logo

Quick View

OR SEARCH CITATIONS

My Activity
Publications
CONTENT TYPES

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Chem. Res. Toxicol. 2014, 27, 10, 1837-1852
RETURN TO ISSUEPREVArticleNEXT

Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase ι Involved in Translesion DNA Synthesis

View Author Information
†Division of Pharmacology, Department of Molecular Cell Biology, and ‡Department of Physiology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do 440-746, Republic of Korea
§ Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, United States
∥ Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States
*(J.-Y.C.) Tel: +82-31-299-6193. Fax: +82-31-299-6209. E-mail: [email protected].
Cite this: Chem. Res. Toxicol. 2014, 27, 10, 1837-1852
Publication Date (Web):August 27, 2014
https://doi.org/10.1021/tx5002755
Copyright © 2014 American Chemical Society
RIGHTS & PERMISSIONS
Authors ChoiceACS AuthorChoice
Article Views
717
Altmetric
-
Citations
9
LEARN ABOUT THESE METRICS

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

PDF (5 MB)
Supporting Info (1)»
SUBJECTS:

Abstract

High Resolution Image
Download MS PowerPoint Slide

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N2-ethyl(Et)G, O6-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, N2-EtG, O6-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg2+ (but not with Mn2+), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg2+). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg2+ or Mn2+, except for that opposite N2-EtG with Mn2+ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg2+), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn2+ than with Mg2+. The results indicate that the R96G variation severely impairs most of the Mg2+- and Mn2+-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg2+-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.

Introduction

ARTICLE SECTIONS
Jump To
DNA damage is constantly generated from endogenous and exogenous sources in cells and poses a major obstacle to vital cellular processes of replication and transcription, possibly leading to mutation and cell death. To cope with the constant threat of DNA damage, cells are equipped with a sophisticated network of DNA damage response systems, including DNA repair mechanisms, damage tolerance processes, and cell-cycle checkpoints.(1, 2) Such systems should ideally have high fidelity, efficiency, and coordination with each other to preserve genome integrity, but these properties are not perfect nor the same for all lesions. Sometimes the attempts at repair can result in genomic errors and cell apoptosis. Inherited defects in human DNA damage response machineries (e.g., XPC, POLH, ATM) cause the faulty repair, damage tolerance, and checkpoints and commonly result in severe cancer predisposition disorders along with other different disease phenotypes.(3, 4) Reduced DNA repair capacity and related genetic variations have been shown to be associated with enhanced cancer risks in human individuals.(5-8) Along the same line, it can be speculated that the differential cellular capacity for DNA damage tolerance influences the final biological outcomes from residual genomic lesions and thus could be a determining factor for mutation and cancer predisposition in individuals.
Persistent unrepaired DNA lesions can interfere with DNA replication, which can lead to replication fork stalling and copying errors. As a prompt response to the lesion-blocked replication fork, cells are able to utilize a DNA damage tolerance system involving specialized translesion polymerases, mostly belonging to the Y-Family, which overcomes DNA lesions and performs translesion synthesis (TLS). TLS is a potentially mutagenic process due to the low fidelity of TLS polymerases with lesions in many cases, while serving to avoid the permanent cell cycle arrest and cell death. Indeed, each Y-Family polymerase can carry out its unique TLS, varying in both efficiency and fidelity depending on the type, size, and location of the lesion.(9) Therefore, individual TLS polymerases may play distinctive roles, i.e., protective (error-free), provocative (error-prone), or neutral, in mutagenesis induced by each specific DNA lesion in cells. For instance, both pol κ and REV1 can perform error-free and efficient TLS at bulky N2-guanine (G) lesions, such as benzo[a]pyrene-derived N2-G adducts but pol η and pol ι perform relatively error-prone TLS (albeit yielding different types of errors) at those adducts,(10-13) suggesting error-free roles for the former two pols but error-prone roles for the latter two pols in TLS (at least regarding bulky carcinogen-bound N2-G adducts). In these circumstances, it can be postulated that the overall cellular TLS capacity, comprising behaviors of multiple individual polymerases employed against carcinogen-specific DNA lesions, will determine the levels of lesion-derived mutations in the newly synthesized genome and thus play a role in preventing or facilitating mutagenesis resulting from genotoxic carcinogens in cells, which could further relate to cancer susceptibility in individuals.
DNA polymerase (pol) ι, a member of human Y-family DNA polymerases, has been known to perform TLS at various DNA lesions although it has the lowest fidelity in DNA synthesis among polymerases. Pol ι is inherently very error-prone in nucleotide insertion, particularly opposite undamaged template bases G and T, respectively, yielding misinsertion of either dTTP or dGTP at a frequency of about 0.1 and 1 (relative to the correct nucleotide insertion), which is ascribed to its unique active site and related non-Watson–Crick base pairing.(14-16) Pol ι is able to mediate relatively mutagenic but occasionally accurate replicative bypass with a variety of DNA lesions, including minor-groove N2-G adducts, major-groove O6-G adducts, 8-oxo-7,8-dihydroG (8-oxoG), pyrimidine dimers, and abasic lesions, with different nucleotide selectivities according to lesion types. Pol ι inserts both dCTP and dTTP opposite N2-G and O6-G adducts, with a slight preference of either dCTP or dTTP, respectively.(17) Pol ι slightly prefers dCTP over dGTP for insertion opposite 8-oxoG, prefers dATP opposite the 3′ T (but both dGTP and dTTP opposite 5′ T) of (6–4) TT photoproducts, and slightly prefers to insert dTTP and dGTP opposite abasic lesions.(18, 19) Pol ι is distinctively known to prefer Mn2+ over Mg2+ ions as a metal in polymerase catalysis and is maximally active at low concentrations (0.05–0.25 mM) of Mn2+.(20)
Substantial evidence suggests a possible implication of pol ι in cancer in mammals. Pol ι deficiency results in a higher susceptibility to UV-induced skin cancers in mice under pol η-null conditions,(21, 22) and the loss of pol ι increases urethane-induced lung mutagenesis and tumorigenesis in C57BL/6J pol ι-knockout mice.(23) Dysregulation of pol ι is also found in many types of cancers. Pol ι is overexpressed in various types of cancerous tissues or cells including breast, prostate, uterus, stomach, rectal, esophageal, and bladder cancers,(24-27) which might hypothetically lead to a mutator phenotype due to an elevated error-prone DNA replication. Two germline POLI single nucleotide variations (SNVs), which might result in a missense change at codon 532 or 731 located in the ubiquitin-binding motifs, have been associated with a significantly higher risk of some subsets of lung and prostate cancers, respectively, although their specific mechanisms have not been elucidated yet.(28, 29) On the basis of these circumstances, we can infer that pol ι would serve a protective (or sometimes facilitative) role in genomic mutagenesis induced by genotoxic agents in cells, and the altered status of pol ι function by genetic variation might affect individual risks of mutation and cancer from exposure to certain genotoxic carcinogens.
The human POLI gene encodes the pol ι protein consisting of 740 amino acids according to NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank/), which has the additional N-terminal 25 residues to the formerly erroneously designated open-reading frame (ORF) amino acid sequence. For such a reason, most of the previous biochemical and structural experiments on pol ι were performed using the sequence information on the N-terminal truncated (25-amino acids-shorter) ORF. The catalytic core of pol ι is positioned in the N-terminal region (amino acids 26–445), and its ternary complex crystal structure has been determined.(14) However, biochemical properties of the new wild-type pol ι, as well as the effect of the extra 25 amino acids (containing 12 acidic residues) added at the N-terminus, on pol ι function, have not been reported. Until the present time, a total of ∼122 germline variations in POLI gene have been described for human individuals in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP), but the functional impacts of these genetic variations have not been biochemically evaluated yet. Biochemical approaches to evaluate the effects of germline genetic variations on pol ι function are indispensable for understanding and predicting their mechanistic basis and biological outcomes either before or after studying their clinical associations. In this study, we focused on the nonsynonymous coding POLI gene variations, which are located in the polymerase core domains and the N-terminal 25 amino acids region, and predicted to have damaging effects by in silico prediction analysis tools, e.g., SIFT and PolyPhen,(30-32) because they would be likely to affect the catalytic function of pol ι and change its TLS function. In order to characterize the putatively functional genetic variations of human pol ι, we investigated the biochemical effects of six selected missense or deletion genetic variations on the enzymatic properties of human pol ι regarding both normal DNA synthesis and bypass of various DNA lesions. We performed the experiments with “standing-start” full-length primer extensions, steady-state kinetics, and pol-DNA binding in the presence of either Mg2+ or Mn2+ ions, using wild-type recombinant human pol ι (1–445 amino acids) and six variants with primer-annealed oligonucleotide DNA templates containing an undamaged G, N2-ethyl(Et)G, O6-methyl(Me)G, 8-oxoG, or abasic site. Here we describe two germline genetic variations that can alter in vitro enzyme function of pol ι in nucleotide incorporation with normal and lesion DNA substrates, as well as DNA substrate binding. These observations are discussed in the context of understanding the possible mechanistic and functional aspects of altered TLS with pol ι variants.

Experimental Procedures

ARTICLE SECTIONS
Jump To

Materials

T4 polynucleotide kinase, restriction endonucleases, and dNTPs were purchased from New England Biolabs (Ipswich, MA). [γ-32P]ATP (specific activity 3 × 103 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Biospin columns were purchased from Bio-Rad (Hercules, CA). A protease inhibitor cocktail was obtained from Roche Applied Science (Indianapolis, IN). The vector pBG101 was gratefully obtained from the Center for Structural Biology, Vanderbilt University. The pCR2.1-TOPO TA cloning kit was from Invitrogen (Carlsbad, CA), and the QuickChange mutagenesis kit was from Stratagene (La Jolla, CA). FPLC columns were purchased from GE Healthcare (Uppsala, Sweden).

DNA Substrates

24-Mer (5′-GCC TCG AGC CAG CCG CAG ACG CAG-3′) and 36-mer (3′-CGG AGC TCG GTC GGC GTC TGC GTC XCT CCT GCG GCT-5′; X = G, O6-MeG, or tetrahydrofuran (abasic site analogue)) oligonucleotides containing a G, O6-MeG, or abasic site (stable tetrahydrofuran derivative) were obtained from Midland Certified Reagent Co. (Midland, TX). A 36-mer (X = N2-EtG) containing N2-EtG was prepared as previously described.(33) A 36-mer (X = 8-oxoG) containing an 8-oxoG, and an 18-FAM-mer (5′-(FAM)-AGC CAG CCG CAG ACG CAG-3′; FAM = 6-carboxyfluorescein) were obtained from Bioneer (Daejeon, Korea). Primers (24-mers) were 5′ end-labeled using T4 polynucleotide kinase with [γ-32P]ATP and annealed with 36-mer templates to make duplex primer-template DNA substrates for use in polymerase activity assays. 18-FAM-mer primers were annealed with 36-G-mer templates for use in DNA binding assays.

Selection of Human POLI Gene Variations Having Potentially Functional Impact

We searched for human POLI gene variations that are highly likely to alter enzyme function. First, we screened the naturally occurring germ-line genetic variations in the protein-coding sequence of the POLI gene from the public database dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP). We selected four candidate variations to be likely dysfunctional based on three criteria: (i) nonsynonymous coding variations that cause a missense or nonsense change, (ii) variations located in the polymerase core domain (amino acid residues 1 to 445), and (iii) missense variations predicted to be deleterious or damaging on protein function by SIFT and Polyphen.(30-32) We also included two more candidate variations, which cause the initiation codon change or an amino acid deletion and thus might exert functional changes. Thus, we selected six variants (i.e., a deletion, an initiator codon, and four missense variants) and performed detailed biochemical analyses using the corresponding recombinant protein pol ι1–445 proteins purified from Escherichia coli. Current information for the six POLI gene variations included in this study is summarized in Table 1, based on public databases, e.g., dbSNP, 1000 genomes (http://browser.1000genomes.org).
Table 1. POLI Gene Variations Studied
     predictionb
rs IDanucleotide changeamino acid changeprotein domainminor allele frequencySIFTPolyPhen-2
rs199757163c.3G > AM1_A25del (Δ1–25)c 0.001dN/AeN/A
rs10584411c.51_53delD17del (ΔD17) 0.747dN/AN/A
rs3218778c.286A > GR96Gfinger0.006fdamagingprobably damaging
rs3218784c.783A > GI261Mthumb0.011ddamagingpossibly damaging
rs3218783c.826G > AE276Kthumb0.0005ddamagingpossibly damaging
rs11558769c.1120T > AY374NPADN/Adamagingbenign
a

A reference SNP identification number provided by dbSNP.

b

Possible functional effects of genetic variations are predicted in silico using SIFT and PolyPhen-2.(30-32)

c

The Greek symbol Δ denotes a deletion.

d

From 1000 Genomes project.

e

Not available.

f

From NIHPDR and PDR90 resources described in dbSNP.

Construction of Expression Vectors for Core Proteins of Wild-type Pol ι and Six Variants

The gene fragments covering the core proteins (amino acids 1–445) of wild-type pol ι and the Δ1–25 variant were obtained by PCR amplification from human testis cDNA (Clontech, Mountain View, CA) as template using AccuTaq LA DNA polymerase (Sigma, St. Louis, MO) with a forward primer (5′-GAA TCC ATG GAG AAG CTG GGG GTG G-3′ for wild-type or 5′-GAA TCC ATG GAG TCG GCA GAG GGT GTG-3′ for Δ1–25) and a reverse primer (5′-CTA CTT AGC AGT ATT TAG TGC-3′). Each of the resulting PCR products of 1.3 kb POLI core was cloned into the vector pCR2.1-TOPO, and the nucleotide sequences of pol ι gene inserts for wild-type and Δ1–25 were confirmed. From the nucleotide sequencing of wild-type gene inserts, we obtained two kinds of vectors encoding the wild-type or ΔD17 variant pol ι(1–445), indicating that ΔD17 variation is very common in the human population. Each of the POLI gene fragments were then cloned into the BamHI and EcoRI sites of the vector pBG101, which can generate the pBG101-wtPOLI1–445, and two vectors encoding the Δ1–25 or ΔD17 pol ι(1–445) variant. Each of the four different mutations in the POLI gene was created by a QuickChange mutagenesis kit with the pBG101-wtPOLI1–445 vector as template. The oligonucleotide primers for introducing the point mutations in POLI were 5′-GGT TAC CTG CAA CTA TGA AGC TGG GAA ACT TGG AG-3′ for R96G, 5′-CTT ATT CAT AGT TTG AAT CAC ATG AAG GAA ATA CCT GGT ATT GGC-3′ for I261M, 5′-CCA AAT GTC TTA AAG CAC TGG GTA TCA ATA GTG TGC G-3′ for E276K, 5′-GTG AGA TTA ATA ATC CGT CGG AAT TCC TCT GAG AAG C-3′ for Y374N, and the corresponding antiparallel primer for each mutation. All four substitutions were confirmed by nucleotide sequence analyses of the constructed vectors.

Expression and Purification of Recombinant Proteins

The wild-type and variant forms of recombinant pol ι core proteins were expressed in E. coli strain BL21(DE3) cells. E. coli harboring each vector for the recombinant protein were grown in Luria–Bertani broth supplemented with kanamycin (50 μg mL–1) at 37 °C, with aeration, to an OD600 of 0.6. Isopropyl-β-d-thiogalactopyranoside was added to 0.2 mM, and incubation was continued for 14 h at 16 °C. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, containing 300 mM NaCl, 10% glycerol (v/v), 5 mM β-mercaptoethanol, 1 mg lysozyme mL–1, and protease inhibitor cocktail), cooled on ice for 30 min, and then lysed by sonication (12 × 10 s duration with a Branson digital sonifier microtip, (VWR, West Chester, PA), 45% amplitude, with intermittent cooling time). The cell lysate was clarified by centrifugation at 4 × 104 × g for 60 min at 4 °C. The resulting supernatant was loaded onto a 1 mL GSTrap 4B column, and the column was washed with 20 mL of Buffer A (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 10% glycerol (v/v), and 5 mM β-mercaptoethanol). GST-tagged pol ι core bound on the column was cleaved by Prescission Protease for 14 h at 4 °C. Cleaved pol ι core fractions (eluted with Buffer A) were collected and diluted 6-fold with buffer B (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10% glycerol (v/v), and 5 mM β-mercaptoethanol). Pol ι core was further purified to near homogeneity with the use of a Mono S column and a 50 mM to 2 M NaCl gradient in buffer B. Pol ι core was eluted at ∼250 mM NaCl. Protein concentrations were estimated using a Bradford protein assay, and the quality of purified proteins was assessed by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining (Figure S1, Supporting Information).

DNA Polymerization Assays and Steady-State Kinetic Analysis

Standard DNA polymerase reactions of 8 μL were performed in 50 mM Tris-HCl (pH 7.5) buffer containing 50 mM NaCl, 5 mM dithiothreitol, 100 μg mL–1 bovine serum albumin (BSA) (w/v), and 10% glycerol (v/v) with 100 nM primer-template substrate at 37 °C. Reactions were initiated by the addition of dNTPs with MgCl2 (5 mM final concentration) or MnCl2 (0.15 mM final concentration) to preincubated enzyme/DNA mixtures and terminated with six volumes of a solution of 20 mM EDTA in 95% formamide (v/v). For steady-state kinetic analysis, the primer-template was extended in the presence of 0.1–33 nM pol ι enzyme (or up to 400 nM enzyme for R96G) with increasing concentrations of individual dNTPs for 10 min, when the maximum amount of extension products was ≤20% of total DNA substrates. Products were resolved using a 16% polyacrylamide (w/v) gel electrophoresis system containing 8 M urea and visualized using a Bio-Rad Personal Molecular Imager and Quantity One software (Bio-Rad). The product formation rates (as a function of dNTP concentration) were plotted to estimate the kinetic parameters Km and kcat by nonlinear regression fitting to the Michaelis–Menten equation using Graph Pad Prism 5.0 software (GraphPad, San Diego, CA). Misinsertion frequency (fins) opposite G or G adducts was calculated as fins = (kcat/Km)dNTP/(kcat/Km)dCTP.(34)

Fluorescence Polarization Experiments

The 2 nM 18-FAM-mer primer annealed with unmodified 36-mer template was incubated with varying concentrations of pol ι, and fluorescence polarization (FP) was measured with a Biotek Synergy NEO plate reader (Winooski, VT) using excitation and emission wavelengths of 485 and 528 nm, respectively. The polymerase-DNA binding reaction was done in the presence of 50 mM HEPES buffer (pH 7.5) containing 10 mM potassium acetate, 10 mM KCl, 0.1 mM EDTA, 2 mM β-mercaptoethanol, and 0.1 mg mL–1 BSA in the presence of MgCl2 or MnCl2 (final 0.15 or 1 mM concentration), as modified from a previous study.(35) FP data were plotted vs enzyme concentration and fit to a quadratic equation to estimate Kd,DNA using the equation: P = P0 + (Pmax – P0)((Dt + Et + Kd,DNA) – ((Dt + Et + Kd,DNA)2 – (4DtEt))1/2)/(2Dt), where P is the measured change in polarization (in units of millipolarization (mP)), P0 is initial polarization (DNA alone), Pmax is maximum polarization, Dt is DNA concentration, Et is enzyme concentration, and Kd,DNA is the equilibrium dissociation constant for enzyme binding to DNA.

Results

ARTICLE SECTIONS
Jump To

Overall Study Approach

The aim of this study was to analyze the potentially dysfunctional germ-line genetic variants of human pol ι and identify functional pol ι variants. To achieve this, we first screened for human POLI genetic variations likely to alter the enzymatic function of pol ι from the dbSNP database. We utilized the new annotated open reading frame (ORF) sequence of the POLI gene as described in GenBank accession number NM_007195 (http://www.ncbi.nlm.nih.gov/genbank/), which encodes a pol ι protein of 740 amino acids with an additional 25 amino acids at the N-terminus compared to the previous wild-type, in that the previous ORF of pol ι was erroneously annotated to start at a site 75 bases downstream of the actual translational initiation site.(36) We picked four candidate variations by searching for nonsynonymous coding variations that are located in the polymerase core domains (finger, palm, thumb, and little finger) and are also predicted as damaging with prediction tools (SIFT and PolyPhen). The SIFT algorithm utilizes a sequence homology-based approach to classify amino acid substitutions, which is based on the evolutionary conservation of the amino acids within protein families,(30) while the PolyPhen algorithm uses both sequence- and structure-based prediction.(31, 32) The R96G variation (close to the incoming nucleotide at the finger domain) and the I261M and E276K variations (in the thumb domain) were predicted to be damaging by both SIFT and PolyPhen, while the Y374N variation in the little finger domain was predicted to be damaging only by SIFT (Table 1 and Figure 1). We also selected two more candidate variations that would result in an altered translation initiation or an amino acid deletion at the N-terminal extension of 25 amino acids (Table 1 and Figure 1), one of which is the initiator codon variation (c.3G > A) to mutate the start codon (ATG) to a nonstart codon (ATA) and thus theoretically yield the variant protein deleted of the first 25 N-terminal residues (Δ1–25) that was previously referred to the wild-type enzyme. After that, we investigated the biochemical impact of six genetic variations on the enzymatic features of pol ι in both normal and translesion DNA synthesis at G and various DNA lesions. To effectively observe the alterations of polymerase function in six selected pol ι variants of the polymerase core domains, we utilized the core protein (amino acids 1–445) of pol ι, which contains all the polymerase core domains critical to polymerase activity. The previous core protein (26–445) was also shown to have the similar polymerase activity to the previous full-length pol ι (26–740).(37) A set of experiments, including “standing-start” full-length primer extensions, steady-state kinetics of nucleotide incorporation opposite the lesions, and pol ι-DNA binding assays, was carried out successively using the recombinant core (1–445 amino acids) of pol ι enzymes and oligonucleotides containing a normal G or each of four bypassable DNA lesions, i.e., N2-EtG, O6-MeG, 8-oxoG, or abasic site at a defined site. We also compared the effects of two metals—Mn2+ and Mg2+—in these experiments because pol ι is known to prefer Mn2+ to Mg2+ in polymerase catalysis.(20)

Figure 1

Figure 1. Locations of genetic pol ι variations. Structure of human pol ι(26–445) (PDB code, 2FLL) bound to primer/template DNA and incoming nucleotide is shown using Pymol. Pol ι(26–445) is shown in cartoon ribbons, and the primer/template DNA and nucleotide are shown in gray sticks. The finger, palm, thumb, and PAD domains are colored yellow, red, green, and blue, respectively. The amino acid residues (in purple spheres) of genetic pol ι variants are indicated. The structural domains of pol ι are shown in the upper schematic diagram using DOG (version 2.0),(60) where positions of amino acids related to six studied variations are indicated.

