efficient hFEN1 substrate recognition and catalysis.
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    59. 59
      Kuznetsova, A. A.; Fedorova, O. S.; Kuznetsov, N. A. Kinetic Features of 3′-5′ Exonuclease Activity of Human AP-Endonuclease APE1. Molecules 2018, 23 (9), 2101,  DOI: 10.3390/molecules23092101
      59
      Kinetic features of 3'-5' exonuclease activity of human AP-endonuclease APE1
      Kuznetsova, Alexandra A.; Fedorova, Olga S.; Kuznetsov, Nikita A.
      Molecules (2018), 23 (9), 2101/1-2101/14CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5' to the damage. In addn. to the AP-endonuclease activity, APE1 possesses 3'-5' exonuclease activity, which presumably is responsible for cleaning up nonconventional 3' ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3'-end nucleotide removal in the 3'-5' exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5' dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3' end of the short oligonucleotide. The impact of the 3'-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3'-5' exonuclease activity was detd. Kinetic data revealed that the rate-limiting step of 3' nucleotide removal by APE1 in the 3'-5' exonuclease process is the release of the detached nucleotide from the enzyme's active site.
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    60. 60
      Warshel, A.; Levitt, M. Theoretical Studies of Enzymic Reactions: Dielectric, Electrostatic and Steric Stabilization of the Carbonium Ion in the Reaction of Lysozyme. J. Mol. Biol. 1976, 103 (2), 227– 249,  DOI: 10.1016/0022-2836(76)90311-9
      60
      Theoretical studies of enzymic reactions: dielectric, electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme
      Warshel, A.; Levitt, M.
      Journal of Molecular Biology (1976), 103 (2), 227-49CODEN: JMOBAK; ISSN:0022-2836.
      A general method for detailed study of enzymic reactions is presented. The method considers the complete enzyme-substrate complex together with the surrounding solvent and evaluates all the different quantum mech. and classical energy factors that can affect the reaction pathway. These factors include the quantum mech. energies assocd. with bond cleavage and charge redistribution of the substrate and the classical energies of steric and electrostatic interactions between the substrate and the enzyme. The electrostatic polarization of the enzyme atoms and the orientation of the dipoles of the surrounding H2O mols. is simulated by a microscopic dielec. model. The solvation energy resulting from this polarization is considerable and must be included in any realistic calcn. of chem. reactions involving anything more than an isolated mol. in vacuo. Without it, acidic groups can never become ionized and the charge distribution on the substrate will not be reasonable. The same dielec. model can also be used to study the reaction of the substrate in soln. In this way the reaction in soln. can be compared with the enzymic reaction. The stability of the carbonium ion intermediate formed in the cleavage of a glycosidic bond by lysozyme was studied. Electrostatic stabilization is an important factor in increasing the rate of the reaction step that leads to the formation of the carbonium ion intermediate. Steric factors, such as the strain of the substrate on binding to lysozyme, do not seem to contribute significantly.
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    61. 61
      Brunk, E.; Rothlisberger, U. Mixed Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulations of Biological Systems in Ground and Electronically Excited States. Chem. Rev. 2015, 115 (12), 6217– 6263,  DOI: 10.1021/cr500628b
      61
      Mixed Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulations of Biological Systems in Ground and Electronically Excited States
      Brunk, Elizabeth; Rothlisberger, Ursula
      Chemical Reviews (Washington, DC, United States) (2015), 115 (12), 6217-6263CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The quantum nature of electrons and nuclei is manifested in countless biol. events, including the rearrangements of electrons in biochem. reactions, electron and proton tunneling, coupled proton-electron transfers, photoexcitations, and long-lived quantum coherences and quantum entanglement. Quantum mech. (QM) phenomena are thus at the core of fundamental biol. processes. The introduction of mixed quantum mech./mol. mech. (QM/MM) methods that allow one to treat electronic quantum phenomena in complex classical environments represented a seminal step toward the quantum mech. treatment of realistic biol. systems. Subsequent extension of the QM/MM approach from adiabatic simulations in the electronic ground state to nonadiabatic dynamics in electronically excited states added an addnl. layer of complexity by making it necessary to account for the quantum nature of nuclear degrees of freedom. The present review examines the current state of the art of QM/MM mol. dynamics approaches in ground and electronically excited states and their applications to biol. problems.
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    62. 62
      Stevens, D. R.; Hammes-Schiffer, S. Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations. J. Am. Chem. Soc. 2018, 140 (28), 8965– 8969,  DOI: 10.1021/jacs.8b05177
      62
      Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations
      Stevens, David R.; Hammes-Schiffer, Sharon
      Journal of the American Chemical Society (2018), 140 (28), 8965-8969CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The enzyme human DNA polymerase η (Pol η) is crit. for bypassing lesions during DNA replication. In addn. to the two Mg2+ ions aligning the active site, expts. suggest that a third Mg2+ ion could play an essential catalytic role. Herein the role of this third metal ion is investigated with quantum mech./mol. mech. (QM/MM) free energy simulations of the phosphoryl transfer reaction and a proposed self-activating proton transfer from the incoming nucleotide to the pyrophosphate leaving group. The simulations with only two metal ions in the active site support a sequential mechanism, with phosphoryl transfer followed by relatively fast proton transfer. The simulations with three metal ions in the active site suggest that the third metal ion may play a catalytic role through electrostatic interactions with the leaving group. These electrostatic interactions stabilize the product, making the phosphoryl transfer reaction more thermodynamically favorable with a lower free energy barrier relative to the activated state corresponding to the deprotonated 3'OH nucleophile, and also inhibit the subsequent proton transfer.
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    63. 63
      Field, M. J.; Bash, P. A.; Karplus, M. A Combined Quantum Mechanical and Molecular Mechanical Potential for Molecular Dynamics Simulations. J. Comput. Chem. 1990, 11 (6), 700– 733,  DOI: 10.1002/jcc.540110605
      63
      A combined quantum mechanical and molecular mechanical potential for molecular dynamics simulations
      Field, Martin J.; Bash, Paul A.; Karplus, Martin
      Journal of Computational Chemistry (1990), 11 (6), 700-33CODEN: JCCHDD; ISSN:0192-8651.
      A combined quantum mech. (QM) and mol. mech. (MM) potential has been developed for the study of reactions in condensed phases. For the quantum mech. calcns. semiempirical methods of the MNDO and AM1 type are used, while the mol. mechanics part is treated with the HARMM force field. Specific prescriptions are given for the interactions between the QM and MM portions of the system; cases in which the QM and MM methodol. is applied to parts of the same mol. or to different mols. are considered. The details of the method and a range of test calcns., including comparisons with ab initio and exptl. results, are given. In many cases satisfactory results are obtained. However, there are limitations to this type of approach, some of which arise from the AM1 or MNDO methods themselves and others from the present QM/MM implementation. This suggests that it is important to test the applicability of the method to each particular case prior to its use. Possible areas of improvement in the methodol. are discussed.
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    64. 64
      Needleman, S. B.; Wunsch, C. D. A General Method Applicable to Search for Similarities in Amino Acid Sequence of Two Proteins. J. Mol. Biol. 1970, 48 (3), 443– 453,  DOI: 10.1016/0022-2836(70)90057-4
      64
      General method applicable to the search for similarities in the amino acid sequence of two proteins
      Needleman, Saul B.; Wunsch, Christian D.
      Journal of Molecular Biology (1970), 48 (3), 443-53CODEN: JMOBAK; ISSN:0022-2836.
      A computer adaptable method for finding similarities in the amino acid sequences of two proteins has been developed, making it possible to det. whether significant homology exists between the proteins. This information is used to trace their possible evolutionary development. The max. match is a no. dependent upon the similarity of the sequences. One of its definitions is the largest no. of amino acids of one protein that can be matched with those of a second protein allowing for all possible interruptions in either of the sequences. While the interruptions give rise to a very large no. of comparisons, the method efficiently excludes from consideration those comparisons that cannot contribute to the max. match. Comparisons are made from the smallest unit of significance, a pair of amino acids, one from each protein.
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    65. 65
      Szymanski, M. R.; Yu, W.; Gmyrek, A. M.; White, M. A.; Molineux, I. J.; Lee, J. C.; Yin, Y. W. A Domain in Human EXOG Converts Apoptotic Endonuclease to DNA-Repair Exonuclease. Nat. Commun. 2017, 8, 14959,  DOI: 10.1038/ncomms14959
      65
      A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease
      Szymanski Michal R; Yu Wangsheng; Yin Y Whitney; Szymanski Michal R; Yu Wangsheng; White Mark A; Lee J Ching; Yin Y Whitney; Gmyrek Aleksandra M; White Mark A; Lee J Ching; Molineux Ian J
      Nature communications (2017), 8 (), 14959 ISSN:.
      Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 ÅA resolution, respectively. A Wing domain, absent in other ββα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic 'tape-measure'. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase γ.
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    66. 66
      Wu, C.-C.; Lin, J. L. J.; Yang-Yen, H.-F.; Yuan, H. S. A Unique Exonuclease ExoG Cleaves between RNA and DNA in Mitochondrial DNA Replication. Nucleic Acids Res. 2019, 47 (10), 5405– 5419,  DOI: 10.1093/nar/gkz241
      66
      A unique exonuclease ExoG cleaves between RNA and DNA in mitochondrial DNA replication
      Wu, Chyuan-Chuan; Lin, Jason L. J.; Yang-Yen, Hsin-Fang; Yuan, Hanna S.
      Nucleic Acids Research (2019), 47 (10), 5405-5419CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5' end of nascent DNA. A subsequent 5'-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5'-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA-DNA junctions to remove the 5'-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound resp. with DNA, RNA/DNA hybrid and RNA-DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA-DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the mol. basis of ExoG functioning as a unique 5'-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.
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    67. 67
      Zhang, J.; Mccabe, K. A.; Bell, C. E. Crystal Structures of λ Exonuclease in Complex with DNA Suggest an Electrostatic Ratchet Mechanism for Processivity. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (29), 11872– 11877,  DOI: 10.1073/pnas.1103467108
      67
      Crystal structures of λ exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity
      Zhang, Jinjin; McCabe, Kimberly A.; Bell, Charles E.
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (29), 11872-11877, S11872/1-S11872/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5'-ended strand to form 5' mononucleotides and a long 3' overhang. The crystal structure of λ exonuclease revealed a toroidal homo-trimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topol. linkage of the trimer to the DNA was proposed. Here, we have detd. the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resoln. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5'-ended strand insert into the active site of one subunit of the trimer, while the 3'-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5' phosphate of the DNA binds to a pos. charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg2+ ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5' phosphate to the pos. charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.
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    68. 68
      Zhang, J.; Pan, X.; Bell, C. E. Crystal Structure of λ Exonuclease in Complex with DNA and Ca2+. Biochemistry 2014, 53 (47), 7415– 7425,  DOI: 10.1021/bi501155q
      68
      Crystal Structure of λ Exonuclease in Complex with DNA and Ca2+
      Zhang, Jinjin; Pan, Xinlei; Bell, Charles E.
      Biochemistry (2014), 53 (47), 7415-7425CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Bacteriophage λ exonuclease (λexo) is a ring-shaped homotrimer that resects double-stranded DNA ends in the 5'-3' direction to generate a long 3'-overhang that is a substrate for recombination. λexo is a member of the type II restriction endonuclease-like superfamily of proteins that use a Mg2+-dependent mechanism for nucleotide cleavage. A previous structure of λexo in complex with DNA and Mg2+ was detd. using a nuclease-defective K131A variant to trap a stable complex. This structure revealed the detailed coordination of the two active site Mg2+ ions but did not show the interactions involving the side chain of the conserved active site Lys-131 residue. Here, we have detd. the crystal structure of wild-type (WT) λexo in complex with the same DNA substrate, but in the presence of Ca2+ instead of Mg2+. Surprisingly, there is only one Ca2+ bound in the active site, near the position of MgA in the structure with Mg2+. The scissile phosphate is displaced by 2.2 Å relative to its position in the structure with Mg2+, and the network of interactions involving the attacking water mol. is broken. Thus, the structure does not represent a catalytic configuration. However, the crystal structure does show clear electron d. for the side chain of Lys-131, which is held in place by interactions with Gln-157 and Glu-129. By combining the K131A-Mg2+ and WT-Ca2+ structures, we constructed a composite model to show the likely interactions of Lys-131 during catalysis. The implications with regard to the catalytic mechanism are discussed.