High Resolution Image
Download MS PowerPoint Slide

Primer Extension across G and DNA Lesions with All Four dNTPs by Wild-Type and Variant Pol ι Enzymes in the Presence of MgCl2

To evaluate the possible changes in Mg2+-dependent DNA polymerase activities of six human pol ι variants at undamaged and damaged DNA templates, we performed “standing-start” primer extensions with the wild-type and variant pol ι proteins using 24-mer/36-mer duplexes containing a G, N2-EtG, O6-MeG, 8-oxoG, or abasic site at position 25 of the template in the presence of all four dNTPs and 5 mM MgCl2 (Figure 2). Those four DNA adducts, which were previously found to be bypassed relatively efficiently by human pol ι,(11, 17, 19, 38) were selected as the favored substrate lesions of pol ι. Wild-type pol ι extended about half of the primers past G and yielded mainly one-base extended 25-mer products with some traces of 26- and 27-mers (with a 50 nM enzyme concentration), and this pattern was also observed with the ΔD17, I261M, E276K, and Y374N variants. However, the Δ1–25 variant readily synthesized products mainly up to 27- and 28-mers and seemed much more effective than the wild-type enzyme. In contrast, the R96G variant generated almost no extension at G even with a 50 nM enzyme concentration, indicating severe impairment of polymerase activity due to this amino acid substitution. For translesion synthesis at N2-EtG, O6-MeG, 8-oxoG, or an abasic lesion, those six variants showed a similar trend of results as that observed with an undamaged G template, although the extents of bypass synthesis across each lesions differed from one another. With each of those four lesions, the Δ1–25 variant yielded considerably more one-base or up to four-base extended products than the wild-type, while the R96G variant yielded only a trace of one-base or almost no extension at the lesions. Similar patterns were also observed in the presence of 1 mM MgCl2 (at which pol ι was observed to be maximally active with normal DNA substrates tested, results not shown). These results indicate that R96G and Δ1–25 pol ι variants might have a defective and hyperactive Mg2+-dependent TLS ability, respectively.

Figure 2

Figure 2. Extension of 32P-labeled primers opposite G, O6-MeG, N2-EtG, 8-oxoG, and an abasic site by human wild-type pol ι (1–445) and variants in the presence of Mg2+. The primer (24-mer) was annealed with each of the five different 36-mer templates containing an unmodified G, O6-MeG, N2-EtG, 8-oxoG, or abasic site placed at the 25th position from the 3′-end. Reactions were done in the presence of 5 mM MgCl2 for 15 min with DNA substrate (100 nM primer/template), all four dNTPs (50 μM each), and increasing concentrations of pol ι (0–50 nM) as indicated. The extension products were separated by denaturing gel electrophoresis and imaged using a phosphorimager.

High Resolution Image
Download MS PowerPoint Slide

Primer Extension across G and DNA Lesions with All Four dNTPs by Wild-Type and Variant Pol ι Enzymes in the Presence of MnCl2

To examine the effect of Mn2+ as a prosthetic group (instead of Mg2+) on normal and translesion polymerase activities by six variants, standing-start primer extension experiments were done with the wild-type and variant pol ι proteins using 24-mer primers annealed to 36-mer templates containing G, N2-EtG, O6-MeG, 8-oxoG, or an abasic site in the presence of all four dNTPs and 0.15 mM MnCl2 (Figure 3). Wild-type pol ι extended most 24-mer primers across G and four lesions and yielded 25- or up to 28-mer products (10 nM enzyme concentration with 0.15 mM Mn2+, Figure 3A), more effectively than with 5 mM Mg2+ (Figure 2A), indicating a catalytic preference of pol ι for Mn2+ compared to Mg2+, as expected from the previous literature.(20) The primer extension results for each pol ι protein across G and four lesions in the presence of Mn2+ were almost similar to those with Mg2+ except for the case of the Δ1–25 variant. Opposite G and four DNA lesions, the R96G variant generated extension products considerably lower in extent than the wild-type in the presence of Mn2+ (Figure 3A), as similarly observed with Mg2+ (Figure 2A), although the decrease of R96G bypass extent was less opposite template N2-EtG than the other templates. However, the Δ1–25 variant extended the primers across G and four lesions to a similar extent as the wild-type pol ι in the presence of Mn2+ (Figure 3), in contrast to the increase in Mg2+-dependent polymerase activity of this variant (Figure 2).

Figure 3

Figure 3. Extension of 32P-labeled primers opposite G, O6-MeG, N2-EtG, 8-oxoG, and an abasic site by human wild-type pol ι (1–445) and variants in the presence of Mn2+. Primer (24-mer) was annealed with each of the five different 36-mer templates containing an unmodified G, O6-MeG, N2-EtG, 8-oxoG, or abasic site placed at the 25th position from the 3′-end. Reactions were done in the presence of 0.15 mM MnCl2 for 15 min with DNA substrate (100 nM primer/template), all four dNTPs (50 μM each), and increasing concentrations of pol ι (0–10 nM) as indicated. The extension products were separated by denaturing gel electrophoresis and imaged using a phosphorimager.

High Resolution Image
Download MS PowerPoint Slide

Steady-State Kinetics of Nucleotide Incorporation Opposite DNA Lesions by the Wild-Type and Variant Pol ι Enzymes in the Presence of MgCl2

To analyze the efficiency and fidelity of six pol ι variants for Mg2+-dependent nucleotide insertion opposite G and four DNA lesions, we determined steady-state kinetic parameters for incorporation of single nucleotides into 24-mer/36-mer duplexes opposite a G or each of four lesions in the presence of 5 mM MgCl2 by six variants in comparison to wild-type pol ι (Tables 2–4). The values of kcat/Km and misinsertion frequency (f = (kcat/Km)incorrect dNTP/(kcat/Km)correct dNTP) were employed as semiquantitative measures for the nucleotide insertion efficiency and fidelity of a distributive pol ι, as applied in previous work.(19) Wild-type pol ι inserted single nucleotides opposite each template with the efficiency order (based on the maximum kcat/Km) abasic lesion > O6-MeG > G > 8-oxoG > N2-EtG. Wild-type pol ι inserted the correct dCTP in slight preference to dTTP opposite G and N2-EtG but misinserted dTTP and dGTP (in preference to dCTP) opposite O6-MeG and 8-oxoG, respectively, while inserting nucleoside triphosphates opposite an abasic site in the preferential order of dGTP > dTTP > dATP > dCTP. The ΔD17, I261M, E276K, and Y374N variants inserted dCTP in preference to dTTP opposite undamaged G, with the values of kcat/Km similar to those of wild-type pol ι. However, the R96G variant showed ∼40-fold reductions in kcat/Km values for dCTP and dTTP insertion opposite G compared to wild-type pol ι, while the Δ1–25 variant showed 6- to 7-fold increases in those values. Similar trends of results were observed with four DNA lesion templates, with some alterations in nucleotide preference for some cases. The R96G variant displayed about 8-, 33-, 50-, and 72-fold reductions in kcat/Km values for dCTP insertion, respectively, opposite N2-EtG, 8-oxoG, O6-MeG, and an abasic site compared to wild-type pol ι, with 5- and 10-fold increases in misinsertion frequencies for A and T opposite 8-oxoG but a 3-fold decrease in misinsertion frequency for T opposite O6-MeG. However, the Δ1–25 variant displayed 6- to 14-fold increases in kcat/Km for correct dCTP insertion opposite four lesions compared to wild-type pol ι, with misinsertion frequencies similar to the wild-type protein. Interestingly, both the Δ1–25 and R96G variants slightly preferred dTTP over dGTP for insertion opposite an abasic site, as opposed to the case of the wild-type pol ι, which prefers to insert dGTP (Table 4).
Table 2. Steady-State Kinetic Parameters for dNTP Incorporation Opposite G, O6-MeG, and N2-EtG by Wild-Type and Variant hPols ι(1-445) in the Presence of 5 mM Mg2+
templatepol ι(1–445)dNTPKm (μM)kcat (s–1)kcat/Km (s–1 mM–1)finsarelative efficiencyb
Gwild-typeC1100 ± 3000.021 ± 0.0020.01911
  T1800 ± 4000.0093 ± 0.00070.00520.27 
 Δ1–25C370 ± 470.042 ± 0.0020.1115.8
  T1000 ± 1000.037 ± 0.0020.0370.34 
 ΔD17C1900 ± 3000.046 ± 0.0020.02411.3
  T2900 ± 10000.013 ± 0.0020.00450.19 
 R96GC3100 ± 8000.0015 ± 0.00010.0004810.025
  T820 ± 230c0.00015 ± 0.000010.000180.38 
 I261MC1500 ± 1000.034 ± 0.00080.02311.2
  T2400 ± 6000.0078 ± 0.00070.00330.14 
 E276KC1700 ± 2000.03 ± 0.0010.01810.95
  T4700 ± 16000.014 ± 0.0020.00300.17 
 Y374NC1900 ± 2000.022 ± 0.0010.01210.63
  T3400 ± 9000.0079 ± 0.00080.00230.19 
O6-MeGwild-typeC1800 ± 5000.033 ± 0.0040.01811
  T940 ± 1700.027 ± 0.0020.0291.6 
 Δ1–25C620 ± 1800.10 ± 0.010.1618.9
  T170 ± 500.043 ± 0.0030.251.6 
 ΔD17C2800 ± 6000.055 ± 0.0050.02011.1
  T1400 ± 3000.088 ± 0.0060.0633.2 
 R96GC3000 ± 8000.0011 ± 0.00010.0003610.02
  T3200 ± 6000.0010 ± 0.00010.000320.89 
 I261MC2200 ± 8000.034 ± 0.0040.01811.0
  T1100 ± 2000.039 ± 0.0020.0351.9 
 E276KC1800 ± 4000.029 ± 0.0020.01610.89
  T1600 ± 3000.042 ± 0.0030.0261.6 
 Y374NC2200 ± 5000.048 ± 0.0050.02211.2
  T1500 ± 4000.043 ± 0.0040.0291.3 
N2-EtGwild-typeC2800 ± 4000.018 ± 0.0010.006411
  T2700 ± 2000.012 ± 0.00030.00440.69 
 Δ1–25C550 ± 600.048 ± 0.0010.087114
  T770 ± 1100.033 ± 0.0010.0430.49 
 ΔD17C3000 ± 3000.028 ± 0.00010.009311.5
  T2800 ± 2000.020 ± 0.0010.00710.76 
 R96GC5400 ± 16000.0040 ± 0.00050.0007410.12
  T1600 ± 1000.00049 ± 0.000090.000310.41 
 I261MC2000 ± 3000.015 ± 0.0010.007511.2
  T1900 ± 7000.012 ± 0.0020.00630.84 
 E276KC3700 ± 5000.026 ± 0.0020.007011.1
  T3700 ± 4000.021 ± 0.0010.00570.81 
 Y374NC1400 ± 2000.017 ± 0.0010.01211.9
  T2100 ± 7000.013 ± 0.0020.00620.52 
a

Misinsertion frequency, calculated by dividing kcat/Km for dNTP incorporation by the kcat/Km for dCTP incorporation opposite template base. All values are presented to two significant digits.

b

Relative efficiency, calculated by dividing kcat/Km of each pol ι(1–445) for dCTP incorporation opposite template base by kcat/Km of wild-type pol ι(1–445) for dCTP incorporation opposite template base.

c

The apparent Km value, determined under the condition where the amount of enzyme is greater than the amount of DNA and thus is not strictly steady-state.

Table 3. Steady-State Kinetic Parameters for dNTP Incorporation Opposite 8-oxoG by Wild-Type and Variant hPols ι(1-445) in the Presence of 5 mM Mg2+
pol ι(1–445)dNTPKm (μM)kcat (s–1)kcat/Km (s–1 mM–1)finsarelative efficiencyb
wild-typeA1300 ± 2000.00081 ± 0.000050.000620.077 
 T1600 ± 3000.0027 ± 0.00020.00170.27 
 G230 ± 400.0032 ± 0.00010.0141.7 
 C1600 ± 3000.013 ± 0.0010.008111
Δ1–25A530 ± 800.0034 ± 0.000170.00640.13 
 T510 ± 700.0074 ± 0.00030.0150.31 
 G120 ± 100.0061 ± 0.000140.0511.1 
 C1200 ± 1000.057 ± 0.0020.04815.9
ΔD17A1000 ± 2000.00088 ± 0.000050.000880.14 
 T2400 ± 2000.0046 ± 0.00010.00190.30 
 G530 ± 500.0062 ± 0.00020.0121.9 
 C4000 ± 9000.025 ± 0.0030.006310.78
R96GA840 ± 30c0.000072 ± 0.0000080.0000860.36 
 T290 ± 700.00018 ± 0.000010.000622.6 
 G160 ± 20c0.00010 ± 0.0000030.000632.6 
 C1900 ± 4000.00045 ± 0.000040.0002410.030
I261MA820 ± 1100.00075 ± 0.000030.000920.21 
 T1400 ± 3000.0028 ± 0.00020.00200.47 
 G320 ± 200.0044 ± 0.00010.0143.3 
 C2100 ± 3000.0090 ± 0.00050.004310.53
E276KA1500 ± 1000.0010 ± 0.000020.000660.11 
 T2200 ± 2000.0055 ± 0.00020.00250.43 
 G920 ± 300.012 ± 0.00010.0132.2 
 C3800 ± 2000.022 ± 0.0010.005810.72
Y374NA1000 ± 1000.00061 ± 0.000030.000610.086 
 T1200 ± 2000.0023 ± 0.00010.00190.27 
 G260 ± 200.0030 ± 0.00010.0131.8 
 C1700 ± 8000.012 ± 0.0020.007110.88
a

Misinsertion frequency, calculated by dividing kcat/Km for dNTP incorporation by the kcat/Km for dCTP incorporation opposite 8-oxoG. All values are presented to two significant digits.

b

Relative efficiency, calculated by dividing kcat/Km of each pol ι(1–445) for dCTP incorporation opposite 8-oxoG by kcat/Km of wild-type pol ι(1–445) for dCTP incorporation opposite 8-oxoG.

c

The apparent Km value, determined under the condition where the amount of enzyme is greater than the amount of DNA and thus is not strictly steady-state.

Table 4. Steady-State Kinetic Parameters for dNTP Incorporation Opposite Abasic Site by Wild-Type and Variant hPols ι(1-445) in the Presence of 5 mM Mg2+
pol ι(1–445)dNTPKm (μM)kcat (s–1)kcat/Km (s–1 mM–1)dNTP selectivity ratioarelative efficiencyb
wild-typeA1100 ± 300.081 ± 0.0070.0740.46 
 T600 ± 900.058 ± 0.0020.0970.61 
 G770 ± 1100.12 ± 0.010.1611
 C830 ± 5500.027 ± 0.0050.0330.21 
Δ1–25A160 ± 100.15 ± 0.0020.940.72 
 T140 ± 200.18 ± 0.011.31 
 G230 ± 500.21 ± 0.010.910.705.7
 C680 ± 1000.14 ± 0.010.210.16 
ΔD17A770 ± 2000.081 ± 0.0060.110.61 
 T760 ± 500.10 ± 0.0020.130.72 
 G910 ± 900.17 ± 0.010.1811.1
 C2100 ± 8000.074 ± 0.0100.0350.19 
R96GA1700 ± 5000.0035 ± 0.00030.00210.26 
 T820 ± 640.0066 ± 0.00020.00801 
 G1600 ± 4000.0048 ± 0.00080.00300.380.019
 C1800 ± 4000.0012 ± 0.00010.000670.084 
I261MA690 ± 600.053 ± 0.0020.0770.64 
 T480 ± 900.051 ± 0.0030.110.92 
 G670 ± 800.080 ± 0.0030.1210.75
 C860 ± 2500.031 ± 0.0020.0360.30 
E276KA1400 ± 1000.086 ± 0.0040.0640.49 
 T1000 ± 1000.079 ± 0.0020.0790.61 
 G950 ± 1100.13 ± 0.010.1310.81
 C2000 ± 3000.054 ± 0.0020.0270.21 
Y374NA850 ± 2100.047 ± 0.0040.0560.58 
 T600 ± 900.056 ± 0.0030.0930.97 
 G1000 ± 2000.096 ± 0.0070.09610.60
 C1200 ± 1000.032 ± 0.0030.0270.28 
a

dNTP selectivity ratio, calculated by dividing kcat/Km of each dNTP incorporation by the highest kcat/Km for dNTP incorporation opposite the abasic site. All values are presented to two significant digits.

b

Relative efficiency, calculated by dividing kcat/Km of each pol ι(1–445) for dGTP incorporation opposite the abasic site by kcat/Km of wild-type pol ι(1–445) for dGTP incorporation opposite the abasic site.

Steady-State Kinetics of Nucleotide Incorporation Opposite DNA Lesions by the Wild-Type and Variant Pol ι Enzymes in the Presence of MnCl2

To evaluate the efficiency and fidelity in Mn2+-dependent nucleotide insertion opposite G and DNA lesions by six pol ι variants, we determined steady-state kinetic parameters for nucleotide incorporation opposite a G or each of four lesions in the presence of 0.15 mM MnCl2 by six variants in comparison to the wild-type (Tables 5–7). The kcat/Km values of wild-type pol ι for Mn2+-dependent dCTP and dTTP insertion opposite G were 3 orders of magnitude higher than those of the wild-type protein for Mg2+-dependent insertion. For Mn2+-dependent nucleotide insertion, most of the variants (including the Δ1–25 variant) showed the kcat/Km values similar to those of wild-type, whereas the R96G variant displayed 29- to 41-fold reduction in kcat/Km compared to wild-type (Table 5). Similar trends of results were observed with four DNA lesion templates, except for the case with the template N2-EtG and the R96G variant. The R96G variant showed about 16-, 8-, and 5-fold decreases in kcat/Km values for dCTP insertion, respectively, opposite O6-MeG, 8-oxoG, and the abasic site compared to those of the wild-type protein, while showing a 9-fold increase in activity opposite N2-EtG compared to wild-type, indicating that the R96G variation might substantially impair Mn2+-dependent TLS opposite O6-MeG, 8-oxoG, and an abasic site but facilitate that only opposite N2-EtG. These steady-state kinetic data might in large part explain the relatively proficient Mn2+-dependent bypass of the R96G variant opposite N2-EtG compared to the other lesions (Figure 3).
Table 5. Steady-State Kinetic Parameters for dNTP Incorporation Opposite G, O6-MeG, and N2-EtG by Wild-Type and Variant hPols ι(1-445) in the Presence of 0.15 mM Mn2+
templatepol ι(1–445)dNTPKm (μM)kcat (s–1)kcat/Km (s–1 mM–1)finsarelative efficiencyb
Gwild-typeC1.5 ± 0.10.11 ± 0.0027311
  T3.3 ± 0.20.094 ± 0.002280.38 
 Δ1–25C0.52 ± 0.050.036 ± 0.00077210.99
  T1.2 ± 0.10.055 ± 0.001460.64 
 ΔD17C1.4 ± 0.0030.12 ± 0.0038611.2
  T3.6 ± 0.20.12 ± 0.002330.38 
 R96GC11 ± 10.028 ± 0.0012.510.034
  T11 ± 10.0075 ± 0.00030.680.27 
 I261MC1.3 ± 0.080.068 ± 0.00095210.71
  T3.0 ± 0.20.068 ± 0.001230.44 
 E276KC1.4 ± 0.080.051 ± 0.00063610.49
  T3.4 ± 0.10.047 ± 0.0005140.39 
 Y374NC2.6 ± 0.20.14 ± 0.0035410.74
  T3.8 ± 0.20.095 ± 0.001250.46 
O6-MeGwild-typeC3.9 ± 0.10.10 ± 0.0012611
  T3.2 ± 0.20.069 ± 0.001220.85 
 Δ1–25C1.6 ± 0.090.039 ± 0.00052410.93
  T1.4 ± 0.20.027 ± 0.001190.79 
 ΔD17C3.8 ± 0.20.18 ± 0.0034711.8
  T2.5 ± 0.40.087 ± 0.003350.74 
 R96GC12 ± 30.019 ± 0.0021.610.062
  T4.3 ± 0.60.0073 ± 0.00031.71.1 
 I261MC2.3 ± 0.10.075 ± 0.0013311.3
  T4.0 ± 0.50.036 ± 0.0019.00.27 
 E276KC2.5 ± 0.10.044 ± 0.00061810.69
  T3.2 ± 0.40.038 ± 0.001120.67 
 Y374NC3.7 ± 0.30.077 ± 0.0012110.81
  T2.0 ± 0.30.050 ± 0.002251.2 
N2-EtGwild-typeC27 ± 20.034 ± 0.0011.311
  T80 ± 50.045 ± 0.0010.560.44 
 Δ1–25C7.5 ± 1.10.011 ± 0.0011.511.2
  T29 ± 20.018 ± 0.00050.620.42 
 ΔD17C13 ± 10.025 ± 0.0011.911.5
  T93 ± 120.044 ± 0.0030.470.24 
 R96GC3.7 ± 0.10.045 ± 0.00041219.2
  T26 ± 90.0058 ± 0.00080.220.018 
 I261MC16 ± 20.023 ± 0.0011.411.1
  T87 ± 110.042 ± 0.0020.480.34 
 E276KC20 ± 50.018 ± 0.0020.9010.69
  T92 ± 130.042 ± 0.0030.460.51 
 Y374NC33 ± 30.039 ± 0.021.210.92
  T82 ± 40.035 ± 0.0010.430.36 
a

Misinsertion frequency, calculated by dividing kcat/Km for each dNTP incorporation by the kcat/Km for dCTP incorporation opposite template base. All values are presented to two significant digits.

b

Relative efficiency, calculated by dividing kcat/Km of each pol ι(1–445) for dCTP incorporation opposite template base by kcat/Km of wild-type pol ι(1–445) for dCTP incorporation opposite template base.