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    69. 69
      Cheng, K.; Xu, H.; Chen, X.; Wang, L.; Tian, B.; Zhao, Y.; Hua, Y. Structural Basis for DNA 5′-End Resection by RecJ. eLife 2016, 5, e14294  DOI: 10.7554/eLife.14294
      69
      Structural basis for DNA 5'-end resection by RecJ
      Cheng, Kaiying; Xu, Hong; Chen, Xuanyi; Wang, Liangyan; Tian, Bing; Zhao, Ye; Hua, Yuejin
      eLife (2016), 5 (), e14294/1-e14294/21CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      The resection of DNA strand with a 5' end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5' nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5'-phosphate-binding pocket above the active site dets. the 5'-3' polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180 ° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination.
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    70. 70
      Zhang, F.; Shi, J.; Chen, S. H.; Bian, C.; Yu, X. The PIN Domain of EXO1 Recognizes Poly(ADP-Ribose) in DNA Damage Response. Nucleic Acids Res. 2015, 43 (22), 10782– 10794,  DOI: 10.1093/nar/gkv939
      70
      The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response
      Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun
      Nucleic Acids Research (2015), 43 (22), 10782-10794CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Following DNA double-strand breaks, poly(ADPribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a no. of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We obsd. that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response.
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    71. 71
      Genna, V.; Carloni, P.; De Vivo, M. A Strategically Located Arg/Lys Residue Promotes Correct Base Paring during Nucleic Acid Biosynthesis in Polymerases. J. Am. Chem. Soc. 2018, 140 (9), 3312– 3321,  DOI: 10.1021/jacs.7b12446
      71
      A Strategically Located Arg/Lys Residue Promotes Correct Base Paring During Nucleic Acid Biosynthesis in Polymerases
      Genna, Vito; Carloni, Paolo; De Vivo, Marco
      Journal of the American Chemical Society (2018), 140 (9), 3312-3321CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Polymerases (Pols) synthesize the double-stranded nucleic acids in the Watson-Crick (W-C) conformation, which is crit. for DNA and RNA functioning. Yet, the mol. basis to catalyze the W-C base pairing during Pol-mediated nucleic acids biosynthesis remains unclear. Here, through bioinformatics analyses on a large data set of Pol/DNA structures, we first describe the conserved presence of one pos. charged residue (Lys or Arg), which is similarly located near the enzymic two-metal active site, always interacting directly with the incoming substrate (d)NTP. Incidentally, we noted that some Pol/DNA structures showing the alternative Hoogsteen base pairing were often solved with this specific residue either mutated, displaced, or missing. We then used quantum and classical simulations coupled to free-energy calcns. to illustrate how, in human DNA Pol-η, the conserved Arg61 favors W-C base pairing through defined interactions with the incoming nucleotide. Taken together, these structural observations and computational results suggest a structural framework in which this specific residue is crit. for stabilizing the incoming (d)NTP nucleotide and base pairing during Pol-mediated nucleic acid biosynthesis. These results may benefit enzyme engineering for nucleic acid processing and encourage new drug discovery strategies to modulate Pols function.
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    72. 72
      Palermo, G.; Miao, Y.; Walker, R. C.; Jinek, M.; McCammon, J. A. CRISPR-Cas9 Conformational Activation as Elucidated from Enhanced Molecular Simulations. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (28), 7260– 7265,  DOI: 10.1073/pnas.1707645114
      72
      CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations
      Palermo, Giulia; Miao, Yinglong; Walker, Ross C.; Jinek, Martin; McCammon, J. Andrew
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (28), 7260-7265CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      CRISPR-Cas9 has become a facile genome editing technol., yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive mol. simulations in an enhanced sampling regime, using a Gaussian-accelerated mol. dynamics (GaMD) methodol., which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a pos. charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose exptl. characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage. These results provide at.-level information on the mol. mechanism of CRISPR-Cas9 that will inspire future exptl. investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.
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    73. 73
      Mulholland, A. J.; Roitberg, A. E.; Tuñón, I. Enzyme Dynamics and Catalysis in the Mechanism of DNA Polymerase. Theor. Chem. Acc. 2012, 131, 1286,  DOI: 10.1007/s00214-012-1286-8
      There is no corresponding record for this reference.
    74. 74
      Riccardi, L.; Genna, V.; De Vivo, M. Metal–Ligand Interactions in Drug Design. Nat. Rev. Chem. 2018, 2 (7), 100– 112,  DOI: 10.1038/s41570-018-0018-6
      74
      Metal-ligand interactions in drug design
      Riccardi, Laura; Genna, Vito; De Vivo, Marco
      Nature Reviews Chemistry (2018), 2 (7), 100-112CODEN: NRCAF7; ISSN:2397-3358. (Nature Research)
      The fast-growing body of exptl. data on metalloenzymes and organometallic compds. is fostering the exploitation of metal-ligand interactions for the design of new drugs. Atomistic understanding of the metal-ligand interactions will help us identify potent metalloenzyme inhibitors and metallodrugs. Static docking calcns. have proved effective in identifying hit compds. that target metalloproteins. However, the flexibility, dynamics and electronic structure of metal-centered complexes pose difficult challenges for shaping metal-ligand interactions in structure-based drug design. In this respect, once-prohibitive quantum mechanics-based strategies and extensive mol. simulations are rapidly becoming practical approaches for fast-paced drug discovery. These methods account for ligand exchange and structural flexibility at metal-centered complexes and provide good ests. of the thermodn. and kinetics of metal-aided drug binding. This Perspective examines the successes, limitations and new avenues for modeling metalloenzyme inhibitors and metallodrugs to further explore and expand the unconventional chem. space of these distinctive drugs.
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    75. 75
      De Vivo, M. Bridging Quantum Mechanics and Structure-Based Drug Design. Front. Biosci., Landmark Ed. 2011, 16, 1619– 1633,  DOI: 10.2741/3809
      75
      Bridging quantum mechanics and structure-based drug design
      De Vivo, Marco
      Frontiers in Bioscience, Landmark Edition (2011), 16 (5), 1619-1633CODEN: FRBIF6; ISSN:1093-4715. (Frontiers in Bioscience)
      A review. The last decade has seen great advances in the use of quantum mechanics (QM) to solve biol. problems of pharmaceutical relevance. For instance, enzymic catalysis is often investigated by means of the so-called QM/MM approach, which uses QM and mol. mechanics (MM) methods to det. the (free) energy landscape of the enzymic reaction mechanism. Here, I will discuss a few representative examples of QM and QM/MM studies of important metalloenzymes of pharmaceutical interest (i.e. metallophosphatases and metallo-beta-lactamases). This review article aims to show how QM-based methods can be used to elucidate ligand-receptor interactions. The challenge is then to exploit this knowledge for the structure-based design of new and potent inhibitors, such as transition state (TS) analogs that resemble the structure and physicochem. properties of the enzymic TS. Given the results and potential expressed to date by QM-based methods in studying biol. problems, the application of QM in structure-based drug design will likely increase, making of these once-prohibitive computations a routinely used tool for drug design.
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    76. 76
      Genna, V.; Marcia, M.; De Vivo, M. A Transient and Flexible Cation−π Interaction Promotes Hydrolysis of Nucleic Acids in DNA and RNA Nucleases. J. Am. Chem. Soc. 2019, 141 (27), 10770– 10776,  DOI: 10.1021/jacs.9b03663
      76
      A Transient and Flexible Cation-π Interaction Promotes Hydrolysis of Nucleic Acids in DNA and RNA Nucleases
      Genna, Vito; Marcia, Marco; Vivo, Marco De
      Journal of the American Chemical Society (2019), 141 (27), 10770-10776CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Metal-dependent DNA and RNA nucleases are enzymes that cleave nucleic acids with great efficiency and precision. These enzyme-mediated hydrolytic reactions are fundamental for the replication, repair, and storage of genetic information within the cell. Here, extensive classical and quantum-based free-energy mol. simulations show that a cation-π interaction is transiently formed in situ at the metal core of Bacteriophage-λ Exonuclease (Exo-λ), during catalysis. This noncovalent interaction (Lys131-Tyr154) triggers nucleophile activation for nucleotide excision. Then, our simulations also show the oscillatory dynamics and swinging of the newly formed cation-π dyad, whose conformational change may favor proton release from the cationic Lys131 to the bulk soln., thus restoring the precatalytic protonation state in Exo-λ. Altogether, we report on the novel mechanistic character of cation-π interactions for catalysis. Structural and bioinformatic analyses support that flexible orientation and transient formation of mobile cation-π interactions may represent a common catalytic strategy to promote nucleic acid hydrolysis in DNA and RNA nucleases.
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    77. 77
      Yan, C.; Dodd, T.; He, Y.; Tainer, J. A.; Tsutakawa, S. E.; Ivanov, I. Transcription Preinitiation Complex Structure and Dynamics Provide Insight into Genetic Diseases. Nat. Struct. Mol. Biol. 2019, 26 (6), 397– 406,  DOI: 10.1038/s41594-019-0220-3
      77
      Transcription preinitiation complex structure and dynamics provide insight into genetic diseases
      Yan, Chunli; Dodd, Thomas; He, Yuan; Tainer, John A.; Tsutakawa, Susan E.; Ivanov, Ivaylo
      Nature Structural & Molecular Biology (2019), 26 (6), 397-406CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
      Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly's global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE-p62 interactions that link core-PIC to TFIIH.p62 rigging interlaces p34, p44 and XPD while capping the DNA-binding and ATP-binding sites of XPD. PIC kinks and locks substrate DNA, creating neg.supercoiling within the Pol II cleft to facilitate promoter opening. Mapping disease mutations assocd. with xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome onto defined communities reveals clustering into three mechanistic classes that affect TFIIH helicase functions, protein interactions and interface dynamics.
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    78. 78
      Casalino, L.; Palermo, G.; Spinello, A.; Rothlisberger, U.; Magistrato, A. All-Atom Simulations Disentangle the Functional Dynamics Underlying Gene Maturation in the Intron Lariat Spliceosome. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (26), 6584– 6589,  DOI: 10.1073/pnas.1802963115
      78
      All-atom simulations disentangle the functional dynamics underlying gene maturation in the intron lariat spliceosome
      Casalino, Lorenzo; Palermo, Giulia; Spinello, Angelo; Rothlisberger, Ursula; Magistrato, Alessandra
      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (26), 6584-6589CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The spliceosome (SPL) is a majestic macromol. machinery composed of five small nuclear RNAs and hundreds of proteins. SPL removes noncoding introns from precursor mRNAs (pre-mRNAs) and ligates coding exons, giving rise to functional mRNAs. Building on the first SPL structure solved at near-at.-level resoln., here we elucidate the functional dynamics of the intron lariat spliceosome (ILS) complex through multi-microsecond-long mol.-dynamics simulations of ∼1,000,000 atoms models. The ILS essential dynamics unveils (i) the leading role of the Spp42 protein, which heads the gene maturation by tuning the motions of distinct SPL components, and (ii) the crit. participation of the Cwf19 protein in displacing the intron lariat/U2 branch helix. These findings provide unprecedented details on the SPL functional dynamics, thus contributing to move a step forward toward a thorough understanding of eukaryotic pre-mRNA splicing.