Table 6. Steady-State Kinetic Parameters for dNTP Incorporation Opposite 8-oxoG by Wild-Type and Variant hPols ι(1-445) in the Presence of 0.15 mM Mn2+
pol ι(1–445)dNTPKm (μM)kcat (s–1)kcat/Km (s–1 mM–1)finsarelative efficiencyb
wild-typeA2.0 ± 0.30.015 ± 0.0017.50.83 
 T30 ± 20.046 ± 0.0021.50.17 
 G0.90 ± 0.070.014 ± 0.0002161.8 
 C4.2 ± 0.20.038 ± 0.0019.011
Δ1–25A17 ± 10.021 ± 0.00031.20.092 
 T5.2 ± 0.30.028 ± 0.0015.40.42 
 G0.21 ± 0.10.0082 ± 0.0004393.0 
 C1.8 ± 0.20.023 ± 0.0011311.4
ΔD17A2.0 ± 0.20.017 ± 0.00038.50.77 
 T29 ± 10.055 ± 0.0015.30.48 
 G0.53 ± 0.060.013 ± 0.0003252.3 
 C3.5 ± 0.20.040 ± 0.00061111.2
R96GA3.3 ± 0.30.0017 ± 0.000040.520.43 
 T13 ± 10.0046 ± 0.00010.350.29 
 G1.8 ± 0.20.0038 ± 0.00012.11.8 
 C8.5 ± 0.50.010 ± 0.00021.210.13
I261MA1.4 ± 0.20.015 ± 0.0004111.0 
 T25 ± 30.026 ± 0.0011.00.091 
 G0.66 ± 0.070.012 ± 0.0003181.6 
 C3.1 ± 0.10.034 ± 1.00031111.2
E276KA2.2 ± 0.30.022 ± 0.001101.4 
 T24 ± 40.044 ± 0.0031.80.26 
 G0.60 ± 0.040.016 ± 0.0002273.9 
 C3.2 ± 0.20.022 ± 0.00046.910.77
Y374NA1.4 ± 0.10.015 ± 0.0003111.0 
 T29 ± 60.025 ± 0.0030.860.078 
 G0.72 ± 0.060.018 ± 0.0003252.3 
 C3.6 ± 0.10.040 ± 0.00031111.2
a

Misinsertion frequency, calculated by dividing kcat/Km for each dNTP incorporation by the kcat/Km for dCTP incorporation opposite 8-oxoG. All values are presented to two significant digits.

b

Relative efficiency, calculated by dividing kcat/Km of each pol ι(1–445) for dCTP incorporation opposite 8-oxoG by kcat/Km of wild-type pol ι(1–445) for dCTP incorporation opposite 8-oxoG.

Table 7. Steady-State Kinetic Parameters for dNTP Incorporation Opposite Abasic Site by Wild-Type and Variant hPols ι(1-445) in the Presence of 0.15 mM Mn2+
pol ι(1–445)dNTPKm (μM)kcat (s–1)kcat/Km (s–1 mM–1)dNTP selectivity ratioarelative efficiencyb
wild-typeA0.68 ± 0.040.012 ± 0.0001180.18 
 T1.5 ± 0.10.064 ± 0.001430.43 
 G0.29 ± 0.060.029 ± 0.00110011
 C2.8 ± 0.10.066 ± 0.001240.24 
Δ1–25A0.50 ± 0.060.010 ± 0.003200.22 
 T1.1 ± 0.10.024 ± 0.001220.24 
 G0.099 ± 0.0050.0091 ± 0.00019210.92
 C0.98 ± 0.080.043 ± 0.001440.48 
ΔD17A0.83 ± 0.130.016 ± 0.001190.31 
 T1.8 ± 0.20.048 ± 0.001270.44 
 G0.34 ± 0.040.021 ± 0.00046210.62
 C1.8 ± 0.30.11 ± 0.004610.98 
R96GA1.9 ± 0.30.011 ± 0.00045.80.76 
 T1.9 ± 0.20.0088 ± 0.00024.60.61 
 G0.49 ± 0.060.0037 ± 0.00017.610.076
 C3.4 ± 0.30.015 ± 0.00044.40.58 
I261MA0.35 ± 0.050.021 ± 0.001600.50 
 T1.3 ± 0.090.026 ± 0.0004200.17 
 G0.27 ± 0.020.032 ± 0.00112011.2
 C2.5 ± 0.40.050 ± 0.002200.17 
E276KA0.72 ± 0.110.017 ± 0.001240.39 
 T2.7 ± 0.30.028 ± 0.0007100.16 
 G0.34 ± 0.020.021 ± 0.00026210.62
 C2.2 ± 0.10.041 ± 0.0005190.31 
Y374NA0.71 ± 0.090.026 ± 0.001370.38 
 T2.0 ± 0.20.082 ± 0.002410.42 
 G0.39 ± 0.030.038 ± 0.0019710.97
 C3.3 ± 0.30.047 ± 0.001140.14 
a

dNTP selectivity ratio, calculated by dividing kcat/Km for each dNTP incorporation by the highest kcat/Km for dNTP incorporation opposite the abasic site. All values are presented to two significant digits.

b

Relative efficiency, calculated by dividing kcat/Km of each pol ι(1–445) for dGTP incorporation opposite the abasic site by kcat/Km of wild-type pol ι(1–445) for dGTP incorporation opposite the abasic site.

Binding of the Wild-Type Pol ι(1–445) and the Variants Δ1–25 and R96G to DNA Substrate

To analyze the binding affinities of two dysfunctional pol ι variants, Δ1–25 and R96G, for the primer-template DNA substrate in the presence of either Mg2+ or Mn2+, we performed fluorescence polarization experiments. The equilibrium dissociation constants (Kd,DNA) of wild-type pol ι and the Δ1–25 and R96G variants were estimated by fitting the fluorescence polarization values of fluorescein-labeled DNA substrates (18-FAM-mer primers annealed to unmodified 36-G-mer templates) as a function of the pol ι concentration to a quadratic equation (Table 8). Wild-type pol ι bound weakly to DNA with a Kd,DNA of 490 and 840 nM, respectively, at 0.15 and 1 mM Mg2+concentrations, while binding relatively tightly to DNA with a Kd,DNA of 68 nM at 0.15 mM Mn2+ (but not at 1 mM Mn2+). The R96G variant had Kd,DNA values 2- to 3-fold higher than wild-type in the presence of either metal, indicating a slight decrease in DNA binding affinity of pol ι by this variant. In contrast, the Δ1–25 variant bound DNA much more tightly (20- to 29-fold) than the wild-type protein in the presence of 0.15 or 1 mM Mg2+. Similarly, the Δ1–25 variant also had a DNA-binding affinity (Kd,DNA = 140 nM) ∼10-fold lower than that of wild-type in the presence of 5 mM Mg2+ (results not shown). This large difference in the DNA-binding affinities was reduced to only 4-fold in the presence of 0.15 mM of Mn2+. These data indicate that the N-terminal region (residues 1–25), rich in negatively charged amino acids, might severely interfere with DNA substrate binding of wild-type pol ι, but this interference could be totally ablated by the deletion of N-terminal 25 amino acids or could be substantially overcome by the low level of Mn2+. These features might at least in part explain the substantial increase in polymerase activity of the Δ1–25 variant versus the wild-type pol ι seen in the presence of Mg2+ (Figure 2, Tables 2–4) but not with Mn2+ (Figure 3, Tables 5–7). Here we also note that the Kd,DNA value (68 nM) of wild-type pol ι with Mn2+ would yield a relatively high dissociation rate, koff (∼0.68 s–1), which might make pol ι very distributive.
Table 8. Kd Values of Wild-Type hPol ι (1-445), and the Δ1–25 and R96G Variants for 18-FAM-mer/36-G-mer DNA Substrate in the Presence of MnCl2 or MgCl2
 Kd (nM) of pol ι(1–445)
MnCl2 or MgCl2wild-typeΔ1–25R96G
0.15 mM MnCl268 ± 1117 ± 3220 ± 50
0.15 mM MgCl2490 ± 7017 ± 3900 ± 200
1 mM MnCl2840 ± 25088 ± 161800 ± 400
1 mM MgCl2840 ± 32041 ± 71800 ± 600

Discussion

ARTICLE SECTIONS
Jump To
In this study we examined the biochemical properties of six nonsynonymous coding variants of human pol ι in comparison with the wild-type, based on the newly annotated ORF sequence information. Four missense and two deletion variations were selected for this study because they were expected to cause functional alterations on pol ι on the basis of the nature of the changes, positions in the pol ι catalytic core, and predicted effects. Our biochemical data revealed that the R96G variation severely impairs the efficiency of pol ι for both normal and translesion syntheses at G, N2-EtG, 8-oxoG, O6-MeG, and an abasic site regardless of the presence of Mn2+ or Mg2+ (except for the case of a Mn2+-facilitated C insertion opposite N2-EtG), whereas the Δ1–25 deletion variation, which is equivalent to the previously misannotated wild-type, considerably increases pol ι efficiency for both normal and translesion syntheses in the presence of Mg2+ (but not with Mn2+). The Δ1–25 variation greatly (20- to 29-fold) increased the DNA-binding affinity of pol ι in the presence of Mg2+, which was much less prominent (only about ∼4-fold) in the presence of a low concentration of Mn2+. We also note that the poor DNA-binding affinity of the wild-type pol ι was considerably improved by the addition of a low level of Mn2+ when compared to that of Mg2+. In this study we report that two rare, nonsynonymous POLI genetic variations can affect the TLS activities of human pol ι in opposite directions in vitro and that wild-type pol ι is substantially more sluggish in activity than expected from analysis of the previous wild-type protein (Δ1–25) in the presence of Mg2+, unlike in the presence of Mn2+.
To the best of our knowledge, this is the first study analyzing biochemical alterations in germline genetic variants and the newly denoted wild-type version of human pol ι, using in vitro polymerase activity, enzyme kinetics, and binding assays. Two of the six POLI genetic variations studied here were found to significantly affect the in vitro enzymatic function of pol ι in translesion DNA synthesis and DNA substrate binding. These functional genetic variations of pol ι appear to be rare, similar to dysfunctional pol κ variants we reported recently,(35) in that minor allele frequencies (MAFs) of the Δ1–25 and R96G pol ι variations are ∼0.1% and 0.6%, respectively, in public databases (Table 1). The biological significance of rare frequency genetic variations should not be overlooked in that recent reports suggest the possible relevance of rare genetic variations to complex human diseases. Rare genetic variations have been proposed as a potential source of missing disease heritability that has not been fully explained by common genetic variations,(39) and the recent experimental reports support this view by providing evidence that rare genetic variations are abundantly present in human populations and are more likely to be misfunctional than common variations.(40, 41) Only 19 nonsynonymous germline variations were described in 2008 (including two reported cancer-related SNPs(28, 29)), but now a total of 91 nonsynonymous germline variations have been listed in dbSNP, most of which seem to be rare in that MAFs of 89 variations are either <1% or unavailable yet. About 46 types of missense and nonsense somatic POLI gene mutations have also been described from various human cancer tissues such as endometrium, large intestine, and lung in the COSMIC database (www.sanger.ac.uk/genetics/CGP/cosmic/), but their functional effects have not been revealed yet. Although we focused on only six selected “putatively functional” coding variants, our biochemical investigation can be a useful initial step to find pol ι variations that have functional impacts and understanding their mechanistic implications. Eleven additional nonsynonymous coding POLI variations, located in polymerase core domains and also putatively deleterious, have been added in dbSNP since our study began, and those functional candidates are also under our investigation.
The six germline pol ι variants characterized in this study can be classified into three types according to the changes of relative polymerase efficiencies opposite G and the lesions in the presence of the added metal (Mg2+ or Mn2+) compared to wild-type (Tables 2–7). The first type is the defective variant (R96G), which is severely impaired in both Mg2+- and Mn2+-dependent polymerase efficiencies for both normal synthesis and lesion bypass. Surprisingly, this particular variant exhibited a substantial improvement in the Mn2+-dependent N2-EtG bypass, i.e., large increases (9-fold and 24-fold, respectively) in both efficiency and fidelity for Mn2+-dependent dCTP insertion opposite N2-EtG compared to wild-type enzyme (Table 5), from which we may speculate the likelihood of a beneficial effect of this variation to efficiently and faithfully bypass such a lesion at the expense of general diminution in polymerase function. The second type is the hyperactive variant (Δ1–25). This N-terminal truncation variant displayed considerable enhancement only in Mg2+-dependent polymerase efficiency but without any alteration in Mn2+-dependent efficiency. The last type is the “wild-type-like” variants (Δ17, I261M, E276K, and Y374N), which retain both normal and TLS polymerase efficiencies similar to those of wild-type in the presence of either Mg2+ or Mn2+. The fidelity of nucleotide insertion opposite G and the lesions does not appear to be altered in most variants except for the R96G variant when compared to wild-type (Tables 2–7). The R96G variant had a reduced fidelity in Mg2+-dependent 8-oxoG bypass due to a greater reduction of insertion efficiency for correct dCTP than for the other nucleotides, as well as an improved fidelity in Mn2+-dependent N2-EtG bypass (vide supra). Although all four studied missense variations (R96G, I261M, E276K, and Y374N) were predicted to be damaging by SIFT and/or PolyPhen, only the former variant was found to be deleterious, but the latter three variants were found to have nearly neutral effects on pol ι function in our study, indicating the substantial false-positive nature of in silico prediction. False-positive errors of 20 and 9% have been reported with SIFT and Polyphen, respectively.(42) Taken together, our results suggest the necessity and importance of biochemical approaches to verify the functional alterations in genetic variants, although in silico predictions may still be useful for screening putatively damaging genetic variations for functional studies.
Three-dimensional structures of the catalytic core of pol ι have been determined in complex with various DNA substrates with/without incoming nucleotides, which would be useful for the mechanistic understanding of genetic variants (Figure 1).(14, 37, 38, 43-45) The catalytic core of pol ι contains the palm, fingers, thumb, and polymerase-associated domain (PAD) domains, forming the unique narrow active site that is not conducive to Watson–Crick base pairing.(14, 44) The mechanistic basis for our finding that the R96G variation caused a severe reduction in pol ι catalytic activity can be explained by a structural role of Arg96 in the active site as previously revealed.(14, 44) Thr90, Tyr93, and Arg96 from the fingers domain and Lys239 from the palm domain (the latter three of which are conserved in all Y-family DNA polymerases) function to stabilize the incoming nucleotide by making hydrogen bonds with the triphosphate moiety in the pol ι-DNA-dNTP ternary complex. In particular, Arg96 of pol ι undergoes a substantial conformational change (facing inward) upon nucleotide binding to form hydrogen bonds with the β- and γ-phosphates of the incoming nucleoside triphosphate. Our finding of defective function in the R96G variant is in good agreement with the previous report that Ala substitution of the homologous Arg67 residue in yeast pol η also considerably diminishes its polymerase activity, suggesting that this conserved Arg residue is commonly crucial for catalytic function in Y-family polymerases.(46) However, it is not clear how the R96G variant unexpectedly displays an increased competence in an error-free Mn2+-dependent N2-EtG bypass. Pol ι normally accommodates the N2-EtG in the syn conformation paired with incoming dCTP in the active site.(43) The R96G variation abolishes the long and positively charged side chain on Arg96 and thus might lead to an altered conformation that hinders nucleoside triphosphate binding and catalysis for most of the templates but is perhaps well suited only for Mn2+-assisted pairing between a template N2-EtG (syn) and an incoming dCTP (anti) in the active site pocket.
We established, for the first time, that the Δ1–25 variant (i.e., the former wild-type protein) has a considerably higher Mg2+-dependent polymerase activity (but with no alteration in Mn2+-dependent polymerase activity) and a much higher DNA binding affinity than wild-type pol ι. This unexpected biochemical trait is certainly due to the absence of the N-terminal extension of 25 amino acids (containing 12 acidic residues). As far as we know, all reported pol ι structures have been resolved only from the catalytic core fragment (amino acids 26–445) lacking the N-terminal 25 amino acids, and thus, the structural role of the N-terminal extension is not known yet. However, it is evident from our observations that this negatively charged N-terminal extension can interfere with both DNA substrate binding and Mg2+-dependent polymerase activity of pol ι, but these hindrances can be largely weakened by a low level of Mn2+. Only Mn2+ (at low concentration, 150 μM) but not Mg2+ seems to considerably overcome the inherently poor DNA binding trait of the wild-type pol ι, possibly by masking or neutralizing the negative charged N-terminal region of this enzyme and thus reducing the potential electrostatic repulsion from the negatively charged phosphosugar backbone of DNA substrates. Pol ι is also known to have a greater preference for Mn2+ over Mg2+ as a divalent metal for polymerase catalysis and shows maximal polymerase activity at a low concentration of Mn2+ (with the optimum around 50–250 μM).(20) Taken together, we can postulate that the wild-type pol ι may require a low level of Mn2+ rather than Mg2+ as a metal for performing proper polymerase function for two reasons: (i) mediating efficient catalysis and (ii) facilitating tight DNA substrate binding, although the structural basis for such a scenario has not been revealed yet. These biochemical traits of wild-type pol ι are likely to be advantageous in that cells might be able to control the error-prone polymerase function of pol ι by varying the local concentration of metal near DNA damage sites in cell nucleus, which might permit the proficient TLS events by pol ι only in the presence of a low level of Mn2+. Although the intracellular level of Mn2+ is physiologically very low (0.1 to 40 μM),(47-49) that of Mn2+ could be increased in some pathological cell conditions (e.g., Mn2+ overexposure or misregulated Mn2+ homeostasis),(50, 51) which might stimulate the activity of error-prone pol ι, as well as other Mn2+-dependent DNA-processing enzymes in the nucleus, and thus induce genomic instability. Mn2+ has also been known to alter both catalytic efficiency and fidelity of other DNA polymerases including pol β, pol λ, and pol μ, albeit at high concentrations.(52-54) The effects might be related to their active site features with regard to the cofactor Mn2+, which has a slightly smaller ionic radius and a more relaxed coordination than Mg2+, and might allow different interactions with nucleoside triphosphate, DNA, and catalytic residues in the polymerase active site, which could alter polymerase function.(55, 56)
It is very conceivable that the cellular pol ι-mediated TLS capacity could be substantially diminished or enhanced in individuals having these two identified dysfunctional POLI gene variations. If a cell possessed only the R96G pol ι variant (i.e., homozygote), then most of pol ι-mediated TLS events (except for the increased events of accurate N2-EtG bypass) would decrease in cells. Conversely, if the cell had only the Δ1–25 pol ι variant, then pol ι-mediated Mg2+-dependent TLS events would increase in cells. We might expect that high replication errors occur with hyperactive pol ι in normal DNA replication because pol ι is inherently error-prone in its general nature. However, predicting how these two POLI gene variations would lead to overall TLS-associated mutation outcomes in cells is not straightforward due to the complex TLS properties of pol ι and the cellular existence of other competitive TLS polymerases. Eukaryotic TLS process is carried out not by a single TLS polymerase but by a set of specialized TLS polymerases recruited at a site of DNA damage, although the extent of their individual participation varies depending on the lesion type. The mutation consequence from a particular DNA lesion in cells can be governed by the total set of enzymatic behavior of multiple TLS polymerases, mainly Y-family polymerases, utilized for the lesion substrate. Unlike Y-family pols η and κ, which are generally considered to be specialized for efficient and error-free bypasses at their cognate lesions, UV-induced pyrimidine dimers and bulky N2-G adducts, respectively, it is not clear what kind of DNA lesions could be the cognate substrate lesions for pol ι. There is much kinetic and structural evidence suggesting that some DNA lesions such as N2-EtG, O6-MeG, 8-oxoG, and abasic sites might be among the favored substrate lesions for pol ι, in that this enzyme can incorporate nucleotides opposite those lesions as efficiently as (or slightly less efficiently than) opposite an unmodified G by utilizing its unique active site and/or Hoogsteen base pairing but with different nucleotide selectivities.(12, 17, 19, 37, 38, 43, 45) Pol ι seems to mediate relatively error-free bypass with some minor-groove N2-G lesions such as N2-EtG, while performing a relatively error-prone bypass opposite major-groove O6-alkylG adducts such as O6-MeG.(12, 17, 37, 43) In a support of this view, pol ι (as well as pol κ) has been implicated in the error-free bypass of N2-carboxyMeG and N2-carboxyEtG lesions in mouse cells.(57) Pol ι has also been implicated to play a protective role from oxidative DNA damage, possibly by mediating both the error-free TLS and the base excision repair of 8-oxoG.(38, 58, 59) However, the fidelity of 8-oxoG bypass by pol ι seems to be sequence context-dependent because the incorrect dGTP was incorporated more favorably than dCTP opposite template 8-oxoG in our different sequence context as observed here. Experimental evidence support the dual and conflicting “Janus” roles of pol ι in cancers: a protective role of pol ι in mouse lung and skin carcinogenesis(22, 23) and a hypermutagenic role of pol ι, upregulated in various human cancer tissues.(24-27) Under these circumstances, it can be hypothesized that individual humans who possess the R96G or the Δ1–25 pol ι variation might have an altered but complex risk for mutation and cancer to various carcinogen exposures, depending on the DNA damage status in their target tissues. In this aspect, it is necessary to perform further studies to verify the in vivo impact of those functional pol ι variants in both cellular and organismal contexts. It is also worth examining other nonsynonymous coding variations located in protein interaction domains of pol ι to bind other proteins such as PCNA and ubiquitin, in that they might alter cellular TLS events such as polymerase switching and coordination. It is also plausible that noncoding functional variations in the gene regulatory regions of pol ι might be able to alter pol ι expression levels in cells and thus influence TLS events, although this aspect was out of scope for this study.
In conclusion, our results suggest that two germline genetic variations in human POLI gene may either hinder or promote the TLS capability of pol ι with various DNA lesions in vitro, possibly leading to different and distinctive mutation phenotypes in genetically affected cells and individuals, i.e., facilitating or protecting against mutagenesis/carcinogenesis after exposure to genotoxic carcinogens. The verification of two dysfunctional genetic variations for human pol ι in this study may provide insight into our understanding of individual differences in cellular TLS capacities to various carcinogen-derived DNA lesions. Such functional genetic alterations in TLS DNA polymerases might be expected to play some part in determining an individual’s genomic mutational susceptibility to specific carcinogens and the related cancer risk in human populations, although further in vivo or clinical investigations are needed to elucidate these associations.

Supporting Information

ARTICLE SECTIONS
Jump To

Analysis of human pol ι (1–445) wild-type and variant proteins by SDS-polyacrylamide gel electrophoresis (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

Funding Information

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant 2012R1A1A2042391) (to J.-Y.C.), Samsung Biomedical Research Institute grant, #SBRI SMX1132091 (to J.-Y.C.), and the National Institutes of Health Grants R01 CA183895 (to R.L.E.) and R01 ES010375 (to F.P.G.).

The authors declare no competing financial interest.

Terms & Conditions

Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at http://pubs.acs.org/page/copyright/permissions.html.