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    79. 79
      Orellana, L.; Thorne, A. H.; Lema, R.; Gustavsson, J.; Parisian, A. D.; Hospital, A.; Cordeiro, T. N.; Bernadó, P.; Scott, A. M.; Brun-Heath, I.; Lindahl, E.; Cavenee, W. K.; Furnari, F. B.; Orozco, M. Oncogenic Mutations at the EGFR Ectodomain Structurally Converge to Remove a Steric Hindrance on a Kinase-Coupled Cryptic Epitope. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (20), 10009– 10018,  DOI: 10.1073/pnas.1821442116
      79
      Oncogenic mutations at the EGFR ectodomain structurally converge to remove a steric hindrance on a kinase-coupled cryptic epitope
      Orellana, Laura; Thorne, Amy H.; Lema, Rafael; Gustavsson, Johan; Parisian, Alison D.; Hospital, Adam; Cordeiro, Tiago N.; Bernado, Pau; Scott, Andrew M.; Brun-Heath, Isabelle; Lindahl, Erik; Cavenee, Webster K.; Furnari, Frank B.; Orozco, Modesto
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (20), 10009-10018CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Epidermal growth factor receptor (EGFR) signaling is initiated by a large ligand-favored conformational change of the extracellular domain (ECD) from a closed, self-inhibited tethered monomer, to an open untethered state, which exposes a loop required for strong dimerization and activation. In glioblastomas (GBMs), structurally heterogeneous missense and deletion mutations conc. at the ECD for unclear reasons. We explore the conformational impact of GBM missense mutations, combining elastic network models (ENMs) with multiple mol. dynamics (MD) trajectories. Our simulations reveal that the main missense class, located at the I-II interface away from the self-inhibitory tether, can unexpectedly favor spontaneous untethering to a compact intermediate state, here validated by small-angle X-ray scattering (SAXS). Significantly, such intermediate is characterized by the rotation of a large ECD fragment (N-TR1), deleted in the most common GBM mutation, EGFRvIII, and that makes accessible a cryptic epitope characteristic of cancer cells. This observation suggested potential structural equivalence of missense and deletion ECD changes in GBMs. Corroborating this hypothesis, our FACS, in vitro, and in vivo data demonstrate that entirely different ECD variants all converge to remove N-TR1 steric hindrance from the 806-epitope, which we show is allosterically coupled to an intermediate kinase and hallmarks increased oncogenicity. Finally, the detected extraintracellular coupling allows for synergistic cotargeting of the intermediate with mAb806 and inhibitors, which is proved herein.
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    80. 80
      Palermo, G.; Casalino, L.; Magistrato, A.; McCammon, A. J. Understanding the Mechanistic Basis of Non-Coding RNA through Molecular Dynamics Simulations. J. Struct. Biol. 2019, 206 (3), 267– 279,  DOI: 10.1016/j.jsb.2019.03.004
      80
      Understanding the mechanistic basis of non-coding RNA through molecular dynamics simulations
      Palermo, Giulia; Casalino, Lorenzo; Magistrato, Alessandra; Andrew McCammon, J.
      Journal of Structural Biology (2019), 206 (3), 267-279CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)
      A review. Noncoding RNA (ncRNA) has a key role in regulating gene expression, mediating fundamental processes and diseases via a variety of yet unknown mechanisms. Here, we review recent applications of conventional and enhanced Mol. Dynamics (MD) simulations methods to address the mechanistic function of large biomol. systems that are tightly involved in the ncRNA function and that are of key importance in life sciences. This compendium focuses of three biomol. systems, namely the CRISPR-Cas9 genome editing machinery, group II intron ribozyme and the ribonucleoprotein complex of the spliceosome, which edit and process ncRNA. We show how the application of a novel accelerated MD simulations method has been key in disclosing the conformational transitions underlying RNA binding in the CRISPR-Cas9 complex, suggesting a mechanism for RNA recruitment and clarifying the conformational changes required for attaining genome editing. As well, we discuss the use of mixed quantum-classical MD simulations in deciphering the catalytic mechanism of RNA splicing as operated by group II intron ribozyme, one of the largest ncRNA structures crystd. so far. Finally, we debate the future challenges and opportunities in the field, discussing the recent application of MD simulations for unraveling the functional biophysics of the spliceosome, a multi-mega Dalton complex of proteins and small nuclear RNAs that performs RNA splicing in humans. This showcase of applications highlights the current talent of MD simulations to dissect at.-level details of complex biomol. systems instrumental for the design of finely engineered genome editing machines. As well, this review aims at inspiring future investigations of several other ncRNA regulatory systems, such as micro and small interfering RNAs, which achieve their function and specificity using RNA-based recognition and targeting strategies.
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    81. 81
      Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
      81
      ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
      Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
      Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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    82. 82
      Galindo-Murillo, R.; Robertson, J. C.; Zgarbová, M.; Šponer, J.; Otyepka, M.; Jurečka, P.; Cheatham, T. E. Assessing the Current State of Amber Force Field Modifications for DNA. J. Chem. Theory Comput. 2016, 12 (8), 4114– 4127,  DOI: 10.1021/acs.jctc.6b00186
      82
      Assessing the Current State of Amber Force Field Modifications for DNA
      Galindo-Murillo, Rodrigo; Robertson, James C.; Zgarbova, Marie; Sponer, Jiri; Otyepka, Michal; Jurecka, Petr; Cheatham, Thomas E.