Abbreviations

A

adenine

BSA

bovine serum albumin

C

cytosine

Et

ethyl

FAM

6-carboxyfluorescein

G

guanine

Me

methyl

8-oxoG

8-oxo-7,8-dihydroG

PCR

polymerase chain reaction

pol

DNA polymerase

SDS

sodium dodecyl sulfate

T

thymine

TLS

translesion synthesis

References

ARTICLE SECTIONS
Jump To

This article references 60 other publications.

  1. 1
    Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A., and Ellenberger, T. (2006) DNA Repair And Mutagenesis, 2nd ed., American Society for Microbiology Press, Washington, D.C.
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Giglia-Mari, G., Zotter, A., and Vermeulen, W. (2011) DNA damage response Cold Spring Harb. Perspect. Biol. 3, a000745
    [ Crossref], Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Hoeijmakers, J. H. (2009) DNA damage, aging, and cancer N. Engl. J. Med. 361, 1475– 1485
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    3
    DNA, damage, aging, and cancer
    Hoeijmakers, Jan H. J.
    New England Journal of Medicine (2009), 361 (15), 1475-1486CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
    A review. Topics discussed include: DNA damage and aging; the magnitude of DNA damage; the DNA repair toolbox; diseases of nucleotide-excision repair; progeria, aging and the survival response; DNA damage and DNA maintenance in cancer; compromised genome maintenance and cancer therapy.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1GiurbK&md5=95403c4568de46eed137cd5145487fcb
  4. 4
    Jackson, S. P. and Bartek, J. (2009) The DNA-damage response in human biology and disease Nature 461, 1071– 1078
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    4
    The DNA-damage response in human biology and disease
    Jackson, Stephen P.; Bartek, Jiri
    Nature (London, United Kingdom) (2009), 461 (7267), 1071-1078CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    A review. The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite const. assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biol. significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlGktLrK&md5=cca7cfec66852124695ebeb515f255a5
  5. 5
    Kennedy, D. O., Agrawal, M., Shen, J., Terry, M. B., Zhang, F. F., Senie, R. T., Motykiewicz, G., and Santella, R. M. (2005) DNA repair capacity of lymphoblastoid cell lines from sisters discordant for breast cancer J. Natl. Cancer Inst. 97, 127– 132
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    5
    DNA Repair Capacity of Lymphoblastoid Cell Lines From Sisters Discordant for Breast Cancer
    Kennedy, David O.; Agrawal, Meenakshi; Shen, Jing; Terry, Mary Beth; Zhang, Fang Fang; Senie, Ruby T.; Motykiewicz, Grazyna; Santella, Regina M.
    Journal of the National Cancer Institute (2005), 97 (2), 127-132CODEN: JNCIEQ; ISSN:0027-8874. (Oxford University Press)
    Background: Interindividual differences in DNA repair capacity may influence cancer risk. We tested whether the nucleotide excision repair pathway was deficient in breast cancer case patients by analyzing sister pairs. Methods: Cell lines derived from sisters discordant for breast cancer (137 families contg. 158 case patients and 154 control sisters) were obtained from the Metropolitan New York Registry of Breast Cancer Families. Lymphoblastoid cells were treated with benzo[a]pyrene diolepoxide (BPDE) for 30 min and were either harvested immediately or were washed and cultured in complete medium for 4 h to allow DNA repair. Immunofluorescence using a polyclonal anti-BPDE-DNA primary antibody was used to quantify BPDE-DNA adducts. Percent DNA repair capacity was calcd. from the difference between staining immediately after treatment minus that after 4 h of repair, divided by the initial damage and was categorized into quartiles based on control values. Odds ratios and 95% confidence intervals (CIs) were calcd. using conditional logistic regression models adjusted for age at blood donation, body mass index, and smoking. Statistical tests were two-sided. Results: Mean percent DNA repair capacity was lower in breast cancer case patients than in control subjects (difference = 8.6, 95% CI = 4.3 to 13.8, P = .001). Using the quartile with the highest percent DNA repair capacity as the referent group, adjusted odds ratios of breast cancer increased from 1.23 (95% CI = 0.57 to 2.65) to 2.38 (95% CI = 1.17 to 4.86) to 2.99 (95% CI = 1.45 to 6.17) (Ptrend = .002) as DNA repair capacity decreased. Conclusions: Deficient DNA repair capacity is assocd. with increased breast cancer risk.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmsV2jug%253D%253D&md5=bce11c64e855be84af763c4ae0935e1c
  6. 6
    Gorlova, O. Y., Weng, S. F., Zhang, Y., Amos, C. I., Spitz, M. R., and Wei, Q. (2008) DNA repair capacity and lung cancer risk in never smokers Cancer Epidemiol. Biomarkers Prev. 17, 1322– 1328
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    6
    DNA Repair Capacity and Lung Cancer Risk in Never Smokers
    Gorlova, Olga Y.; Weng, Shih-Feng; Zhang, Yiqun; Amos, Christopher I.; Spitz, Margaret R.; Wei, Qingyi
    Cancer Epidemiology, Biomarkers & Prevention (2008), 17 (6), 1322-1328CODEN: CEBPE4; ISSN:1055-9965. (American Association for Cancer Research)
    Besides secondhand smoke exposure, few other risk factors for lung cancer in lifetime never smokers have been identified. We present the ests. of lung cancer risk assocd. with suboptimal DNA repair capacity (DRC) measured by the host-cell reactivation assay in lifetime never smokers using data from 219 cases and 309 matched controls enrolled in a case-control study. Suboptimal DRC level (below the control median) conferred a significantly increased lung cancer risk in never smokers [odds ratio, 1.92; 95% confidence interval (95% CI), 1.3-2.9; P = 0.0024]. There was a 3.38-fold risk for individuals with DRC below the first quartile (95% CI, 1.8-6.3) compared with individuals with DRC above the third quartile. Secondhand smoke exposure in individuals with DRC below the control median was assocd. with a 3.81-fold risk of lung cancer (95% CI, 2.3-6.4). A 2.49-fold (95% CI, 1.1-5.6) risk was noted for the joint effects of lung cancer family history in first-degree relatives and suboptimal DRC. Relatives of probands (cases and controls) with lowest DRC (below the first quartile) were significantly more likely to be diagnosed with lung cancer (odds ratio, 2.69; 95% CI, 1.1-6.7) compared with relatives of probands with the most proficient DRC (above the third quartile). Relatives of probands with suboptimal (below the control median) vs. proficient DRC also had an earlier age at diagnosis with lung cancer, although the only statistically significant difference was in female relatives (55.4 vs. 67.7 years; P = 0.03). (Cancer Epidemiol Biomarkers Prev 2008;17(6):1322-8).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsVKgtro%253D&md5=cc99ad382991c1e38add953e67bdafd7
  7. 7
    Wang, L. E., Gorlova, O. Y., Ying, J., Qiao, Y., Weng, S. F., Lee, A. T., Gregersen, P. K., Spitz, M. R., Amos, C. I., and Wei, Q. (2013) Genome-wide association study reveals novel genetic determinants of DNA repair capacity in lung cancer Cancer Res. 73, 256– 264
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    7
    Genome-Wide Association Study Reveals Novel Genetic Determinants of DNA Repair Capacity in Lung Cancer
    Wang, Li-E.; Gorlova, Olga Y.; Ying, Jun; Qiao, Yawei; Weng, Shih-Feng; Lee, Annette T.; Gregersen, Peter K.; Spitz, Margaret R.; Amos, Christopher I.; Wei, Qingyi
    Cancer Research (2013), 73 (1), 256-264CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    Suboptimal cellular DNA repair capacity (DRC) has been shown to be assocd. with enhanced cancer risk, but genetic variants affecting the DRC phenotype have not been comprehensively investigated. In this study, with the available DRC phenotype data, we analyzed correlations between the DRC phenotype and genotypes detected by the Illumina 317K platform in 1,774 individuals of European ancestry from a Texas lung cancer genome-wide assocn. study. The discovery phase was followed by a replication in an independent set of 1,374 cases and controls of European ancestry. We applied a generalized linear model with single nucleotide polymorphisms as predictors and DRC (a continuous variable) as the outcome. Covariates of age, sex, pack-years of smoking, DRC assay-related variables, and case-control status of the study participants were adjusted in the model. We validated that reduced DRC was assocd. with an increased risk of lung cancer in both independent datasets. Several suggestive loci that contributed to the DRC phenotype were defined in ERCC2/XPD, PHACTR2, and DUSP1. In summary, we detd. that DRC is an independent risk factor for lung cancer, and we defined several genetic loci contributing to DRC phenotype.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjs1Wrsw%253D%253D&md5=53a35941a58c108e64969ba0055668ab
  8. 8
    Alberg, A. J., Jorgensen, T. J., Ruczinski, I., Wheless, L., Shugart, Y. Y., Berthier-Schaad, Y., Kessing, B., Hoffman-Bolton, J., Helzlsouer, K. J., Kao, W. H., Francis, L., Alani, R. M., Smith, M. W., and Strickland, P. T. (2013) DNA repair gene variants in relation to overall cancer risk: a population-based study Carcinogenesis 34, 86– 92
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    8
    DNA repair gene variants in relation to overall cancer risk: a population-based study
    Alberg, Anthony J.; Jorgensen, Timothy J.; Ruczinski, Ingo; Wheless, Lee; Shugart, Yin Yao; Berthier-Schaad, Yvette; Kessing, Bailey; Hoffman-Bolton, Judith; Helzlsouer, Kathy J.; Kao, W. H. Linda; Francis, Lesley; Alani, Rhoda M.; Smith, Michael W.; Strickland, Paul T.
    Carcinogenesis (2013), 34 (1), 86-92CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)
    The hypothesis that germ-line polymorphisms in DNA repair genes influence cancer risk has previously been tested primarily on a cancer site-specific basis. The purpose of this study was to test the hypothesis that DNA repair gene allelic variants contribute to globally elevated cancer risk by measuring assocns. with risk of all cancers that occurred within a population-based cohort. In the CLUE II cohort study established in 1989 in Washington County, MD, this study was comprised of all 3619 cancer cases ascertained through 2007 compared with a sample of 2296 with no cancer. Assocns. were measured between 759 DNA repair gene single nucleotide polymorphisms (SNPs) and risk of all cancers. A SNP in O6-methylguanine-DNA methyltransferase, MGMT, (rs2296675) was significantly assocd. with overall cancer risk [per minor allele odds ratio (OR) 1.30, 95% confidence interval (CI) 1.19-1.43 and P-value: 4.1 × 10-8]. The assocn. between rs2296675 and cancer risk was stronger among those aged ≤54 years old than those who were ≥55 years at baseline (P-for-interaction = 0.021). OR were in the direction of increased risk for all 15 categories of malignancies studied (P < 0.0001), ranging from 1.22 (P = 0.42) for ovarian cancer to 2.01 (P = 0.008) for urinary tract cancers; the smallest P-value was for breast cancer (OR 1.45, P = 0.0002). The results indicate that the minor allele of MGMT SNP rs2296675, a common genetic marker with 37% carriers, was significantly assocd. with increased risk of cancer across multiple tissues. Replication is needed to more definitively det. the scientific and public health significance of this obsd. assocn.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktV2qsg%253D%253D&md5=641623112ece30025dc08da8fe51e2aa
  9. 9
    Choi, J.-Y., Eoff, R. E., and Guengerich, F. P. (2011) Bypass DNA polymerases, In Chemical Carcinogenesis (Penning, T. M., Ed.) pp 345– 373, Humana Press, New York.
    [ Crossref], Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Choi, J.-Y., Angel, K. C., and Guengerich, F. P. (2006) Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase κ J. Biol. Chem. 281, 21062– 21072
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    10
    Translesion Synthesis across Bulky N2-Alkyl Guanine DNA Adducts by Human DNA Polymerase κ
    Choi, Jeong-Yun; Angel, Karen C.; Guengerich, F. Peter
    Journal of Biological Chemistry (2006), 281 (30), 21062-21072CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    DNA polymerase (pol) κ is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a no. of chem. modifications with human pol κ, and the conclusion has been presented, based on limited quant. data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol κ was studied with a series of eight N2-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH2(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, kcat/Km) were similar for insertion of dCTP opposite the lesions and for extension beyond the N2-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with kcat/Km values ∼10-3 less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic anal. showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (kp 5.9-10.3 s-1). The rapid bursts were abolished opposite BPG when α-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols η and ι; pol κ is the most resistant to N2-bulk and the most quant. efficient of these in catalyzing dCTP incorporation opposite bulky guanine N2-adducts, particularly the largest (N2-BPG).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xnt1Gqurg%253D&md5=fe7b66fcfab81d10bfea755750f9a674
  11. 11
    Choi, J.-Y. and Guengerich, F. P. (2005) Adduct size limits efficient and error-free bypass across bulky N2-guanine DNA lesions by human DNA polymerase η J. Mol. Biol. 352, 72– 90
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    11
    Adduct Size Limits Efficient and Error-free Bypass Across Bulky N 2-Guanine DNA Lesions by Human DNA Polymerase η
    Choi, Jeong-Yun; Guengerich, F. Peter
    Journal of Molecular Biology (2005), 352 (1), 72-90CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
    The N2 position of guanine (G) is one of the major sites for DNA modification by various carcinogens. Eight oligonucleotides with varying adduct bulk at guanine N2 were analyzed for catalytic efficiency and fidelity with human DNA polymerase (pol) η, which is involved in translesion synthesis (TLS). Pol η effectively bypassed N2-methyl(Me)G, N2-ethyl(Et)G, N2-isobutyl(Ib)G, N 2-benzyl(Bz)G, and N2-CH2(2-naphthyl)G but was severely blocked at N 2-CH2(9-anthracenyl)G (N2-AnthG) and N2-CH2(6-benzo[a]pyrenyl)G (N2-BPG). Steady-state kinetic anal. showed proportional decreases of kcat/Km in dCTP insertion opposite N2-AnthG and N2-BPG (73 and 320-fold) and also kcat/Km in next-base extension from a C paired with each adduct (15 and 51-fold relative to G). Frequencies of dATP misinsertion and extension beyond mispairs were also proportionally increased (70 and 450-fold; 12 and 44-fold) with N2-AnthG and N2-BPG, indicating the effect of adduct bulk on blocking and misincorporation in TLS by pol η. N2-AnthG and N2-BPG also greatly decreased the pre-steady-state kinetic burst rate (25 and 125-fold) compared to unmodified G. N2-AnthG decreased dCTP binding affinity (2.6-fold) and increased DNA substrate binding affinity. These results and the small kinetic thio effects (Sp-dCTPαS) suggest that the early steps, possibly conformational change, are interfered with by the bulky adducts. In contrast, human pol δ bypassed adducts effectively up to N2-EtG but was strongly blocked by N2-IbG and larger adducts. We conclude that TLS DNA polymerases may be required for the efficient bypass of pol δ-blocking N2-G adducts bulkier than N2-EtG in human cells, and the bulk size can be a major factor for efficient and error-free bypass at these adducts by TLS DNA polymerases.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpsVWltrk%253D&md5=e3a72f9f9476440f6c5470b715653f4c
  12. 12
    Choi, J.-Y. and Guengerich, F. P. (2006) Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι J. Biol. Chem. 281, 12315– 12324
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    12
    Kinetic Evidence for Inefficient and Error-prone Bypass across Bulky N2-Guanine DNA Adducts by Human DNA Polymerase ι
    Choi, Jeong-Yun; Guengerich, F. Peter
    Journal of Biological Chemistry (2006), 281 (18), 12315-12324CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    DNA polymerase (pol) ι has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol ι. Pol ι effectively bypassed N2-Me (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic anal. showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). DTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (Sp-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chem. step is largely interfered with by adducts. Severe inhibition of polymn. opposite N2,N2-diMeG compared with N2-EtG by pol η but not by pol ι is consistent with Hoogsteen base pairing by pol ι. Thus, polymn. by pol ι is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol ι may play a limited role in translesion synthesis on bulky N2-G adducts in cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFKksrk%253D&md5=b0289a6d2fb0c6770ea42735361eadb0
  13. 13
    Choi, J.-Y. and Guengerich, F. P. (2008) Kinetic analysis of translesion synthesis opposite bulky N2- and O6-alkylguanine DNA adducts by human DNA polymerase REV1 J. Biol. Chem. 283, 23645– 23655
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    13
    Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1
    Choi, Jeong-Yun; Guengerich, F. Peter
    Journal of Biological Chemistry (2008), 283 (35), 23645-23655CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addn. to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N2-alkylG adducts (in oligonucleotides) ranging in size from Me to CH2(6-benzo[a]pyrenyl) (BP) and O6-alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O6-PobG, which caused almost complete blockage. Steady-state kinetic parameters (kcat/Km) were similar for insertion of dCTP opposite G and N2-G adducts but were severely reduced opposite the O6-G adducts. REV1 showed apparent pre-steady-state burst kinetics for dCTP incorporation only opposite N2-BPG and little, if any, opposite G, N2-benzyl (Bz)G, or O6-BzG. The maximal polymn. rate (kpol 0.9 s-1) opposite N2-BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N2-BPG-adducted DNA 3-fold more tightly than unmodified G-contg. DNA. These results and the lack of an elemental effect ((Sp)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N2-BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N2-adduct at G but not to O6-adducts.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVWktr7E&md5=66e2872a86dced5324948771bdfd6f96
  14. 14
    Nair, D. T., Johnson, R. E., Prakash, S., Prakash, L., and Aggarwal, A. K. (2004) Replication by human DNA polymerase-iota occurs by Hoogsteen base-pairing Nature 430, 377– 380
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    14
    Replication by human DNA polymerase-ι occurs by Hoogsteen base-pairing
    Nair, Deepak T.; Johnson, Robert E.; Prakash, Satya; Prakash, Louise; Aggarwal, Aneel K.
    Nature (London, United Kingdom) (2004), 430 (6997), 377-380CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Almost all DNA polymerases show a strong preference for incorporating the nucleotide that forms the correct Watson-Crick base pair with the template base. In addn., the catalytic efficiencies with which any given polymerase forms the four possible correct base pairs are roughly the same. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, is an exception to these rules. hPolι incorporates the correct nucleotide opposite a template adenine with a several hundred to several thousand fold greater efficiency than it incorporates the correct nucleotide opposite a template thymine, whereas its efficiency for correct nucleotide incorporation opposite a template guanine or cytosine is intermediate between these two extremes. Here the authors present the crystal structure of hPolι bound to a template primer and an incoming nucleotide. The structure reveals a polymerase that is 'specialized' for Hoogsteen base-pairing, whereby the templating base is driven to the syn conformation. Hoogsteen base-pairing offers a basis for the varied efficiencies and fidelities of hPolι opposite different template bases, and it provides an elegant mechanism for promoting replication through minor-groove purine adducts that interfere with replication.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXls1eqs7s%253D&md5=dc936aea2d3b57abf2726a820100f8eb
  15. 15
    Choi, J.-Y., Lim, S., Eoff, R. L., and Guengerich, F. P. (2009) Kinetic analysis of base-pairing preference for nucleotide incorporation opposite template pyrimidines by human DNA polymerase ι J. Mol. Biol. 389, 264– 274
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    15
    Kinetic Analysis of Base-Pairing Preference for Nucleotide Incorporation Opposite Template Pyrimidines by Human DNA Polymerase ι
    Choi, Jeong-Yun; Lim, Seonhee; Eoff, Robert L.; Guengerich, F. Peter
    Journal of Molecular Biology (2009), 389 (2), 264-274CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    DNA polymerase (pol) ι, a member of the mammalian Y-family of DNA polymerases involved in translesion DNA synthesis, has been previously suggested to peculiarly utilize Hoogsteen base pairing for DNA synthesis opposite template purines, unlike pols η and κ, which utilize Watson-Crick (W-C) base pairing. To investigate the possible roles of Hoogsteen, W-C, and wobble base-pairing modes in the selection of nucleotides opposite template pyrimidines by human pol ι, we carried out kinetic analyses of incorporation of modified purine nucleoside triphosphates including 7-deazapurines, inosine, 2-aminopurine, 2,6-diaminopurine, and 6-chloropurine, which affect H-bonding in base-pair formation opposite template pyrimidines. Carbon substitution at the N7 atom of purine nucleoside triphosphates, which disrupts Hoogsteen base pairing, only slightly inhibited DNA synthesis opposite template pyrimidines by pol ι, which was not substantially different from human pols η and κ. Opposite template T, only the relative wobble stabilities (inferred from the potential nos. of H-bonding, steric, and electrostatic interactions but not measured) of base pairs were pos. correlated to the relative efficiencies of nucleotide incorporation by pol ι but not the relative W-C or Hoogsteen stabilities, unlike pols η and κ. In contrast, opposite C, only the relative W-C stabilities of base pairs were pos. correlated to the relative efficiencies of nucleotide incorporation by pol ι, as with pols η and κ. These results suggest that pol ι might not indispensably require Hoogsteen base pairing for DNA synthesis opposite pyrimidines but rather might prefer wobble base pairing in the selection of nucleotides opposite T and W-C base pairing opposite C.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtVSqsLw%253D&md5=3eb7a5d578036c7548000737d62681da
  16. 16
    Kirouac, K. N. and Ling, H. (2009) Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase iota EMBO J. 28, 1644– 1654
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    16
    Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase ι
    Kirouac, Kevin N.; Ling, Hong
    EMBO Journal (2009), 28 (11), 1644-1654CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)
    Human DNA polymerase ι (polι) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of polι complexed with DNA contg. a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by polι. The template thymine remains in an anti conformation irresp. of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template 'U-turn' is stabilized by polι and the Me group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping expts. indicate that the finger domain is responsible for polι's high error rates on pyrimidines and dets. the incorporation specificity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlvValsbw%253D&md5=f5526a9c72a7abafe604f81e13f7b309
  17. 17
    Choi, J.-Y., Chowdhury, G., Zang, H., Angel, K. C., Vu, C. C., Peterson, L. A., and Guengerich, F. P. (2006) Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases J. Biol. Chem. 281, 38244– 38256
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    17
    Translesion Synthesis across O6-Alkylguanine DNA Adducts by Recombinant Human DNA Polymerases
    Choi, Jeong-Yun; Chowdhury, Goutam; Zang, Hong; Angel, Karen C.; Vu, Choua C.; Peterson, Lisa A.; Guengerich, F. Peter
    Journal of Biological Chemistry (2006), 281 (50), 38244-38256CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O6-Me and -benzyl (Bz) G. Recombinant human polymerase (pol) δ also copied past these two lesions but was totally blocked by O6-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols ι and κ produced mainly only 1-base incorporation opposite O6-MeG and O6-BzG and had very low activity in copying O6-PobG. Human pol η copied past all three adducts. Steady-state kinetic anal. showed similar efficiencies of insertion opposite the O6-alkylG adducts for dCTP and dTTP with pol η and κ; pol ι showed a strong preference for dTTP. pol η, ι, and κ showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O6-MeG but little, if any, for O6-BzG or O6-PobG. Anal. of the pol η O6-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry anal. of all of the pol η primer extension products indicated multiple components, mainly with C or T inserted opposite O6-alkylG but with no deletions in the cases of O6-MeG and O6-PobG. With pol η and O6-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O6-BzG). The results with pol η may be relevant to some mutations previously reported with O6-alkylG adducts in mammalian cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht12nu7bE&md5=a745f9c97b4ccce6a6c57fab972500a0
  18. 18
    Vaisman, A., Frank, E. G., Iwai, S., Ohashi, E., Ohmori, H., Hanaoka, F., and Woodgate, R. (2003) Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase iota DNA Repair 2, 991– 1006
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    18
    Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase ι
    Vaisman, Alexandra; Frank, Ekaterina G.; Iwai, Shigenori; Ohashi, Eiji; Ohmori, Haruo; Hanaoka, Fumio; Woodgate, Roger
    DNA Repair (2003), 2 (9), 991-1006CODEN: DRNEAR; ISSN:1568-7864. (Elsevier Science B.V.)
    Humans possess four Y-family polymerases: pols η, ι, κ and the Rev1 protein. The pivotal role that polη plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding polη), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols ι, κ and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of polι to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that polι can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, polι efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of ∼102 over the incorporation of incorrect nucleotides, while incorporation opposite the 5'T is highly mutagenic. Polκ has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions of polι and polκ result in a significant increase in bypass of T-T CPDs when compared to polι alone. Our data suggest that under certain conditions and sequence contexts, polι can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochem. activities may, therefore, be of biol. significance esp. in XP-V cells lacking the primary T-T CPD bypassing enzyme, polη.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXntVGhtL0%253D&md5=8f000b050e2d676e6f9f91ab26cc9348
  19. 19
    Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F. P. (2010) Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases alpha, delta, eta, iota, kappa, and REV1 J. Mol. Biol. 404, 34– 44
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    19
    Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases α, δ, η, ι, κ, and REV1
    Choi, Jeong-Yun; Lim, Seonhee; Kim, Eun-Jin; Jo, Ara; Guengerich, F. Peter
    Journal of Molecular Biology (2010), 404 (1), 34-44CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides contg. a synthetic abasic lesion (THF analog) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, resp.) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlKhtb%252FP&md5=e5890e1ff1bc45ca9bf085982bf7e43c
  20. 20
    Frank, E. G. and Woodgate, R. (2007) Increased catalytic activity and altered fidelity of human DNA polymerase ι in the presence of manganese J. Biol. Chem. 282, 24689– 24696
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    20
    Increased Catalytic Activity and Altered Fidelity of Human DNA Polymerase ι in the Presence of Manganese
    Frank, Ekaterina G.; Woodgate, Roger
    Journal of Biological Chemistry (2007), 282 (34), 24689-24696CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg2+ is the physiol. cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol λ, may preferentially utilize Mn2+ in vitro. Here we report on the effects of Mn2+ and Mg2+ on the enzymic properties of human DNA polymerase ι (pol ι). Pol ι exhibited the greatest activity in the presence of low levels of Mn2+ (0.05-0.25 mM). Peak activity in the presence of Mg2+ was obsd. in the range of 0.1-0.5 mM and was significantly reduced at concns. >2 mM. Steady-state kinetic analyses revealed that Mn2+ increases the catalytic activity of pol ι by ∼30-60,000-fold through a dramatic decrease in the Km value for nucleotide incorporation. Interestingly, whereas pol ι preferentially misinserts G opposite T by a factor of ∼1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mM Mg2+, resp., the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mM Mn2+. Low levels of Mn2+ also dramatically increased the ability of pol ι to traverse a variety of DNA lesions in vitro. Titrn. expts. revealed a strong preference of pol ι for Mn2+ even when Mg2+ is present in a >10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol ι in vivo may actually be Mn2+ rather than Mg2+, as tacitly assumed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpt1SlsLk%253D&md5=f29f919801291f0902131c752edf5bb3