      Journal of Chemical Theory and Computation (2016), 12 (8), 4114-4127CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      The utility of mol. dynamics (MD) simulations to model biomol. structure, dynamics, and interactions has witnessed enormous advances in recent years due to the availability of optimized MD software and access to significant computational power, including GPU multicore computing engines and other specialized hardware. This has led researchers to routinely extend conformational sampling times to the microsecond level and beyond. The extended sampling time has allowed the community not only to converge conformational ensembles through complete sampling but also to discover deficiencies and overcome problems with the force fields. Accuracy of the force fields is a key component, along with sampling, toward being able to generate accurate and stable structures of biopolymers. The Amber force field for nucleic acids has been used extensively since the 1990s, and multiple artifacts have been discovered, cor., and reassessed by different research groups. We present a direct comparison of two of the most recent and state-of-the-art Amber force field modifications, bsc1 and OL15, that focus on accurate modeling of double-stranded DNA. After extensive MD simulations with five test cases and two different water models, we conclude that both modifications are a remarkable improvement over the previous bsc0 force field. Both force field modifications show better agreement when compared to exptl. structures. To ensure convergence, the Drew-Dickerson dodecamer (DDD) system was simulated using 100 independent MD simulations, each extended to at least 10 μs, and the independent MD simulations were concatenated into a single 1 ms long trajectory for each combination of force field and water model. This is significantly beyond the time scale needed to converge the conformational ensemble of the internal portions of a DNA helix absent internal base pair opening. Considering all of the simulations discussed in the current work, the MD simulations performed to assess and validate the current force fields and water models aggregate over 14 ms of simulation time. The results suggest that both the bsc1 and OL15 force fields render av. structures that deviate significantly less than 1 Å from the av. exptl. structures. This can be compared to similar but less exhaustive simulations with the CHARMM 36 force field that aggregate to the ∼90 μs time scale and also perform well but do not produce structures as close to the DDD NMR av. structures (with root-mean-square deviations of 1.3 Å) as the newer Amber force fields. On the basis of these analyses, any future research involving double-stranded DNA simulations using the Amber force fields should employ the bsc1 or OL15 modification.
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    83. 83
      Dans, P. D.; Ivani, I.; Hospital, A.; Portella, G.; González, C.; Orozco, M. How Accurate Are Accurate Force-Fields for B-DNA?. Nucleic Acids Res. 2017, 45 (7), 4217– 4230,  DOI: 10.1093/nar/gkw1355
      83
      How accurate are accurate force-fields for B-DNA?
      Dans, Pablo D.; Ivani, Ivan; Hospital, Adam; Portella, Guillem; Gonzalez, Carlos; Orozco, Modesto
      Nucleic Acids Research (2017), 45 (7), 4217-4230CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Last generation of force-fields are raising expectations on the quality of mol. dynamics (MD) simulations of DNA, as well as to the belief that theor. models can substitute exptl. ones in several cases. However these claims are based on limited benchmarks, where MD simulations have shown the ability to reproduce already existing 'exptl. models', which in turn, have an unclear accuracy to represent DNA conformation in soln. In this work we explore the ability of different force-fields to predict the structure of two new B-DNA dodecamers, detd. herein by means of 1H NMR. The study allowed us to check directly for exptl. NMR observables on duplexes previously not solved, and also to assess the reliability of 'exptl. structures'. We obsd. that tech. details in the annealing procedures can induce non-negligible local changes in the final structures. We also found that while not all theor. simulations are equally reliable, those obtained using last generation of AMBER force-fields (BSC1 and BSC0OL15) show predictive power in the multi-microsecond timescale and can be safely used to reproduce global structure of DNA duplexes and fine sequence-dependent details.
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    84. 84
      Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and Testing of a General Amber Force Field. J. Comput. Chem. 2004, 25 (9), 1157– 1174,  DOI: 10.1002/jcc.20035
      84
      Development and testing of a general Amber force field
      Wang, Junmei; Wolf, Romain M.; Caldwell, James W.; Kollman, Peter A.; Case, David A.
      Journal of Computational Chemistry (2004), 25 (9), 1157-1174CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      We describe here a general Amber force field (GAFF) for org. mols. GAFF is designed to be compatible with existing Amber force fields for proteins and nucleic acids, and has parameters for most org. and pharmaceutical mols. that are composed of H, C, N, O, S, P, and halogens. It uses a simple functional form and a limited no. of atom types, but incorporates both empirical and heuristic models to est. force consts. and partial at. charges. The performance of GAFF in test cases is encouraging. In test I, 74 crystallog. structures were compared to GAFF minimized structures, with a root-mean-square displacement of 0.26 Å, which is comparable to that of the Tripos 5.2 force field (0.25 Å) and better than those of MMFF 94 and CHARMm (0.47 and 0.44 Å, resp.). In test II, gas phase minimizations were performed on 22 nucleic acid base pairs, and the minimized structures and intermol. energies were compared to MP2/6-31G* results. The RMS of displacements and relative energies were 0.25 Å and 1.2 kcal/mol, resp. These data are comparable to results from Parm99/RESP (0.16 Å and 1.18 kcal/mol, resp.), which were parameterized to these base pairs. Test III looked at the relative energies of 71 conformational pairs that were used in development of the Parm99 force field. The RMS error in relative energies (compared to expt.) is about 0.5 kcal/mol. GAFF can be applied to wide range of mols. in an automatic fashion, making it suitable for rational drug design and database searching.
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    85. 85
      Singh, U. C.; Kollman, P. A. An Approach to Computing Electrostatic Charges for Molecules. J. Comput. Chem. 1984, 5 (2), 129– 145,  DOI: 10.1002/jcc.540050204
      85
      An approach to computing electrostatic charges for molecules
      Singh, U. Chandra; Kollman, Peter A.
      Journal of Computational Chemistry (1984), 5 (2), 129-45CODEN: JCCHDD; ISSN:0192-8651.
      An algorithm is described for deriving net at. charges from ab-initio quantum-mech. calcns. by using a least-squares fit of the quantum-mech. calcd. electrostatic potential to that of the partial-charge model. Applications were made to the mols. H2O, CH3OH, (CH3)2O, H2CO, NH3, (CH3O)2PO2-, deoxyribose, ribose, adenine, 9-CH3 adenine, thymine, 1-CH3 thymine, guanine, 9-CH3 guanine, cytosine, 1-CH3 cytosine, uracil, and 1-CH3 uracil. Inclusion of lone pairs, their location, and charge is discussed.
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    86. 86
      Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. A Well-Behaved Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges: The RESP Model. J. Phys. Chem. 1993, 97 (40), 10269– 10280,  DOI: 10.1021/j100142a004
      86
      A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model
      Bayly, Christopher I.; Cieplak, Piotr; Cornell, Wendy; Kollman, Peter A.
      Journal of Physical Chemistry (1993), 97 (40), 10269-80CODEN: JPCHAX; ISSN:0022-3654.