  21. 21
    Dumstorf, C. A., Clark, A. B., Lin, Q., Kissling, G. E., Yuan, T., Kucherlapati, R., McGregor, W. G., and Kunkel, T. A. (2006) Participation of mouse DNA polymerase iota in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer Proc. Natl. Acad. Sci. U.S.A. 103, 18083– 18088
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    21
    Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer
    Dumstorf, Chad A.; Clark, Alan B.; Lin, Qingcong; Kissling, Grace E.; Yuan, Tao; Kucherlapati, Raju; McGregor, W. Glenn; Kunkel, Thomas A.
    Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (48), 18083-18088CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    DNA polymerase ι (pol ι) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol ι performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol η, pol ι, or both. A deficiency in mouse DNA polymerase η (pol η) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol ι, indicating that pol ι participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was obsd. in wild-type cells that was diminished in pol η- and pol ι-deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asym. manner. To det. whether pol L status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol η, pol ι, or both. Although pol ι deficiency alone had no effect, UV-induced skin tumors in pol η-deficient mice developed 4 wk earlier in mice concomitantly deficient in pol ι. Collectively, these data reveal functions for pol ι in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlWjt7%252FK&md5=3d751997055f965126d667ce1a0fd573
  22. 22
    Ohkumo, T., Kondo, Y., Yokoi, M., Tsukamoto, T., Yamada, A., Sugimoto, T., Kanao, R., Higashi, Y., Kondoh, H., Tatematsu, M., Masutani, C., and Hanaoka, F. (2006) UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota Mol. Cell. Biol. 26, 7696– 7706
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    22
    UV-B radiation induces epithelial tumors in mice lacking DNA polymerase η and mesenchymal tumors in mice deficient for DNA polymerase
    Ohkumo, Tsuyoshi; Kondo, Yuji; Yokoi, Masayuki; Tsukamoto, Tetsuya; Yamada, Ayumi; Sugimoto, Taiki; Kanao, Rie; Higashi, Yujiro; Kondoh, Hisato; Tatematsu, Masae; Masutani, Chikahide; Hanaoka, Fumio
    Molecular and Cellular Biology (2006), 26 (20), 7696-7706CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)
    DNA polymerase η (Pol η) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol η is involved in the somatic hypermutation of Ig genes, but the cancer predisposition of Polh-/- mice has not been examd. until very recently. Another translesion synthesis polymerase, Pol ι, a Pol η paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol ι is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol ι deficiency does not influence the UV sensitivity of mouse fibroblasts irresp. of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histol. diagnosis after chronic treatment with UV light reveals that Pol ι deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not obsd. in Polh-/- mice. These results suggest the involvement of the Pol η and Pol ι proteins in UV-induced skin carcinogenesis.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFCgurrF&md5=2199a5c9d1becb57bcbcb69b42d4d514
  23. 23
    Iguchi, M., Osanai, M., Hayashi, Y., Koentgen, F., and Lee, G. H. (2014) The error-prone DNA polymerase iota provides quantitative resistance to lung tumorigenesis and mutagenesis in mice Oncogene 33, 3612– 3617
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    23
    The error-prone DNA polymerase ι provides quantitative resistance to lung tumorigenesis and mutagenesis in mice
    Iguchi, M.; Osanai, M.; Hayashi, Y.; Koentgen, F.; Lee, G.-H.
    Oncogene (2014), 33 (27), 3612-3617CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)
    Opposite undamaged nucleotide T, DNA polymerase ι (Polι) preferentially incorporates G rather than A, violating the Watson-Crick rule. Although the actual biol. role of Polι remains enigmatic, we have identified its coding gene as a candidate for pulmonary adenoma resistance 2 (Par2), a mouse quant. trait locus modulating chem. induced lung tumor susceptibility. Notably, the most tumor-sensitive Par2 allele possessed by the 129X1/SvJ mouse is assocd. with a loss-of-function mutation in Polι. To det. whether the nonfunctional Polι is responsible for the 129X1/SvJ-specific Par2 phenotype, we knocked out Polι in a C57BL/6J mouse carrying a less tumor-sensitive Par2 allele. Disruption of the C57BL/6J Polι conferred 129X1/SvJ-like sensitivity on the C57BL/6J Par2 locus and increased the in vivo mutation frequency in the lung, providing definitive proof that Polι causes the Par2 effect and inhibits tumorigenesis and mutagenesis, despite its extreme replication infidelity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1ylsbbM&md5=b8fe6b0bba17659a2f15e6bbc137e025
  24. 24
    Yang, J., Chen, Z., Liu, Y., Hickey, R. J., and Malkas, L. H. (2004) Altered DNA polymerase iota expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis Cancer Res. 64, 5597– 5607
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    24
    Altered DNA polymerase ι expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis
    Yang, Jin; Chen, Zhiwen; Liu, Yang; Hickey, Robert J.; Malkas, Linda H.
    Cancer Research (2004), 64 (16), 5597-5607CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    The recently discovered human enzyme DNA polymerase ι (pol ι) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol ι in DNA lesion bypass, there is no in vivo evidence to confirm this action. It has been reported here that pol ι expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. Author also demonstrated that UV treatment of breast cancer cells addnl. increases pol ι expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol ι expression is an early event after UV-mediated DNA damage. That pol ι may play a role in the higher mutation frequencies obsd. in breast cancer cells was suggested when a redn. in mutation frequency was found after pol ι was immunodepleted from nuclear exts. of the cells. Anal. of the UV-induced mutation spectra revealed that >90% were point mutations. The anal. also demonstrated a decreased C → T nucleotide transition and an increased C → A transversion rate. Overall, the data strongly suggest that pol ι may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmslalurw%253D&md5=6e57151073b48272a3d2a3cbeea31bcf
  25. 25
    Zhou, J., Zhang, S., Xie, L., Liu, P., Xie, F., Wu, J., Cao, J., and Ding, W. Q. (2012) Overexpression of DNA polymerase iota (Poliota) in esophageal squamous cell carcinoma Cancer Sci. 103, 1574– 1579
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    25
    Overexpression of DNApolymerase iota (Polι) in esophageal squamous cell carcinoma
    Zhou, Jundong; Zhang, Shuyu; Xie, Ling; Liu, Pengfei; Xie, Fang; Wu, Jinchang; Cao, Jianping; Ding, Wei-Qun
    Cancer Science (2012), 103 (8), 1574-1579CODEN: CSACCM; ISSN:1349-7006. (Wiley-Blackwell)
    The present study investigated the transcriptional regulation of low-fidelity translesion DNA synthesis (TLS) polymerases in human esophageal carcinoma. Significantly higher mRNA expression of polymerase zeta (Polξ), RAD18,polymerase iota (Polι), and polymerase kappa (Polκ) was found in esophageal carcinomas. The increased expression of Polι in tumor samples was further confirmed by immunohistochem. The promoter of POLI that encodes Polι was found to be hypomethylated, although the overexpression of this gene was unlikely to be assocd. with methylation in tumors. We further identified Sp1 and Oct-1 binding sites present in the POLI promoter. We obsd. that the binding affinity of Sp1 to the POLI promoter was significantly increased in cancerous tissues and that Sp1 activated POLI gene transcription in cultured cell lines. The present study demonstrates overexpression of the TLS genes in esophageal carcinoma and identifies a key role for Sp1 in upregulating POLI gene expression. (Cancer Sci 2012; 103: 1574-1579).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtV2isbfP&md5=2556a4bfa7f27c03fd1e6bd94761a598
  26. 26
    Albertella, M. R., Lau, A., and O’Connor, M. J. (2005) The overexpression of specialized DNA polymerases in cancer DNA Repair 4, 583– 593
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    26
    The overexpression of specialized DNA polymerases in cancer
    Albertella, Mark R.; Lau, Alan; O'Connor, Mark J.
    DNA Repair (2005), 4 (5), 583-593CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)
    Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on noncanonical templates, such as undamaged DNA or on noncognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, the authors examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases β, λ, ι and κ, the authors also investigate the expression of the replicative polymerases α, δ and ε. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase β (pol β) was overexpressed at both the mRNA and protein level in approx. one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols λ, and ι were also overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol β. In contrast, pol κ was rarely overexpressed in tumors but was commonly underexpressed in many samples. Downregulation of pol β expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialized DNA polymerases, and particularly pol β, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitize tumors to chemotherapy.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXivFyru7s%253D&md5=c63ba33c74a51ba6193f37c730b59cb3
  27. 27
    Yuan, F., Xu, Z., Yang, M., Wei, Q., Zhang, Y., Yu, J., Zhi, Y., Liu, Y., Chen, Z., and Yang, J. (2013) Overexpressed DNA polymerase iota regulated by JNK/c-Jun contributes to hypermutagenesis in bladder cancer PLoS One 8, e69317
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    27
    Overexpressed DNA polymerase iota regulated by JNK/c-jun contributes to hypermutagenesis in bladder cancer
    Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin
    PLoS One (2013), 8 (7), e69317CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Human DNA polymerase iota (pol i) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol i may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol i and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation expt., EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol i in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol i, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, assocd. with elevated c-Jun activity, the overexpression of pol i is pos. correlated with the clin. tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol i-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol i by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol i or c-Jun in mutagenesis.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1GisLfK&md5=05f2e0ad776f497c2859b1eb4d20ac3e
  28. 28
    Luedeke, M., Linnert, C. M., Hofer, M. D., Surowy, H. M., Rinckleb, A. E., Hoegel, J., Kuefer, R., Rubin, M. A., Vogel, W., and Maier, C. (2009) Predisposition for TMPRSS2-ERG fusion in prostate cancer by variants in DNA repair genes Cancer Epidemiol. Biomarkers Prev. 18, 3030– 3035
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    28
    Predisposition for TMPRSS2-ERG Fusion in Prostate Cancer by Variants in DNA Repair Genes
    Luedeke, Manuel; Linnert, Carmen M.; Hofer, Matthias D.; Surowy, Harald M.; Rinckleb, Antje E.; Hoegel, Josef; Kuefer, Rainer; Rubin, Mark A.; Vogel, Walther; Maier, Christiane
    Cancer Epidemiology, Biomarkers & Prevention (2009), 18 (11), 3030-3035CODEN: CEBPE4; ISSN:1055-9965. (American Association for Cancer Research)
    The somatic fusion of TMPRSS2 to ETS oncogenes is a common event in prostate cancer (PCa). We hypothesized that defects in DNA repair may lead to an increase of chromosomal rearrangements and thus to the occurrence of ETS oncogene fusion. We have previously conducted a genome-wide linkage anal. in TMPRSS2-ERG fusion-pos. PCa families, revealing potential susceptibility loci on chromosomes 5q14, 9q21, 10q26, 11q24, 12q15, 13q12, 18q, and Xq27. In the present study, nine candidate genes from these regions were selected from the context of DNA repair and screened for mutations in TMPRSS2-ERG fusion-pos. families. Thirteen nonsynonymous variants, 5 of which had a minor allele frequency of <0.05, were genotyped in 210 familial cases, 47 of which with a known TMPRSS2-ERG status, 329 sporadic cases, and 512 controls. Significant assocn. of TMPRSS2-ERG fusion-pos. PCa was found with rare variants in the genes for POLI [variant F532S: P = 0.0011; odds ratios (OR), 4.62; 95% confidence interval (95% CI), 1.84-11.56] and ESCO1 (variant N191S: P = 0.0034; OR, 4.27; 95% CI, 1.62-11.28). Addnl. findings, regardless of TMPRSS2-ERG status, were the overrepresentation of a rare BRCA2 variant (V2728I: P = 0.03; OR, 6.16; 95% CI, 1.19-32.00) in familial PCa and of a common allele of RMI1 (variant N455S: P = 0.02; OR, 1.33; 95% CI, 1.04-1.70) in unselected PCa cases. The DNA repair genes POLI and ESCO1 are proposed as susceptibility genes for TMPRSS2-ERG fusion-pos. PCa that warrant further investigation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtl2kt7%252FE&md5=5086c48e52382132b2f59b86bec6464a
  29. 29
    Sakiyama, T., Kohno, T., Mimaki, S., Ohta, T., Yanagitani, N., Sobue, T., Kunitoh, H., Saito, R., Shimizu, K., Hirama, C., Kimura, J., Maeno, G., Hirose, H., Eguchi, T., Saito, D., Ohki, M., and Yokota, J. (2005) Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk Int. J. Cancer 114, 730– 737
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    29
    Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk
    Sakiyama, Tokuki; Kohno, Takashi; Mimaki, Sachiyo; Ohta, Tsutomu; Yanagitani, Noriko; Sobue, Tomotaka; Kunitoh, Hideo; Saito, Ryusei; Shimizu, Kimiko; Hirama, Chie; Kimura, Junko; Maeno, Go; Hirose, Hiroshi; Eguchi, Takashi; Saito, Daizo; Ohki, Misao; Yokota, Jun
    International Journal of Cancer (2005), 114 (5), 730-737CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
    Single nucleotide polymorphisms (SNPs) were searched for in 36 genes involved in diverse DNA repair pathways, and 50 nonsynonymous (assocd. with amino acid changes) SNPs identified were assessed for assocns. with lung cancer risk by a case-control study consisting of 752 adenocarcinoma cases, 250 squamous cell carcinoma cases and 685 controls. An SNP, Arg72Pro, of the TP53 gene encoding a DNA damage response protein showed the strongest assocn. with squamous cell carcinoma risk (OR Pro/Pro vs. Arg/Arg = 2.2), while 2 other SNPs, Phe257Ser of the REV1 gene encoding a translesion DNA polymerase and Ile658Val of the LIG4 gene encoding a DNA double-strand break repair protein, also showed assocns. (OR Ser/Ser vs. Phe/Phe = 2.0 and OR Ile/Val vs. Ile/Ile = 0.4, resp.). An SNP, Thr706Ala, in the POLI gene encoding another translesion DNA polymerase was assocd. with adenocarcinoma and squamous cell carcinoma risk, particularly in individuals of ages <61 years (OR Ala/Ala + Ala/Thr vs. Thr/Thr = 1.5 and 2.4, resp.). POLI is the human counterpart of PolI, a strong candidate for the Par2 (pulmonary adenoma resistance 2) gene responsible for adenoma/adenocarcinoma susceptibility in mice. The present results suggest that these 4 SNPs function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXitl2quro%253D&md5=e9444dac3ccb43ae6c758429d4895da4
  30. 30
    Ng, P. C. and Henikoff, S. (2001) Predicting deleterious amino acid substitutions Genome Res. 11, 863– 874
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    30
    Predicting deleterious amino acid substitutions
    Ng, Pauline C.; Henikoff, Steven
    Genome Research (2001), 11 (5), 863-874CODEN: GEREFS; ISSN:1088-9051. (Cold Spring Harbor Laboratory Press)
    Many missense substitutions are identified in single nucleotide polymorphism (SNP) data and large-scale random mutagenesis projects. Each amino acid substitution potentially affects protein function. We have constructed a tool that uses sequence homol. to predict whether a substitution affects protein function. SIFT, which sorts intolerant from tolerant substitutions, classifies substitutions as tolerated or deleterious. A higher proportion of substitutions predicted to be deleterious by SIFT gives an affected phenotype than substitutions predicted to be deleterious by substitution scoring matrixes in three test cases. Using SIFT before mutagenesis studies could reduce the no. of functional assays required and yield a higher proportion of affected phenotypes. SIFT may be used to identify plausible disease candidates among the SNPs that cause missense substitutions.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjs1Wmu7w%253D&md5=529639c792d6286a23b3f1b6e41f1147
  31. 31
    Ramensky, V., Bork, P., and Sunyaev, S. (2002) Human non-synonymous SNPs: server and survey Nucleic Acids Res. 30, 3894– 3900
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    31
    Human non-synonymous SNPs: server and survey
    Ramensky, Vasily; Bork, Peer; Sunyaev, Shamil
    Nucleic Acids Research (2002), 30 (17), 3894-3900CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Human single nucleotide polymorphisms (SNPs) represent the most frequent type of human population DNA variation. One of the main goals of SNP research is to understand the genetics of the human phenotype variation and esp. the genetic basis of human complex diseases. Non-synonymous coding SNPs (nsSNPs) comprise a group of SNPs that, together with SNPs in regulatory regions, are believed to have the highest impact on phenotype. Here we present a World Wide Web server to predict the effect of an nsSNP on protein structure and function. The prediction method enabled anal. of the publicly available SNP database HGVbase, which gave rise to a dataset of nsSNPs with predicted functionality. The dataset was further used to compare the effect of various structural and functional characteristics of amino acid substitutions responsible for phenotypic display of nsSNPs. We also studied the dependence of selective pressure on the structural and functional properties of proteins. We found that in our dataset the selection pressure against deleterious SNPs depends on the mol. function of the protein, although it is insensitive to several other protein features considered. The strongest selective pressure was detected for proteins involved in transcription regulation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xms1Klt7k%253D&md5=edc88439061ba8dc0d48665b707dc944
  32. 32
    Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S., and Sunyaev, S. R. (2010) A method and server for predicting damaging missense mutations Nat. Methods 7, 248– 249
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    32
    A method and server for predicting damaging missense mutations
    Adzhubei, Ivan A.; Schmidt, Steffen; Peshkin, Leonid; Ramensky, Vasily E.; Gerasimova, Anna; Bork, Peer; Kondrashov, Alexey S.; Sunyaev, Shamil R.
    Nature Methods (2010), 7 (4), 248-249CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    A method and corresponding software tool, PolyPhen-2, for predicting damaging effects of missense mutations is presented. HumDiv-trained PolyPhen-2 should be used to evaluate rare alleles at loci potentially involved in complex phenotypes, for dense mapping of regions identified by genome-wide assocn. studies and for anal. of natural selection from sequence data, in which even mildly deleterious alleles must be treated as damaging.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjvFKqu78%253D&md5=aafc33a76fbca3184c501aae083f6f75
  33. 33
    Choi, J.-Y. and Guengerich, F. P. (2004) Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase J. Biol. Chem. 279, 19217– 19229
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    33
    Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase
    Choi, Jeong-Yun; Guengerich, F. Peter
    Journal of Biological Chemistry (2004), 279 (18), 19217-19229CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease- (T7-). RT and T7- effectively bypassed N2-methyl(Me)G and readily extended primers but were strongly blocked by N2-ethyl(Et)G, N2-isobutylG, N2-benzylG, and N2-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7- showed a decrease of 103 in kcat/Km for dCTP incorporation opposite N2-MeG and a further large decrease opposite N2-EtG. Misincorporation frequency was increased 102-103-fold by a Me group and another ∼103-fold by an Et group. DATP was preferentially incorporated opposite bulky N2-alkylG mols. N2-MeG attenuated the pre-steady-state kinetic bursts with RT and T7-, and N2-EtG eliminated the bursts. Large elemental effects with thio-dCTP(αS) were obsd. with N2-EtG (6- and 72-fold decreases) but were much less with N2-MeG, indicating that the N2-Et group may affect the rate of the chem. step (phosphodiester bond formation). Similar values of Kd(dCTP) and Kd(DNA) and koff rates of DNA substrates from RT and T7- indicate that ground-state binding and dissocn. rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chem. step, demonstrating the role of bulk in DNA lesions.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjsVKmtbc%253D&md5=3b433b8a493242dd1bda212dce1a1808
  34. 34
    Boosalis, M. S., Petruska, J., and Goodman, M. F. (1987) DNA polymerase insertion fidelity: gel assay for site-specific kinetics J. Biol. Chem. 262, 14689– 14696
    [ PubMed], [ CAS], Google Scholar
    34
    DNA polymerase insertion fidelity. Gel assay for site-specific kinetics
    Boosalis, Michael S.; Petruska, John; Goodman, Myron F.
    Journal of Biological Chemistry (1987), 262 (30), 14689-96CODEN: JBCHA3; ISSN:0021-9258.
    A quant. assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase-α holoenzyme complex and M13 primer-template DNA. The Km and Vmax values are reported for correct insertion of adenine (A) and misinsertion of guanine (G), cytosine (C), and thymine (T) opposite a single template T site. The misinsertion frequencies were 2 × 10-4 for G-T and 5 × 10-5 for both C-T and T-T relative to normal A-T base pairs. The dissocn. const. of the polymerase-DNA-dNTP (deoxynucleotide triphosphate) complex, as measured by Km, played a dominant role in detg. the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 μM), the Km was 1100-fold greater for misinsertion of G opposite T (4.2 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 mM). These Km differences indicated that in the enzyme binding site the stability of A-T base pairs was 4.3 kcal/mol greater than T-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax were relatively small. There was only a 4-fold redn. in Vmax for insertion of G opposite T and an 8-fold redn. for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase-α appeared to be governed primarily by a Km discrimination mechanism.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlais7w%253D&md5=5870530d34c8ab0afe913d4251ab0999
  35. 35
    Song, I., Kim, E. J., Kim, I. H., Park, E. M., Lee, K. E., Shin, J. H., Guengerich, F. P., and Choi, J.-Y. (2014) Biochemical characterization of eight genetic variants of human DNA polymerase κ involved in error-free bypass across bulky N2-guanyl DNA adducts Chem. Res. Toxicol. 27, 919– 930
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    35
    Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky N2-Guanyl DNA Adducts
    Song, Insil; Kim, Eun-Jin; Kim, In-Hyeok; Park, Eun-Mi; Lee, Kyung Eun; Shin, Joo-Ho; Guengerich, F. Peter; Choi, Jeong-Yun
    Chemical Research in Toxicology (2014), 27 (5), 919-930CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky N2-guanyl DNA lesions induced by many carcinogens such as polycyclic arom. hydrocarbons. We analyzed the biochem. properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1-526) protein and the DNA template contg. an N2-CH2(9-anthracenyl)G (N2-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky N2-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtVWgsrg%253D&md5=3b892cadfb539736dddfdb191477c899
  36. 36
    Ketkar, A., Zafar, M. K., Maddukuri, L., Yamanaka, K., Banerjee, S., Egli, M., Choi, J.-Y., Lloyd, R. S., and Eoff, R. L. (2013) Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity Chem. Res. Toxicol. 26, 221– 232
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    36
    Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity
    Ketkar, Amit; Zafar, Maroof K.; Maddukuri, Leena; Yamanaka, Kinrin; Banerjee, Surajit; Egli, Martin; Choi, Jeong-Yun; Lloyd, R. Stephen; Eoff, Robert L.
    Chemical Research in Toxicology (2013), 26 (2), 221-232CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compds. identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC50 values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmvVGntg%253D%253D&md5=6bacbda93b04ec3e2bb56ba5086fb0cf
  37. 37
    Pence, M. G., Choi, J.-Y., Egli, M., and Guengerich, F. P. (2010) Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase ι J. Biol. Chem. 285, 40666– 40672
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    37
    Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι
    Pence, Matthew G.; Choi, Jeong-Yun; Egli, Martin; Guengerich, F. Peter
    Journal of Biological Chemistry (2010), 285 (52), 40666-40672CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    O6-Methylguanine (O6-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiol. ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was detd. for nucleoside triphosphate incorporation opposite O6-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this lab. using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O6-methylG. Misincorporation of dTTP opposite O6-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O6-methylG as the template base and incoming dCTP or dTTP were solved and showed that O6-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O6-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O6-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O6 atoms of O6-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O6-methylG by human pol ι, in contrast to the mispairing modes obsd. previously for O6-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFynsL3F&md5=e785844ad7d4e862280aee325acc4d69
  38. 38
    Kirouac, K. N. and Ling, H. (2011) Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota Proc. Natl. Acad. Sci. U.S.A. 108, 3210– 3215
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    38
    Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota
    Kirouac, Kevin N.; Ling, Hong
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (8), 3210-3215, S3210/1-S3210/3CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biol. role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA contg. an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equil. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the addnl. O8 atom of the lesion base, which influences nucleotide selection, enzymic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXislyjur0%253D&md5=b657fcab4ddbb7f765f3d1911e58b17d
  39. 39
    Manolio, T. A., Collins, F. S., Cox, N. J., Goldstein, D. B., Hindorff, L. A., Hunter, D. J., McCarthy, M. I., Ramos, E. M., Cardon, L. R., Chakravarti, A., Cho, J. H., Guttmacher, A. E., Kong, A., Kruglyak, L., Mardis, E., Rotimi, C. N., Slatkin, M., Valle, D., Whittemore, A. S., Boehnke, M., Clark, A. G., Eichler, E. E., Gibson, G., Haines, J. L., Mackay, T. F., McCarroll, S. A., and Visscher, P. M. (2009) Finding the missing heritability of complex diseases Nature 461, 747– 753
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    39
    Finding the missing heritability of complex diseases
    Manolio, Teri A.; Collins, Francis S.; Cox, Nancy J.; Goldstein, David B.; Hindorff, Lucia A.; Hunter, David J.; McCarthy, Mark I.; Ramos, Erin M.; Cardon, Lon R.; Chakravarti, Aravinda; Cho, Judy H.; Guttmacher, Alan E.; Kong, Augustine; Kruglyak, Leonid; Mardis, Elaine; Rotimi, Charles N.; Slatkin, Montgomery; Valle, David; Whittemore, Alice S.; Boehnke, Michael; Clark, Andrew G.; Eichler, Evan E.; Gibson, Greg; Haines, Jonathan L.; Mackay, Trudy F. C.; McCarroll, Steven A.; Visscher, Peter M.
    Nature (London, United Kingdom) (2009), 461 (7265), 747-753CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    A review. Genome-wide assocn. studies have identified hundreds of genetic variants assocd. with complex human diseases and traits, and have provided valuable insights into their genetic architecture. Most variants identified so far confer relatively small increments in risk, and explain only a small proportion of familial clustering, leading many to question how the remaining, missing heritability can be explained. Here we examine potential sources of missing heritability and propose research strategies, including and extending beyond current genome-wide assocn. approaches, to illuminate the genetics of complex diseases and enhance its potential to enable effective disease prevention or treatment.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1CisbrF&md5=dcdef1c2de0585e1a4d612589311aa19
  40. 40
    Nelson, M. R., Wegmann, D., Ehm, M. G., Kessner, D., St. Jean, P., Verzilli, C., Shen, J., Tang, Z., Bacanu, S. A., Fraser, D., Warren, L., Aponte, J., Zawistowski, M., Liu, X., Zhang, H., Zhang, Y., Li, J., Li, Y., Li, L., Woollard, P., Topp, S., Hall, M. D., Nangle, K., Wang, J., Abecasis, G., Cardon, L. R., Zollner, S., Whittaker, J. C., Chissoe, S. L., Novembre, J., and Mooser, V. (2012) An abundance of rare functional variants in 202 drug target genes sequenced in 14,002 people Science 337, 100– 104
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    40
    An Abundance of Rare Functional Variants in 202 Drug Target Genes Sequenced in 14,002 People
    Nelson, Matthew R.; Wegmann, Daniel; Ehm, Margaret G.; Kessner, Darren; St. Jean, Pamela; Verzilli, Claudio; Shen, Judong; Tang, Zhengzheng; Bacanu, Silviu-Alin; Fraser, Dana; Warren, Liling; Aponte, Jennifer; Zawistowski, Matthew; Liu, Xiao; Zhang, Hao; Zhang, Yong; Li, Jun; Li, Yun; Li, Li; Woollard, Peter; Topp, Simon; Hall, Matthew D.; Nangle, Keith; Wang, Jun; Abecasis, Goncalo; Cardon, Lon R.; Zoellner, Sebastian; Whittaker, John C.; Chissoe, Stephanie L.; Novembre, John; Mooser, Vincent
    Science (Washington, DC, United States) (2012), 337 (6090), 100-104CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Rare genetic variants contribute to complex disease risk; however, the abundance of rare variants in human populations remains unknown. We explored this spectrum of variation by sequencing 202 genes encoding drug targets in 14,002 individuals. We find rare variants are abundant (1 every 17 bases) and geog. localized, so that even with large sample sizes, rare variant catalogs will be largely incomplete. We used the obsd. patterns of variation to est. population growth parameters, the proportion of variants in a given frequency class that are putatively deleterious, and mutation rates for each gene. We conclude that because of rapid population growth and weak purifying selection, human populations harbor an abundance of rare variants, many of which are deleterious and have relevance to understanding disease risk.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpsFKltbg%253D&md5=b560cac006dd86b255383aec1469a4d3
  41. 41
    Zhu, Q., Ge, D., Maia, J. M., Zhu, M., Petrovski, S., Dickson, S. P., Heinzen, E. L., Shianna, K. V., and Goldstein, D. B. (2011) A genome-wide comparison of the functional properties of rare and common genetic variants in humans Am. J. Hum. Genet. 88, 458– 468
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    41
    A Genome-wide Comparison of the Functional Properties of Rare and Common Genetic Variants in Humans
    Zhu, Qian-Qn; Ge, Dong-Lang; Maia, Jessica M.; Zhu, Ming-Fu; Petrovski, Slave; Dickson, Samuel P.; Heinzen, Erin L.; Shianna, Kevin V.; Goldstein, David B.
    American Journal of Human Genetics (2011), 88 (4), 458-468CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
    One of the longest running debates in evolutionary biol. concerns the kind of genetic variation that is primarily responsible for phenotypic variation in species. Here, we address this question for humans specifically from the perspective of population allele frequency of variants across the complete genome, including both coding and noncoding regions. We establish simple criteria to assess the likelihood that variants are functional based on their genomic locations and then use whole-genome sequence data from 29 subjects of European origin to assess the relationship between the functional properties of variants and their population allele frequencies. We find that for all criteria used to assess the likelihood that a variant is functional, the rarer variants are significantly more likely to be functional than the more common variants. Strikingly, these patterns disappear when we focus on only those variants in which the major alleles are derived. These analyses indicate that the majority of the genetic variation in terms of phenotypic consequence may result from a mutation-selection balance, as opposed to balancing selection, and have direct relevance to the study of human disease.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXksFKlsb8%253D&md5=d62d08de43a29231292e1dcf98b381c3
  42. 42
    Ng, P. C. and Henikoff, S. (2006) Predicting the effects of amino acid substitutions on protein function Annu. Rev. Genomics Hum. Genet. 7, 61– 80
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    42
    Predicting the effects of amino acid substitutions on protein function
    Ng, Pauline C.; Henikoff, Steven
    Annual Review of Genomics and Human Genetics (2006), 7 (), 61-80CODEN: ARGHC4; ISSN:1527-8204. (Annual Reviews Inc.)
    A review. Nonsynonymous single nucleotide polymorphisms (nsSNPs) are coding variants that introduce amino acid changes in their corresponding proteins. Because nsSNPs can affect protein function, they are believed to have the largest impact on human health compared with SNPs in other regions of the genome. Therefore, it is important to distinguish those nsSNPs that affect protein function from those that are functionally neutral. Here, the authors provide an overview of amino acid substitution (AAS) prediction methods, which use sequence and/or structure to predict the effect of an AAS on protein function. Most methods predict approx. 25-30% of human nsSNPs to neg. affect protein function, and such nsSNPs tend to be rare in the population. The authors discuss the utility of AAS prediction methods for Mendelian and complex diseases as well as their broader applications for understanding protein function.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1WgsrzJ&md5=713c14f441a4cdde1e5e6cf125d5bef9
  43. 43
    Pence, M. G., Blans, P., Zink, C. N., Hollis, T., Fishbein, J. C., and Perrino, F. W. (2009) Lesion bypass of N2-ethylguanine by human DNA polymerase ι J. Biol. Chem. 284, 1732– 1740
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    43
    Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι
    Pence, Matthew G.; Blans, Patrick; Zink, Charles N.; Hollis, Thomas; Fishbein, James C.; Perrino, Fred W.
    Journal of Biological Chemistry (2009), 284 (3), 1732-1740CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Nucleotide incorporation and extension opposite N2-ethylguanine (N2-ethyl-Gua) by DNA polymerase ι (DNA pol ι) was measured and structures of the DNA polymerase ι-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase ι opposite N2-ethyl-Gua was detd. by steady state kinetic anal. with Mg2+ or Mn2+ as the activating metal. DNA polymerase ι incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase ι opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase ι extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase ι catalyzed reaction is well suited for N2-ethyl-Gua bypass. The structure of DNA polymerase ι with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the Et adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase ι complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase ι polymerase-assocd. domain to accommodate the adduct providing direct evidence that DNA polymerase ι efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtFOhtg%253D%253D&md5=cf9d56e62d17d269f4cc1075a93c1a56
  44. 44
    Nair, D. T., Johnson, R. E., Prakash, L., Prakash, S., and Aggarwal, A. K. (2006) An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site Structure 14, 749– 755
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    44
    An Incoming Nucleotide Imposes an anti to syn Conformational Change on the Templating Purine in the Human DNA Polymerase-ι Active Site
    Nair, Deepak T.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.
    Structure (Cambridge, MA, United States) (2006), 14 (4), 749-755CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    Substrate-induced conformational change of the protein is the linchpin of enzymic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallog. anal. of hPolι binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPolι active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjs1Gksrk%253D&md5=863b779888753f74965fc93fba1265cf
  45. 45
    Nair, D. T., Johnson, R. E., Prakash, L., Prakash, S., and Aggarwal, A. K. (2009) DNA synthesis across an abasic lesion by human DNA polymerase iota Structure 17, 530– 537
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    45
    DNA Synthesis across an Abasic Lesion by Human DNA Polymerase ι
    Nair, Deepak T.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.
    Structure (Cambridge, MA, United States) (2009), 17 (4), 530-537CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    Summary: Abasic sites are among the most abundant DNA lesions formed in human cells, and they present a strong block to replication. DNA polymerase ι (Polι) is one of the few DNA Pols that does not follow the A-rule opposite an abasic site. We present here three structures of human Polι in complex with DNAs contg. an abasic lesion and dGTP, dTTP, or dATP as the incoming nucleotide. The structures reveal a mechanism of translesion synthesis across an abasic lesion that differs from that in other Pols. Both the abasic lesion and the incoming dNTPs are intrahelical and are closely apposed across a constricted active site cleft. The dNTPs partake in distinct networks of hydrogen bonds in the "void" opposite the lesion. These different patterns of hydrogen bonds, as well as stacking interactions, may underlie Polι's small preference for insertion of dGTP over other nucleotides opposite this common lesion.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVCms78%253D&md5=1bbf4b2b98af3f24bc0906ea71bd198b
  46. 46
    Johnson, R. E., Trincao, J., Aggarwal, A. K., Prakash, S., and Prakash, L. (2003) Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases Mol. Cell. Biol. 23, 3008– 3012
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    46
    Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases
    Johnson, Robert E.; Trincao, Jose; Aggarwal, Aneel K.; Prakash, Satya; Prakash, Louise
    Molecular and Cellular Biology (2003), 23 (8), 3008-3012CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)
    Although DNA polymerase η (Polη) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Polη of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equiv. to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, resp.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktV2ltL8%253D&md5=464585f4b76ef7860cd73a2bc3f87e89
  47. 47
    Ash, D. E. and Schramm, V. L. (1982) Determination of free and bound manganese(II) in hepatocytes from fed and fasted rats J. Biol. Chem. 257, 9261– 9264
    [ PubMed], [ CAS], Google Scholar
    47
    Determination of free and bound manganese(II) in hepatocytes from fed and fasted rats
    Ash, David E.; Schramm, Vern L.
    Journal of Biological Chemistry (1982), 257 (16), 9261-4CODEN: JBCHA3; ISSN:0021-9258.
    Free and total Mn(II) contents of hepatocytes from fed and fasted rats were detd. by EPR. The difference spectra of cumulative scans of cell samples and the mother medium allows a sensitivity of 0.1 μM free Mn(II). The free and total Mn(II) contents were 0.71 and 34.4 nmol/mL cell H2O in hepatocytes from fed animals and 0.25 and 35.9 nmol/mL cell H2O, resp., in hepatocytes from fasted animals. There was a decrease in free Mn(II) by a factor of 2.8 with no change in the total cell Mn(II) in response to fasting.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38Xlt1egsL0%253D&md5=f7d1d86f15a3578f7b4900cbc33544ea
  48. 48
    Markesbery, W. R., Ehmann, W. D., Alauddin, M., and Hossain, T. I. (1984) Brain trace element concentrations in aging Neurobiol. Aging 5, 19– 28
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    48
    Brain trace element concentrations in aging
    Markesbery, William R.; Ehmann, William D.; Alauddin, Mohammad; Hossain, T. I. M.
    Neurobiology of Aging (1984), 5 (1), 19-28CODEN: NEAGDO; ISSN:0197-4580.
    Trace element concns. are detd. in various human brain regions over the complete life span using instrumental neutron activation anal. Several different patterns of trace element alteration are obsd. with age. Brain Al, Cl, and Na concns. increase with advancing age, whereas K, P, and Rb decline. Ag, Co, Fe, Sb, and Sc concns. increase up to 40-79 age range then decline. Br, Se, and Zn remain relatively const. throughout adult life. Hg, Mn, and Cs show no consistent trend with age. In infant brains, Br and Cl increase and Al, Cr, Cs, Fe, Mn, P, Rb, Sc, Se, and Zn decrease compared to adults. The essential elements that remain within narrow concn. limits throughout adult life suggest the presence of an efficient homeostatic mechanism for their regulation in the brain, whereas those that are altered with age suggest modifications in control mechanisms or altered relationships with other elements. Increased concns. of nonessential elements may reflect accumulation from our environment, impaired removal, or altered balance with other elements.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXks12qu7c%253D&md5=f8fea6190665b0418d4b99d7a7186e16
  49. 49
    Versieck, J. (1985) Trace elements in human body fluids and tissues Crit. Rev. Clin. Lab. Sci. 22, 97– 184
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    49
    Trace elements in human body fluids and tissues
    Versieck, Jacques
    Critical Reviews in Clinical Laboratory Sciences (1985), 22 (2), 97-184CODEN: CRCLBH; ISSN:0590-8191.
    A review with 633 refs. on trace element anal. and nutrition and the contents of trace elements in human body fluids and tissues.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXkslSitrw%253D&md5=de4d5a49b8e32b042d854a372ad530b3
  50. 50
    Crossgrove, J. and Zheng, W. (2004) Manganese toxicity upon overexposure NMR Biomed. 17, 544– 553
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    50
    Manganese toxicity upon overexposure
    Crossgrove, Janelle; Zheng, Wei
    NMR in Biomedicine (2004), 17 (8), 544-553CODEN: NMRBEF; ISSN:0952-3480. (John Wiley & Sons Ltd.)
    A review on manganese toxicity upon overexposure. Manganese (Mn) is a required element and a metabolic byproduct of the contrast agent mangafodipir trisodium (MnDPDP). The Mn released from MnDPDP is initially sequestered by the liver for first-pass elimination, which allows an enhanced contrast for diagnostic imaging. The administration of i.v. Mn impacts its homeostatic balance in the human body and can lead to toxicity. Human Mn deficiency has been reported in patients on parenteral nutrition and in micronutrient studies. Mn toxicity has been reported through occupational (e.g. welder) and dietary overexposure and is evidenced primarily in the central nervous system, although lung, cardiac, liver, reproductive and fetal toxicity have been noted. Mn neurotoxicity results from an accumulation of the metal in brain tissue and results in a progressive disorder of the extrapyramidal system which is similar to Parkinson's disease. In order for Mn to distribute from blood into brain tissue, it must cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BCB). Brain import, with no evidence of export, would lead to brain Mn accumulation and neurotoxicity. The mechanism for the neurodegenerative damage specific to select brain regions is not clearly understood. Disturbances in iron homeostasis and the valence state of Mn have been implicated as key factors in contributing to Mn toxicity. Chelation therapy with EDTA and supplementation with levodopa are the current treatment options, which are mildly and transiently efficacious. In conclusion, repeated administration of Mn, or compds. that readily release Mn, may increase the risk of Mn-induced toxicity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1Knsr0%253D&md5=6c3a61487421d5f40f0042b16071bc80
  51. 51
    Garcia-Rodriguez, N., Diaz de la Loza Mdel, C., Andreson, B., Monje-Casas, F., Rothstein, R., and Wellinger, R. E. (2012) Impaired manganese metabolism causes mitotic misregulation J. Biol. Chem. 287, 18717– 18729
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    51
    Impaired Manganese Metabolism Causes Mitotic Misregulation
    Garcia-Rodriguez, Nestor; Diaz de la Loza, Maria del Carmen; Andreson, Bethany; Monje-Casas, Fernando; Rothstein, Rodney; Wellinger, Ralf Erik
    Journal of Biological Chemistry (2012), 287 (22), 18717-18729CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn2+ acts as a cofactor for many enzymes and excess of Mn2+ is toxic. Alterations in Mn2+ homeostasis affect metabolic functions and mutations in the human Mn2+/Ca2+ transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn2+ on cell cycle progression and show that an excess of cytosolic Mn2+ alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn2+ pool requires a functional morphol. checkpoint to avoid the formation of polyploid cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsF2msrY%253D&md5=06a56a5f51b508be46aa5c04ca23c97a
  52. 52
    Wang, T. S., Eichler, D. C., and Korn, D. (1977) Effect of Mn2+ on the in vitro activity of human deoxyribonucleic acid polymerase beta Biochemistry 16, 4927– 4934
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    52
    Effect of manganese(2+) ions on the in vitro activity of human deoxyribonucleic acid polymerase β
    Wang, Teresa Shu-Fong; Eichler, Duane C.; Korn, David
    Biochemistry (1977), 16 (22), 4927-34CODEN: BICHAW; ISSN:0006-2960.
    Substitution of Mn2+ for Mg2+ has a profound effect on the kinetic parameters of the human DNA polymerase β reaction. At activated DNA concns. >100 μM in nucleotide, Mg2+ is the preferred cation, but at DNA concns. <100 μM, Mn2+ is preferred. With defined primer-template mols. at low concns. DNA synthesis can be detected only in the presence of Mn2+. The apparent Km for activated DNA with Mn2+ is <10 μM, whereas in reactions with Mg2+, the apparent Km is ∼300 μM. The effects of these divalent cations on the Km values for deoxynucleoside triphosphate substrates are qual. similar. Thus, under appropriate reaction conditions with DNA, Mn2+ may be as effective as Mg2+, strongly preferred or absolutely required for the demonstration of polymerase β activity. A new method was developed for the purifn. of DNA polymerase β from normal adult human liver.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXlvFKhurY%253D&md5=268fb32dc3f3eee6d5c70823905d95ee
  53. 53
    Blanca, G., Shevelev, I., Ramadan, K., Villani, G., Spadari, S., Hübscher, U., and Maga, G. (2003) Human DNA polymerase lambda diverged in evolution from DNA polymerase beta toward specific Mn(++) dependence: a kinetic and thermodynamic study Biochemistry 42, 7467– 7476
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    53
    Human DNA polymerase λ diverged in evolution from DNA polymerase β toward specific Mn++ dependence: A kinetic and thermodynamic study
    Blanca, Giuseppina; Shevelev, Igor; Ramadan, Kristijan; Villani, Giuseppe; Spadari, Silvio; Huebscher, Ulrich; Maga, Giovanni
    Biochemistry (2003), 42 (24), 7467-7476CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The recently discovered human DNA polymerase λ (DNA pol λ) has been implicated in translesion DNA synthesis across abasic sites. One remarkable feature of this enzyme is its preference for Mn2+ over Mg2+ as the activating metal ion, but the mol. basis for this preference is not known. Here, the authors present a kinetic and thermodn. anal. of the DNA polymerase reaction catalyzed by full length human DNA pol λ, showing that Mn2+ favors specifically the catalytic step of nucleotide incorporation. Besides acting as a poor coactivator for catalysis, Mg2+ appeared to bind also to an allosteric site, resulting in the inhibition of the synthetic activity of DNA pol λ and in an increased sensitivity to end product (pyrophosphate) inhibition. Comparison with the closely related enzyme human DNA pol β, as well as with other DNA synthesizing enzymes (mammalian DNA pol α and DNA pol δ, Escherichia coli DNA pol I, and HIV-1 reverse transcriptase) indicated that these features are unique to DNA pol λ. A deletion mutant of DNA pol λ, which contained the highly conserved catalytic core only representing the C-terminal half of the protein, showed biochem. properties comparable to the full length enzyme but clearly different from the close homolog DNA pol β, highlighting the existence of important differences between DNA pol λ and DNA pol β, despite a high degree of sequence similarity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktFymtbo%253D&md5=2238f0022a80a84b197256221d3ee47d
  54. 54
    Dominguez, O., Ruiz, J. F., Lain de Lera, T., Garcia-Diaz, M., Gonzalez, M. A., Kirchhoff, T., Martinez, A. C., Bernad, A., and Blanco, L. (2000) DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells EMBO J. 19, 1731– 1742
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    54
    DNA polymerase mu (Pol μ), homologous to TdT, could act as a DNA mutator in eukaryotic cells
    Dominguez, Orlando; Ruiz, Jose F.; De Lera, Teresa Lain; Garcia-Diaz, Miguel; Gonzalez, Manuel A.; Kirchhoff, Tomas; Martinez-A, Carlos; Bernad, Antonio; Blanco, Luis
    EMBO Journal (2000), 19 (7), 1731-1742CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
    A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol μ), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol μ, overproduced in Escherichia coli in a sol. form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn2+ ions. Interestingly, unlike TdT, the catalytic efficiency of polymn. carried out by Pol μ was enhanced by the presence of a template strand. Using activating Mg2+ ions, template-enhanced polymn. was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn2+ ions, template-enhanced polymn. produced a random insertion of nucleotides. Northern-blotting and in situ anal. showed a preferential expression of Pol μ mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol μ, present in the databases, derived from germinal center B cells. Therefore, Pol μ is a good candidate to be the mutator polymerase responsible for somatic hypermutation of Ig genes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXislymtLc%253D&md5=11e1f0d206762d4f2301bc5d1673bd7f
  55. 55
    Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., and Kraut, J. (1996) Crystal structures of human DNA polymerase β complexed with DNA: implications for catalytic mechanism, processivity, and fidelity Biochemistry 35, 12742– 12761
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    55
    Crystal structures of human DNA polymerase β complexed with DNA: Implications for catalytic mechanism, processivity, and fidelity
    Pelletier, Huguette; Sawaya, Michael R.; Wolfle, William; Wilson, Samuel H.; Kraut, Joseph
    Biochemistry (1996), 35 (39), 12742-12761CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Mammalian DNA polymerase β (pol β) is a small (39 kDa) DNA gap-filling enzyme that comprises an N-terminal 8-kDa domain and a C-terminal 31-kDa domain. Here, crystal structures of human pol β complexed with blunt-ended segments of DNA showed that, although the crystals belonged to a different space group, the DNA was nevertheless bound in the pol β binding channel in the same way as the DNA in previously reported structures of rat pol β complexed with a template-primer and ddCTP. The 8-kDa domain was in 1 of 3 previously obsd. positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small no. of stable conformations. The thumb subdomain was in a more open position in the human pol β-DNA binary complex than in the rat pol β-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that was obsd. in pre-steady-state kinetic studies. Intermol. contacts between the DNA and the 8-kDa domain of a symmetry-related pol β mol. revealed a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addn. to a Lys-rich binding pocket that accommodated a 5'-phosphate end group, the 8-kDa domain also contained a newly discovered helix-hairpin-helix (HhH) motif that bound to DNA in the same way as did a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs was facilitated by a metal cation. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions obsd. in pol β may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol β was particularly striking in that all of the conserved residues were clustered in a short sequence segment, LPGVGXK, where LPGV corresponds to a type II β-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be crit. for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol β may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their resp. DNA lyase activities. A model for productive binding of pol β to a gapped-DNA substrate required a 90° bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlsVymu7s%253D&md5=9ad490433e93546c8f3bd5a23792f5d1
  56. 56
    Yang, W., Lee, J. Y., and Nowotny, M. (2006) Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity Mol. Cell 22, 5– 13
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    56
    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.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFWiurg%253D&md5=71303ca14fea285a44031d5c2eb5b1d3
  57. 57
    Yuan, B., You, C., Andersen, N., Jiang, Y., Moriya, M., O’Connor, T. R., and Wang, Y. (2011) The roles of DNA polymerases κ and ι in the error-free bypass of N2-carboxyalkyl-2′-deoxyguanosine lesions in mammalian cells J. Biol. Chem. 286, 17503– 17511
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    57
    The roles of DNA polymerases κ and ι in the error-free bypass of N2-carboxyalkyl-2'-deoxyguanosine lesions in mammalian cells
    Yuan, Bifeng; You, Changjun; Andersen, Nisana; Jiang, Yong; Moriya, Masaaki; O'Connor, Timothy R.; Wang, Yinsheng
    Journal of Biological Chemistry (2011), 286 (20), 17503-17511CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochem. studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N2-modified 2'-deoxyguanosine derivs. Herein, we used shuttle vector technol. and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) and N2-carboxymethyl-2'-deoxyguanosine (N2-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N2-CEdG lesions. In contrast, no mutation was found after the N2-CEdG- and N2-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N2-CEdG and N2-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtVGjtr4%253D&md5=f55b7b6d4a8bcb103e8b9d4c988c105f
  58. 58
    Kirouac, K. N. and Ling, H. (2011) Poli: Shining light on repair of oxidative DNA lesions and mutations Cell Cycle 10, 1520– 1521
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    58
    Polι: shining light on repair of oxidative DNA lesions and mutations
    Kirouac, Kevin N.; Ling, Hong
    Cell Cycle (2011), 10 (10), 1520-1521CODEN: CCEYAS; ISSN:1538-4101. (Landes Bioscience)
    A review on DNA polymerase ι and its role in repairing oxidative DNA lesions and mutations.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1ChtbnE&md5=0170b601e8847cd1878da5458289a821
  59. 59
    Petta, T. B., Nakajima, S., Zlatanou, A., Despras, E., Couve-Privat, S., Ishchenko, A., Sarasin, A., Yasui, A., and Kannouche, P. (2008) Human DNA polymerase iota protects cells against oxidative stress EMBO J. 27, 2883– 2895
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    59
    Human DNA polymerase iota protects cells against oxidative stress
    Petta, Tirzah Braz; Nakajima, Satoshi; Zlatanou, Anastasia; Despras, Emmanuelle; Couve-Privat, Sophie; Ishchenko, Alexander; Sarasin, Alain; Yasui, Akira; Kannouche, Patricia
    EMBO Journal (2008), 27 (21), 2883-2895CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)
    Human DNA polymerase iota (polι) is a unique member of the Y-family of specialized polymerases that displays a 5'deoxyribose phosphate (dRP) lyase activity. Although polι is well conserved in higher eukaryotes, its role in mammalian cells remains unclear. To investigate the biol. importance of polι in human cells, we generated fibroblasts stably downregulating polι (MRC5-polιKD) and examd. their response to several types of DNA-damaging agents. We show that cell lines downregulating polι exhibit hypersensitivity to DNA damage induced by hydrogen peroxide (H2O2) or menadione but not to ethylmethane sulfonate (EMS), UVC or UVA. Interestingly, exts. from cells downregulating polι show reduced base excision repair (BER) activity. In addn., polι binds to chromatin after treatment of cells with H2O2 and interacts with the BER factor XRCC1. Finally, green fluorescent protein-tagged polι accumulates at the sites of oxidative DNA damage in living cells. This recruitment is partially mediated by its dRP lyase domain and ubiquitin-binding domains. These data reveal a novel role of human polι in protecting cells from oxidative damage.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlalt7fE&md5=364752b1d8a33d4da7327f15c7f3b887
  60. 60
    Ren, J., Wen, L., Gao, X., Jin, C., Xue, Y., and Yao, X. (2009) DOG 1.0: illustrator of protein domain structures Cell Res. 19, 271– 273
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    60
    DOG 1.0: illustrator of protein domain structures
    Ren, Jian; Wen, Longping; Gao, Xinjiao; Jin, Changjiang; Xue, Yu; Yao, Xuebiao
    Cell Research (2009), 19 (2), 271-273CODEN: CREEB6; ISSN:1001-0602. (Nature Publishing Group)
    Software DOG (Domain Graph, version 1.0) to prep. publication-quality figures of protein domain structures is described. The DOG 1.0 software was written in JAVA 1.5 and packed with Install4j 4.0.8.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1ams7k%253D&md5=4f86a979e028e2c41b2275c215bd1052