      The authors present a new approach to generating electrostatic potential (ESP) derived charges for mols. The major strength of electrostatic potential derived charges is that they optimally reproduce the intermol. interaction properties of mols. with a simple two-body additive potential, provided, of course, that a suitably accurate level of quantum mech. calcn. is used to derive the ESP around the mol. Previously, the major weaknesses of these charges have been that they were not easily transferably between common functional groups in related mols., they have often been conformationally dependent, and the large charges that frequently occur can be problematic for simulating intramol. interactions. Introducing restraints in the form of a penalty function into the fitting process considerably reduces the above problems, with only a minor decrease in the quality of the fit to the quantum mech. ESP. Several other refinements in addn. to the restrained electrostatic potential (RESP) fit yield a general and algorithmic charge fitting procedure for generating atom-centered point charges. This approach can thus be recommended for general use in mol. mechanics, mol. dynamics, and free energy calcns. for any org. or bioorg. system.
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    87. 87
      Hess, B. P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation. J. Chem. Theory Comput. 2008, 4 (1), 116– 122,  DOI: 10.1021/ct700200b
      87
      P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation
      Hess, Berk
      Journal of Chemical Theory and Computation (2008), 4 (1), 116-122CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      By removing the fastest degrees of freedom, constraints allow for an increase of the time step in mol. simulations. In the last decade parallel simulations have become commonplace. However, up till now efficient parallel constraint algorithms have not been used with domain decompn. In this paper the parallel linear constraint solver (P-LINCS) is presented, which allows the constraining of all bonds in macromols. Addnl. the energy conservation properties of (P-)LINCS are assessed in view of improvements in the accuracy of uncoupled angle constraints and integration in single precision.
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    88. 88
      Abraham, M. J.; Murtola, T.; Schulz, R.; Pall, S.; Smith, J. C.; Hess, B.; Lindahl, E. Gromacs: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1–2, 19– 25,  DOI: 10.1016/j.softx.2015.06.001
      There is no corresponding record for this reference.
    89. 89
      Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An N·log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98 (12), 10089– 10092,  DOI: 10.1063/1.464397
      89
      Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems
      Darden, Tom; York, Darrin; Pedersen, Lee
      Journal of Chemical Physics (1993), 98 (12), 10089-92CODEN: JCPSA6; ISSN:0021-9606.
      An N·log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolution using fast Fourier transforms. Timings and accuracies are presented for three large cryst. ionic systems.
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    90. 90
      Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103 (19), 8577– 8593,  DOI: 10.1063/1.470117
      90
      A smooth particle mesh Ewald method
      Essmann, Ulrich; Perera, Lalith; Berkowitz, Max L.; Darden, Tom; Lee, Hsing; Pedersen, Lee G.
      Journal of Chemical Physics (1995), 103 (19), 8577-93CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      The previously developed particle mesh Ewald method is reformulated in terms of efficient B-spline interpolation of the structure factors. This reformulation allows a natural extension of the method to potentials of the form 1/rp with p ≥ 1. Furthermore, efficient calcn. of the virial tensor follows. Use of B-splines in the place of Lagrange interpolation leads to analytic gradients as well as a significant improvement in the accuracy. The authors demonstrate that arbitrary accuracy can be achieved, independent of system size N, at a cost that scales as N log(N). For biomol. systems with many thousands of atoms and this method permits the use of Ewald summation at a computational cost comparable to that of a simple truncation method of 10 Å or less.
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    91. 91
      Matta, C. F.; Bader, R. F. W. Atoms-in-molecules study of the genetically encoded amino acids. III. Bond and atomic properties and their correlations with experiment including mutation-induced changes in protein stability and genetic coding. Proteins: Struct., Funct., Genet. 2003, 52 (3), 360– 399,  DOI: 10.1002/prot.10414
      91
      Atoms-in-molecules study of the genetically encoded amino acids. III. Bond and atomic properties and their correlations with experiment including mutation-induced changes in protein stability and genetic coding
      Matta, Cherif F.; Bader, Richard F. W.
      Proteins: Structure, Function, and Genetics (2003), 52 (3), 360-399CODEN: PSFGEY; ISSN:0887-3585. (Wiley-Liss, Inc.)
      This article presents a study of the mol. charge distributions of the genetically encoded amino acids (AA), one that builds on the previous detn. of their equil. geometries and the demonstrated transferability of their common geometrical parameters. The properties of the charge distributions are characterized and given quant. expression in terms of the bond and at. properties detd. within the quantum theory of atoms-in-mols. (QTAIM) that defines atoms and bonds in terms of the observable charge d. The properties so defined are demonstrated to be remarkably transferable, a reflection of the underlying transferability of the charge distributions of the main chain and other groups common to the AA. The use of the at. properties in obtaining an understanding of the biol. functions of the AA, whether free or bound in a polypeptide, is demonstrated by the excellent statistical correlations they yield with exptl. physicochem. properties. A property of the AA side chains of particular importance is the charge sepn. index (CSI), a quantity previously defined as the sum of the magnitudes of the at. charges and which measures the degree of sepn. of pos. and neg. charges in the side chain of interest. The CSI values provide a correlation with the measured free energies of transfer of capped side chain analogs, from the vapor phase to aq. soln., yielding a linear regression equation with r2 = 0.94. The at. vol. is defined by the van der Waals isodensity surface and it, together with the CSI, which accounts for the electrostriction of the solvent, yield a linear regression (r2 = 0.98) with the measured partial molar volumes of the AAs. The changes in free energies of transfer from octanol to water upon interchanging 153 pairs of AAs and from cyclohexane to water upon interchanging 190 pairs of AAs, were modeled using only three calcd. parameters (representing electrostatic and vol. contributions) yielding linear regressions with r2 values of 0.78 and 0.89, resp. These results are a prelude to the single-site mutation-induced changes in the stabilities of two typical proteins: ubiquitin and staphylococcal nuclease. Strong quadratic correlations (r2 ∼ 0.9) were obtained between ΔCSI upon mutation and each of the two terms ΔΔH and TΔΔS taken from recent and accurate differential scanning calorimetry expts. on ubiquitin. When the two terms are summed to yield ΔΔG, the quadratic terms nearly cancel, and the result is a simple linear fit between ΔΔG and ΔCSI with r2 = 0.88. As another example, the change in the stability of staphylococcal nuclease upon mutation has been fitted linearly (r2 = 0.83) to the sum of a ΔCSI term and a term representing the change in the van der Waals vol. of the side chains upon mutation. The suggested correlation of the polarity of the side chain with the second letter of the AA triplet genetic codon is given concrete expression in a classification of the side chains in terms of their CSI values and their group dipole moments. For example, all amino acids with a pyrimidine base as their second letter in mRNA possess side-chain CSI ≤ 2.8 (with the exception of Cys), whereas all those with CSI > 2.8 possess an purine base. The article concludes with two proposals for measuring and predicting mol. complementarity: van der Waals complementarity expressed in terms of the van der Waals isodensity surface and Lewis complementarity expressed in terms of the local charge concns. and depletions defined by the topol. of the Laplacian of the electron d. A display of the exptl. accessible Laplacian distribution for a folded protein would offer a clear picture of the operation of the "stereochem. code" proposed as the determinant in the folding process.