Cited By

This article is cited by 9 publications.

  1. Jae-Kwon Kim, Mina Yeom, Jin-Kyung Hong, Insil Song, Young-Sam Lee, F. Peter Guengerich, and Jeong-Yun Choi . Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ. Chemical Research in Toxicology 2016, 29 (10) , 1741-1754. DOI: 10.1021/acs.chemrestox.6b00244.
  2. Mina Yeom, In-Hyeok Kim, Jae-Kwon Kim, KyeongJin Kang, Robert L. Eoff, F. Peter Guengerich, and Jeong-Yun Choi . Effects of Twelve Germline Missense Variations on DNA Lesion and G-Quadruplex Bypass Activities of Human DNA Polymerase REV1. Chemical Research in Toxicology 2016, 29 (3) , 367-379. DOI: 10.1021/acs.chemrestox.5b00513.
  3. Lin Li, Hui Tian, Chuanle Cheng, Shuhai Li, Lu Ming, Lei Qi. siRNA of DNA polymerase iota inhibits the migration and invasion in the lung cancer cell A549. Acta Biochimica et Biophysica Sinica 2018, 50 (9) , 929-933. DOI: 10.1093/abbs/gmy089.
  4. Yanwei Li, Lei Bao, Ruiming Zhang, Xiaowen Tang, Qingzhu Zhang, Wenxing Wang. Insights into the error bypass of 1-Nitropyrene DNA adduct by DNA polymerase ι: A QM/MM study. Chemical Physics Letters 2017, 686, 12-17. DOI: 10.1016/j.cplett.2017.08.017.
  5. Ekaterina G. Frank, Mary P. McLenigan, John P. McDonald, Donald Huston, Samantha Mead, Roger Woodgate. DNA polymerase ι: The long and the short of it!. DNA Repair 2017, 58, 47-51. DOI: 10.1016/j.dnarep.2017.08.006.
  6. Shiling Gu, Jingyuan Xiong, Ying Shi, Jia You, Zhenyu Zou, Xiaoying Liu, Huidong Zhang. Error-prone bypass of O 6 -methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1. DNA Repair 2017, 57, 35-44. DOI: 10.1016/j.dnarep.2017.06.021.
  7. Konstantin Y. Kazachenko, Nataliya A. Miropolskaya, Leonid V. Gening, Vyacheslav Z. Tarantul, Alena V. Makarova. Alternative splicing at exon 2 results in the loss of the catalytic activity of mouse DNA polymerase iota in vitro. DNA Repair 2017, 50, 77-82. DOI: 10.1016/j.dnarep.2017.01.001.
  8. Jeong-Yun Choi, Amritaj Patra, Mina Yeom, Young-Sam Lee, Qianqian Zhang, Martin Egli, F. Peter Guengerich. Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι. Journal of Biological Chemistry 2016, 291 (40) , 21063-21073. DOI: 10.1074/jbc.M116.748285.
  9. Yang Liu, Xiaolu Ma, Caixia Guo. Effects of the N terminus of mouse DNA polymerase κ on the bypass of a guanine-benzo[a]pyrenyl adduct. Journal of Biochemistry 2016, 159 (4) , 471-479. DOI: 10.1093/jb/mvv118.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Locations of genetic pol ι variations. Structure of human pol ι(26–445) (PDB code, 2FLL) bound to primer/template DNA and incoming nucleotide is shown using Pymol. Pol ι(26–445) is shown in cartoon ribbons, and the primer/template DNA and nucleotide are shown in gray sticks. The finger, palm, thumb, and PAD domains are colored yellow, red, green, and blue, respectively. The amino acid residues (in purple spheres) of genetic pol ι variants are indicated. The structural domains of pol ι are shown in the upper schematic diagram using DOG (version 2.0),(60) where positions of amino acids related to six studied variations are indicated.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. Extension of 32P-labeled primers opposite G, O6-MeG, N2-EtG, 8-oxoG, and an abasic site by human wild-type pol ι (1–445) and variants in the presence of Mg2+. The primer (24-mer) was annealed with each of the five different 36-mer templates containing an unmodified G, O6-MeG, N2-EtG, 8-oxoG, or abasic site placed at the 25th position from the 3′-end. Reactions were done in the presence of 5 mM MgCl2 for 15 min with DNA substrate (100 nM primer/template), all four dNTPs (50 μM each), and increasing concentrations of pol ι (0–50 nM) as indicated. The extension products were separated by denaturing gel electrophoresis and imaged using a phosphorimager.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. Extension of 32P-labeled primers opposite G, O6-MeG, N2-EtG, 8-oxoG, and an abasic site by human wild-type pol ι (1–445) and variants in the presence of Mn2+. Primer (24-mer) was annealed with each of the five different 36-mer templates containing an unmodified G, O6-MeG, N2-EtG, 8-oxoG, or abasic site placed at the 25th position from the 3′-end. Reactions were done in the presence of 0.15 mM MnCl2 for 15 min with DNA substrate (100 nM primer/template), all four dNTPs (50 μM each), and increasing concentrations of pol ι (0–10 nM) as indicated. The extension products were separated by denaturing gel electrophoresis and imaged using a phosphorimager.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 60 other publications.