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    92. 92
      Peraro, M. D.; Spiegel, K.; Lamoureux, G.; De Vivo, M.; DeGrado, W. F.; Klein, M. L. Modeling the Charge Distribution at Metal Sites in Proteins for Molecular Dynamics Simulations. J. Struct. Biol. 2007, 157 (3), 444– 453,  DOI: 10.1016/j.jsb.2006.10.019
      There is no corresponding record for this reference.
    93. 93
      Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79 (2), 926– 935,  DOI: 10.1063/1.445869
      93
      Comparison of simple potential functions for simulating liquid water
      Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.; Impey, Roger W.; Klein, Michael L.
      Journal of Chemical Physics (1983), 79 (2), 926-35CODEN: JCPSA6; ISSN:0021-9606.
      Classical Monte Carlo simulations were carried out for liq. H2O in the NPT ensemble at 25° and 1 atm using 6 of the simpler intermol. potential functions for the dimer. Comparisons were made with exptl. thermodn. and structural data including the neutron diffraction results of Thiessen and Narten (1982). The computed densities and potential energies agree with expt. except for the original Bernal-Fowler model, which yields an 18% overest. of the d. and poor structural results. The discrepancy may be due to the correction terms needed in processing the neutron data or to an effect uniformly neglected in the computations. Comparisons were made for the self-diffusion coeffs. obtained from mol. dynamics simulations.
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    94. 94
      Bussi, G.; Donadio, D.; Parrinello, M. Canonical Sampling through Velocity Rescaling. J. Chem. Phys. 2007, 126 (1), 014101,  DOI: 10.1063/1.2408420
      94
      Canonical sampling through velocity rescaling
      Bussi, Giovanni; Donadio, Davide; Parrinello, Michele
      Journal of Chemical Physics (2007), 126 (1), 014101/1-014101/7CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      The authors present a new mol. dynamics algorithm for sampling the canonical distribution. In this approach the velocities of all the particles are rescaled by a properly chosen random factor. The algorithm is formally justified and it is shown that, in spite of its stochastic nature, a quantity can still be defined that remains const. during the evolution. In numerical applications this quantity can be used to measure the accuracy of the sampling. The authors illustrate the properties of this new method on Lennard-Jones and TIP4P water models in the solid and liq. phases. Its performance is excellent and largely independent of the thermostat parameter also with regard to the dynamic properties.
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    95. 95
      Parrinello, M.; Rahman, A. Polymorphic Transitions in Single Crystals: A New Molecular Dynamics Method. J. Appl. Phys. 1981, 52 (12), 7182– 7190,  DOI: 10.1063/1.328693
      95
      Polymorphic transitions in single crystals: A new molecular dynamics method
      Parrinello, M.; Rahman, A.
      Journal of Applied Physics (1981), 52 (12), 7182-90CODEN: JAPIAU; ISSN:0021-8979.
      A Lagrangian formulation is introduced; it can be used to make mol. dynamics (MD) calcns. on systems under the most general, externally applied, conditions of stress. In this formulation the MD cell shape and size can change according to dynamic equations given by this Lagrangian. This MD technique was used to the study of structural transitions of a Ni single crystal under uniform uniaxial compressive and tensile loads. Some results regarding the stress-strain relation obtained by static calcns. are invalid at finite temp. Under compressive loading, the model of Ni shows a bifurcation in its stress-strain relation; this bifurcation provides a link in configuration space between cubic and hexagonal close packing. Such a transition could perhaps be obsd. exptl. under extreme conditions of shock.
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    96. 96
      Barducci, A.; Bussi, G.; Parrinello, M. Well-Tempered Metadynamics: A Smoothly Converging and Tunable Free-Energy Method. Phys. Rev. Lett. 2008, 100 (2), 020603  DOI: 10.1103/PhysRevLett.100.020603
      96
      Well-Tempered Metadynamics: A Smoothly Converging and Tunable Free-Energy Method
      Barducci, Alessandro; Bussi, Giovanni; Parrinello, Michele
      Physical Review Letters (2008), 100 (2), 020603/1-020603/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      We present a method for detg. the free-energy dependence on a selected no. of collective variables using an adaptive bias. The formalism provides a unified description which has metadynamics and canonical sampling as limiting cases. Convergence and errors can be rigorously and easily controlled. The parameters of the simulation can be tuned so as to focus the computational effort only on the phys. relevant regions of the order parameter space. The algorithm is tested on the reconstruction of an alanine dipeptide free-energy landscape.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.9b10656.

    • Setup protocols of the force-field-based MD simulations; details on enhanced sampling free energy calculations and additional analyses; PDB analyses, structure, and sequence alignments (PDF)

    • Illustrative movie of the overall catalytic process of hExo1, created by merging PDB structures with fragments of trajectories of equilibrium and metadynamics simulations (MP4)


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