    1. 1
      Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A., and Ellenberger, T. (2006) DNA Repair And Mutagenesis, 2nd ed., American Society for Microbiology Press, Washington, D.C.
      Google Scholar
      There is no corresponding record for this reference.
    2. 2
      Giglia-Mari, G., Zotter, A., and Vermeulen, W. (2011) DNA damage response Cold Spring Harb. Perspect. Biol. 3, a000745
      [ Crossref], Google Scholar
      There is no corresponding record for this reference.
    3. 3
      Hoeijmakers, J. H. (2009) DNA damage, aging, and cancer N. Engl. J. Med. 361, 1475– 1485
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      3
      DNA, damage, aging, and cancer
      Hoeijmakers, Jan H. J.
      New England Journal of Medicine (2009), 361 (15), 1475-1486CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
      A review. Topics discussed include: DNA damage and aging; the magnitude of DNA damage; the DNA repair toolbox; diseases of nucleotide-excision repair; progeria, aging and the survival response; DNA damage and DNA maintenance in cancer; compromised genome maintenance and cancer therapy.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1GiurbK&md5=95403c4568de46eed137cd5145487fcb
    4. 4
      Jackson, S. P. and Bartek, J. (2009) The DNA-damage response in human biology and disease Nature 461, 1071– 1078
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      4
      The DNA-damage response in human biology and disease
      Jackson, Stephen P.; Bartek, Jiri
      Nature (London, United Kingdom) (2009), 461 (7267), 1071-1078CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      A review. The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite const. assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biol. significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlGktLrK&md5=cca7cfec66852124695ebeb515f255a5
    5. 5
      Kennedy, D. O., Agrawal, M., Shen, J., Terry, M. B., Zhang, F. F., Senie, R. T., Motykiewicz, G., and Santella, R. M. (2005) DNA repair capacity of lymphoblastoid cell lines from sisters discordant for breast cancer J. Natl. Cancer Inst. 97, 127– 132
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      5
      DNA Repair Capacity of Lymphoblastoid Cell Lines From Sisters Discordant for Breast Cancer
      Kennedy, David O.; Agrawal, Meenakshi; Shen, Jing; Terry, Mary Beth; Zhang, Fang Fang; Senie, Ruby T.; Motykiewicz, Grazyna; Santella, Regina M.
      Journal of the National Cancer Institute (2005), 97 (2), 127-132CODEN: JNCIEQ; ISSN:0027-8874. (Oxford University Press)
      Background: Interindividual differences in DNA repair capacity may influence cancer risk. We tested whether the nucleotide excision repair pathway was deficient in breast cancer case patients by analyzing sister pairs. Methods: Cell lines derived from sisters discordant for breast cancer (137 families contg. 158 case patients and 154 control sisters) were obtained from the Metropolitan New York Registry of Breast Cancer Families. Lymphoblastoid cells were treated with benzo[a]pyrene diolepoxide (BPDE) for 30 min and were either harvested immediately or were washed and cultured in complete medium for 4 h to allow DNA repair. Immunofluorescence using a polyclonal anti-BPDE-DNA primary antibody was used to quantify BPDE-DNA adducts. Percent DNA repair capacity was calcd. from the difference between staining immediately after treatment minus that after 4 h of repair, divided by the initial damage and was categorized into quartiles based on control values. Odds ratios and 95% confidence intervals (CIs) were calcd. using conditional logistic regression models adjusted for age at blood donation, body mass index, and smoking. Statistical tests were two-sided. Results: Mean percent DNA repair capacity was lower in breast cancer case patients than in control subjects (difference = 8.6, 95% CI = 4.3 to 13.8, P = .001). Using the quartile with the highest percent DNA repair capacity as the referent group, adjusted odds ratios of breast cancer increased from 1.23 (95% CI = 0.57 to 2.65) to 2.38 (95% CI = 1.17 to 4.86) to 2.99 (95% CI = 1.45 to 6.17) (Ptrend = .002) as DNA repair capacity decreased. Conclusions: Deficient DNA repair capacity is assocd. with increased breast cancer risk.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmsV2jug%253D%253D&md5=bce11c64e855be84af763c4ae0935e1c
    6. 6
      Gorlova, O. Y., Weng, S. F., Zhang, Y., Amos, C. I., Spitz, M. R., and Wei, Q. (2008) DNA repair capacity and lung cancer risk in never smokers Cancer Epidemiol. Biomarkers Prev. 17, 1322– 1328
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      6
      DNA Repair Capacity and Lung Cancer Risk in Never Smokers
      Gorlova, Olga Y.; Weng, Shih-Feng; Zhang, Yiqun; Amos, Christopher I.; Spitz, Margaret R.; Wei, Qingyi
      Cancer Epidemiology, Biomarkers & Prevention (2008), 17 (6), 1322-1328CODEN: CEBPE4; ISSN:1055-9965. (American Association for Cancer Research)
      Besides secondhand smoke exposure, few other risk factors for lung cancer in lifetime never smokers have been identified. We present the ests. of lung cancer risk assocd. with suboptimal DNA repair capacity (DRC) measured by the host-cell reactivation assay in lifetime never smokers using data from 219 cases and 309 matched controls enrolled in a case-control study. Suboptimal DRC level (below the control median) conferred a significantly increased lung cancer risk in never smokers [odds ratio, 1.92; 95% confidence interval (95% CI), 1.3-2.9; P = 0.0024]. There was a 3.38-fold risk for individuals with DRC below the first quartile (95% CI, 1.8-6.3) compared with individuals with DRC above the third quartile. Secondhand smoke exposure in individuals with DRC below the control median was assocd. with a 3.81-fold risk of lung cancer (95% CI, 2.3-6.4). A 2.49-fold (95% CI, 1.1-5.6) risk was noted for the joint effects of lung cancer family history in first-degree relatives and suboptimal DRC. Relatives of probands (cases and controls) with lowest DRC (below the first quartile) were significantly more likely to be diagnosed with lung cancer (odds ratio, 2.69; 95% CI, 1.1-6.7) compared with relatives of probands with the most proficient DRC (above the third quartile). Relatives of probands with suboptimal (below the control median) vs. proficient DRC also had an earlier age at diagnosis with lung cancer, although the only statistically significant difference was in female relatives (55.4 vs. 67.7 years; P = 0.03). (Cancer Epidemiol Biomarkers Prev 2008;17(6):1322-8).
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsVKgtro%253D&md5=cc99ad382991c1e38add953e67bdafd7
    7. 7
      Wang, L. E., Gorlova, O. Y., Ying, J., Qiao, Y., Weng, S. F., Lee, A. T., Gregersen, P. K., Spitz, M. R., Amos, C. I., and Wei, Q. (2013) Genome-wide association study reveals novel genetic determinants of DNA repair capacity in lung cancer Cancer Res. 73, 256– 264
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      7
      Genome-Wide Association Study Reveals Novel Genetic Determinants of DNA Repair Capacity in Lung Cancer
      Wang, Li-E.; Gorlova, Olga Y.; Ying, Jun; Qiao, Yawei; Weng, Shih-Feng; Lee, Annette T.; Gregersen, Peter K.; Spitz, Margaret R.; Amos, Christopher I.; Wei, Qingyi
      Cancer Research (2013), 73 (1), 256-264CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      Suboptimal cellular DNA repair capacity (DRC) has been shown to be assocd. with enhanced cancer risk, but genetic variants affecting the DRC phenotype have not been comprehensively investigated. In this study, with the available DRC phenotype data, we analyzed correlations between the DRC phenotype and genotypes detected by the Illumina 317K platform in 1,774 individuals of European ancestry from a Texas lung cancer genome-wide assocn. study. The discovery phase was followed by a replication in an independent set of 1,374 cases and controls of European ancestry. We applied a generalized linear model with single nucleotide polymorphisms as predictors and DRC (a continuous variable) as the outcome. Covariates of age, sex, pack-years of smoking, DRC assay-related variables, and case-control status of the study participants were adjusted in the model. We validated that reduced DRC was assocd. with an increased risk of lung cancer in both independent datasets. Several suggestive loci that contributed to the DRC phenotype were defined in ERCC2/XPD, PHACTR2, and DUSP1. In summary, we detd. that DRC is an independent risk factor for lung cancer, and we defined several genetic loci contributing to DRC phenotype.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjs1Wrsw%253D%253D&md5=53a35941a58c108e64969ba0055668ab
    8. 8
      Alberg, A. J., Jorgensen, T. J., Ruczinski, I., Wheless, L., Shugart, Y. Y., Berthier-Schaad, Y., Kessing, B., Hoffman-Bolton, J., Helzlsouer, K. J., Kao, W. H., Francis, L., Alani, R. M., Smith, M. W., and Strickland, P. T. (2013) DNA repair gene variants in relation to overall cancer risk: a population-based study Carcinogenesis 34, 86– 92
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      8
      DNA repair gene variants in relation to overall cancer risk: a population-based study
      Alberg, Anthony J.; Jorgensen, Timothy J.; Ruczinski, Ingo; Wheless, Lee; Shugart, Yin Yao; Berthier-Schaad, Yvette; Kessing, Bailey; Hoffman-Bolton, Judith; Helzlsouer, Kathy J.; Kao, W. H. Linda; Francis, Lesley; Alani, Rhoda M.; Smith, Michael W.; Strickland, Paul T.
      Carcinogenesis (2013), 34 (1), 86-92CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)
      The hypothesis that germ-line polymorphisms in DNA repair genes influence cancer risk has previously been tested primarily on a cancer site-specific basis. The purpose of this study was to test the hypothesis that DNA repair gene allelic variants contribute to globally elevated cancer risk by measuring assocns. with risk of all cancers that occurred within a population-based cohort. In the CLUE II cohort study established in 1989 in Washington County, MD, this study was comprised of all 3619 cancer cases ascertained through 2007 compared with a sample of 2296 with no cancer. Assocns. were measured between 759 DNA repair gene single nucleotide polymorphisms (SNPs) and risk of all cancers. A SNP in O6-methylguanine-DNA methyltransferase, MGMT, (rs2296675) was significantly assocd. with overall cancer risk [per minor allele odds ratio (OR) 1.30, 95% confidence interval (CI) 1.19-1.43 and P-value: 4.1 × 10-8]. The assocn. between rs2296675 and cancer risk was stronger among those aged ≤54 years old than those who were ≥55 years at baseline (P-for-interaction = 0.021). OR were in the direction of increased risk for all 15 categories of malignancies studied (P < 0.0001), ranging from 1.22 (P = 0.42) for ovarian cancer to 2.01 (P = 0.008) for urinary tract cancers; the smallest P-value was for breast cancer (OR 1.45, P = 0.0002). The results indicate that the minor allele of MGMT SNP rs2296675, a common genetic marker with 37% carriers, was significantly assocd. with increased risk of cancer across multiple tissues. Replication is needed to more definitively det. the scientific and public health significance of this obsd. assocn.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktV2qsg%253D%253D&md5=641623112ece30025dc08da8fe51e2aa
    9. 9
      Choi, J.-Y., Eoff, R. E., and Guengerich, F. P. (2011) Bypass DNA polymerases, In Chemical Carcinogenesis (Penning, T. M., Ed.) pp 345– 373, Humana Press, New York.
      [ Crossref], Google Scholar
      There is no corresponding record for this reference.
    10. 10
      Choi, J.-Y., Angel, K. C., and Guengerich, F. P. (2006) Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase κ J. Biol. Chem. 281, 21062– 21072
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      10
      Translesion Synthesis across Bulky N2-Alkyl Guanine DNA Adducts by Human DNA Polymerase κ
      Choi, Jeong-Yun; Angel, Karen C.; Guengerich, F. Peter
      Journal of Biological Chemistry (2006), 281 (30), 21062-21072CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      DNA polymerase (pol) κ is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a no. of chem. modifications with human pol κ, and the conclusion has been presented, based on limited quant. data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol κ was studied with a series of eight N2-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH2(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, kcat/Km) were similar for insertion of dCTP opposite the lesions and for extension beyond the N2-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with kcat/Km values ∼10-3 less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic anal. showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (kp 5.9-10.3 s-1). The rapid bursts were abolished opposite BPG when α-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols η and ι; pol κ is the most resistant to N2-bulk and the most quant. efficient of these in catalyzing dCTP incorporation opposite bulky guanine N2-adducts, particularly the largest (N2-BPG).
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xnt1Gqurg%253D&md5=fe7b66fcfab81d10bfea755750f9a674
    11. 11
      Choi, J.-Y. and Guengerich, F. P. (2005) Adduct size limits efficient and error-free bypass across bulky N2-guanine DNA lesions by human DNA polymerase η J. Mol. Biol. 352, 72– 90
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      11
      Adduct Size Limits Efficient and Error-free Bypass Across Bulky N 2-Guanine DNA Lesions by Human DNA Polymerase η
      Choi, Jeong-Yun; Guengerich, F. Peter
      Journal of Molecular Biology (2005), 352 (1), 72-90CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
      The N2 position of guanine (G) is one of the major sites for DNA modification by various carcinogens. Eight oligonucleotides with varying adduct bulk at guanine N2 were analyzed for catalytic efficiency and fidelity with human DNA polymerase (pol) η, which is involved in translesion synthesis (TLS). Pol η effectively bypassed N2-methyl(Me)G, N2-ethyl(Et)G, N2-isobutyl(Ib)G, N 2-benzyl(Bz)G, and N2-CH2(2-naphthyl)G but was severely blocked at N 2-CH2(9-anthracenyl)G (N2-AnthG) and N2-CH2(6-benzo[a]pyrenyl)G (N2-BPG). Steady-state kinetic anal. showed proportional decreases of kcat/Km in dCTP insertion opposite N2-AnthG and N2-BPG (73 and 320-fold) and also kcat/Km in next-base extension from a C paired with each adduct (15 and 51-fold relative to G). Frequencies of dATP misinsertion and extension beyond mispairs were also proportionally increased (70 and 450-fold; 12 and 44-fold) with N2-AnthG and N2-BPG, indicating the effect of adduct bulk on blocking and misincorporation in TLS by pol η. N2-AnthG and N2-BPG also greatly decreased the pre-steady-state kinetic burst rate (25 and 125-fold) compared to unmodified G. N2-AnthG decreased dCTP binding affinity (2.6-fold) and increased DNA substrate binding affinity. These results and the small kinetic thio effects (Sp-dCTPαS) suggest that the early steps, possibly conformational change, are interfered with by the bulky adducts. In contrast, human pol δ bypassed adducts effectively up to N2-EtG but was strongly blocked by N2-IbG and larger adducts. We conclude that TLS DNA polymerases may be required for the efficient bypass of pol δ-blocking N2-G adducts bulkier than N2-EtG in human cells, and the bulk size can be a major factor for efficient and error-free bypass at these adducts by TLS DNA polymerases.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpsVWltrk%253D&md5=e3a72f9f9476440f6c5470b715653f4c
    12. 12
      Choi, J.-Y. and Guengerich, F. P. (2006) Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι J. Biol. Chem. 281, 12315– 12324
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      12
      Kinetic Evidence for Inefficient and Error-prone Bypass across Bulky N2-Guanine DNA Adducts by Human DNA Polymerase ι
      Choi, Jeong-Yun; Guengerich, F. Peter
      Journal of Biological Chemistry (2006), 281 (18), 12315-12324CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      DNA polymerase (pol) ι has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol ι. Pol ι effectively bypassed N2-Me (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic anal. showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). DTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (Sp-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chem. step is largely interfered with by adducts. Severe inhibition of polymn. opposite N2,N2-diMeG compared with N2-EtG by pol η but not by pol ι is consistent with Hoogsteen base pairing by pol ι. Thus, polymn. by pol ι is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol ι may play a limited role in translesion synthesis on bulky N2-G adducts in cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFKksrk%253D&md5=b0289a6d2fb0c6770ea42735361eadb0
    13. 13
      Choi, J.-Y. and Guengerich, F. P. (2008) Kinetic analysis of translesion synthesis opposite bulky N2- and O6-alkylguanine DNA adducts by human DNA polymerase REV1 J. Biol. Chem. 283, 23645– 23655
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      13
      Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1
      Choi, Jeong-Yun; Guengerich, F. Peter
      Journal of Biological Chemistry (2008), 283 (35), 23645-23655CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addn. to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N2-alkylG adducts (in oligonucleotides) ranging in size from Me to CH2(6-benzo[a]pyrenyl) (BP) and O6-alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O6-PobG, which caused almost complete blockage. Steady-state kinetic parameters (kcat/Km) were similar for insertion of dCTP opposite G and N2-G adducts but were severely reduced opposite the O6-G adducts. REV1 showed apparent pre-steady-state burst kinetics for dCTP incorporation only opposite N2-BPG and little, if any, opposite G, N2-benzyl (Bz)G, or O6-BzG. The maximal polymn. rate (kpol 0.9 s-1) opposite N2-BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N2-BPG-adducted DNA 3-fold more tightly than unmodified G-contg. DNA. These results and the lack of an elemental effect ((Sp)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N2-BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N2-adduct at G but not to O6-adducts.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVWktr7E&md5=66e2872a86dced5324948771bdfd6f96
    14. 14
      Nair, D. T., Johnson, R. E., Prakash, S., Prakash, L., and Aggarwal, A. K. (2004) Replication by human DNA polymerase-iota occurs by Hoogsteen base-pairing Nature 430, 377– 380
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      14
      Replication by human DNA polymerase-ι occurs by Hoogsteen base-pairing
      Nair, Deepak T.; Johnson, Robert E.; Prakash, Satya; Prakash, Louise; Aggarwal, Aneel K.
      Nature (London, United Kingdom) (2004), 430 (6997), 377-380CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Almost all DNA polymerases show a strong preference for incorporating the nucleotide that forms the correct Watson-Crick base pair with the template base. In addn., the catalytic efficiencies with which any given polymerase forms the four possible correct base pairs are roughly the same. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, is an exception to these rules. hPolι incorporates the correct nucleotide opposite a template adenine with a several hundred to several thousand fold greater efficiency than it incorporates the correct nucleotide opposite a template thymine, whereas its efficiency for correct nucleotide incorporation opposite a template guanine or cytosine is intermediate between these two extremes. Here the authors present the crystal structure of hPolι bound to a template primer and an incoming nucleotide. The structure reveals a polymerase that is 'specialized' for Hoogsteen base-pairing, whereby the templating base is driven to the syn conformation. Hoogsteen base-pairing offers a basis for the varied efficiencies and fidelities of hPolι opposite different template bases, and it provides an elegant mechanism for promoting replication through minor-groove purine adducts that interfere with replication.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXls1eqs7s%253D&md5=dc936aea2d3b57abf2726a820100f8eb
    15. 15
      Choi, J.-Y., Lim, S., Eoff, R. L., and Guengerich, F. P. (2009) Kinetic analysis of base-pairing preference for nucleotide incorporation opposite template pyrimidines by human DNA polymerase ι J. Mol. Biol. 389, 264– 274
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      15
      Kinetic Analysis of Base-Pairing Preference for Nucleotide Incorporation Opposite Template Pyrimidines by Human DNA Polymerase ι
      Choi, Jeong-Yun; Lim, Seonhee; Eoff, Robert L.; Guengerich, F. Peter
      Journal of Molecular Biology (2009), 389 (2), 264-274CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      DNA polymerase (pol) ι, a member of the mammalian Y-family of DNA polymerases involved in translesion DNA synthesis, has been previously suggested to peculiarly utilize Hoogsteen base pairing for DNA synthesis opposite template purines, unlike pols η and κ, which utilize Watson-Crick (W-C) base pairing. To investigate the possible roles of Hoogsteen, W-C, and wobble base-pairing modes in the selection of nucleotides opposite template pyrimidines by human pol ι, we carried out kinetic analyses of incorporation of modified purine nucleoside triphosphates including 7-deazapurines, inosine, 2-aminopurine, 2,6-diaminopurine, and 6-chloropurine, which affect H-bonding in base-pair formation opposite template pyrimidines. Carbon substitution at the N7 atom of purine nucleoside triphosphates, which disrupts Hoogsteen base pairing, only slightly inhibited DNA synthesis opposite template pyrimidines by pol ι, which was not substantially different from human pols η and κ. Opposite template T, only the relative wobble stabilities (inferred from the potential nos. of H-bonding, steric, and electrostatic interactions but not measured) of base pairs were pos. correlated to the relative efficiencies of nucleotide incorporation by pol ι but not the relative W-C or Hoogsteen stabilities, unlike pols η and κ. In contrast, opposite C, only the relative W-C stabilities of base pairs were pos. correlated to the relative efficiencies of nucleotide incorporation by pol ι, as with pols η and κ. These results suggest that pol ι might not indispensably require Hoogsteen base pairing for DNA synthesis opposite pyrimidines but rather might prefer wobble base pairing in the selection of nucleotides opposite T and W-C base pairing opposite C.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtVSqsLw%253D&md5=3eb7a5d578036c7548000737d62681da
    16. 16
      Kirouac, K. N. and Ling, H. (2009) Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase iota EMBO J. 28, 1644– 1654
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      16
      Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase ι
      Kirouac, Kevin N.; Ling, Hong
      EMBO Journal (2009), 28 (11), 1644-1654CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)
      Human DNA polymerase ι (polι) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of polι complexed with DNA contg. a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by polι. The template thymine remains in an anti conformation irresp. of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template 'U-turn' is stabilized by polι and the Me group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping expts. indicate that the finger domain is responsible for polι's high error rates on pyrimidines and dets. the incorporation specificity.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlvValsbw%253D&md5=f5526a9c72a7abafe604f81e13f7b309
    17. 17
      Choi, J.-Y., Chowdhury, G., Zang, H., Angel, K. C., Vu, C. C., Peterson, L. A., and Guengerich, F. P. (2006) Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases J. Biol. Chem. 281, 38244– 38256
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      17
      Translesion Synthesis across O6-Alkylguanine DNA Adducts by Recombinant Human DNA Polymerases
      Choi, Jeong-Yun; Chowdhury, Goutam; Zang, Hong; Angel, Karen C.; Vu, Choua C.; Peterson, Lisa A.; Guengerich, F. Peter
      Journal of Biological Chemistry (2006), 281 (50), 38244-38256CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O6-Me and -benzyl (Bz) G. Recombinant human polymerase (pol) δ also copied past these two lesions but was totally blocked by O6-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols ι and κ produced mainly only 1-base incorporation opposite O6-MeG and O6-BzG and had very low activity in copying O6-PobG. Human pol η copied past all three adducts. Steady-state kinetic anal. showed similar efficiencies of insertion opposite the O6-alkylG adducts for dCTP and dTTP with pol η and κ; pol ι showed a strong preference for dTTP. pol η, ι, and κ showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O6-MeG but little, if any, for O6-BzG or O6-PobG. Anal. of the pol η O6-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry anal. of all of the pol η primer extension products indicated multiple components, mainly with C or T inserted opposite O6-alkylG but with no deletions in the cases of O6-MeG and O6-PobG. With pol η and O6-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O6-BzG). The results with pol η may be relevant to some mutations previously reported with O6-alkylG adducts in mammalian cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht12nu7bE&md5=a745f9c97b4ccce6a6c57fab972500a0
    18. 18
      Vaisman, A., Frank, E. G., Iwai, S., Ohashi, E., Ohmori, H., Hanaoka, F., and Woodgate, R. (2003) Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase iota DNA Repair 2, 991– 1006
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      18
      Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase ι
      Vaisman, Alexandra; Frank, Ekaterina G.; Iwai, Shigenori; Ohashi, Eiji; Ohmori, Haruo; Hanaoka, Fumio; Woodgate, Roger
      DNA Repair (2003), 2 (9), 991-1006CODEN: DRNEAR; ISSN:1568-7864. (Elsevier Science B.V.)
      Humans possess four Y-family polymerases: pols η, ι, κ and the Rev1 protein. The pivotal role that polη plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding polη), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols ι, κ and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of polι to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that polι can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, polι efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of ∼102 over the incorporation of incorrect nucleotides, while incorporation opposite the 5'T is highly mutagenic. Polκ has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions of polι and polκ result in a significant increase in bypass of T-T CPDs when compared to polι alone. Our data suggest that under certain conditions and sequence contexts, polι can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochem. activities may, therefore, be of biol. significance esp. in XP-V cells lacking the primary T-T CPD bypassing enzyme, polη.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXntVGhtL0%253D&md5=8f000b050e2d676e6f9f91ab26cc9348
    19. 19
      Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F. P. (2010) Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases alpha, delta, eta, iota, kappa, and REV1 J. Mol. Biol. 404, 34– 44
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      19
      Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases α, δ, η, ι, κ, and REV1
      Choi, Jeong-Yun; Lim, Seonhee; Kim, Eun-Jin; Jo, Ara; Guengerich, F. Peter
      Journal of Molecular Biology (2010), 404 (1), 34-44CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides contg. a synthetic abasic lesion (THF analog) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, resp.) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlKhtb%252FP&md5=e5890e1ff1bc45ca9bf085982bf7e43c
    20. 20
      Frank, E. G. and Woodgate, R. (2007) Increased catalytic activity and altered fidelity of human DNA polymerase ι in the presence of manganese J. Biol. Chem. 282, 24689– 24696
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      20
      Increased Catalytic Activity and Altered Fidelity of Human DNA Polymerase ι in the Presence of Manganese
      Frank, Ekaterina G.; Woodgate, Roger
      Journal of Biological Chemistry (2007), 282 (34), 24689-24696CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg2+ is the physiol. cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol λ, may preferentially utilize Mn2+ in vitro. Here we report on the effects of Mn2+ and Mg2+ on the enzymic properties of human DNA polymerase ι (pol ι). Pol ι exhibited the greatest activity in the presence of low levels of Mn2+ (0.05-0.25 mM). Peak activity in the presence of Mg2+ was obsd. in the range of 0.1-0.5 mM and was significantly reduced at concns. >2 mM. Steady-state kinetic analyses revealed that Mn2+ increases the catalytic activity of pol ι by ∼30-60,000-fold through a dramatic decrease in the Km value for nucleotide incorporation. Interestingly, whereas pol ι preferentially misinserts G opposite T by a factor of ∼1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mM Mg2+, resp., the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mM Mn2+. Low levels of Mn2+ also dramatically increased the ability of pol ι to traverse a variety of DNA lesions in vitro. Titrn. expts. revealed a strong preference of pol ι for Mn2+ even when Mg2+ is present in a >10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol ι in vivo may actually be Mn2+ rather than Mg2+, as tacitly assumed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpt1SlsLk%253D&md5=f29f919801291f0902131c752edf5bb3
    21. 21
      Dumstorf, C. A., Clark, A. B., Lin, Q., Kissling, G. E., Yuan, T., Kucherlapati, R., McGregor, W. G., and Kunkel, T. A. (2006) Participation of mouse DNA polymerase iota in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer Proc. Natl. Acad. Sci. U.S.A. 103, 18083– 18088
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      21
      Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer
      Dumstorf, Chad A.; Clark, Alan B.; Lin, Qingcong; Kissling, Grace E.; Yuan, Tao; Kucherlapati, Raju; McGregor, W. Glenn; Kunkel, Thomas A.
      Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (48), 18083-18088CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      DNA polymerase ι (pol ι) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol ι performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol η, pol ι, or both. A deficiency in mouse DNA polymerase η (pol η) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol ι, indicating that pol ι participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was obsd. in wild-type cells that was diminished in pol η- and pol ι-deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asym. manner. To det. whether pol L status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol η, pol ι, or both. Although pol ι deficiency alone had no effect, UV-induced skin tumors in pol η-deficient mice developed 4 wk earlier in mice concomitantly deficient in pol ι. Collectively, these data reveal functions for pol ι in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlWjt7%252FK&md5=3d751997055f965126d667ce1a0fd573
    22. 22
      Ohkumo, T., Kondo, Y., Yokoi, M., Tsukamoto, T., Yamada, A., Sugimoto, T., Kanao, R., Higashi, Y., Kondoh, H., Tatematsu, M., Masutani, C., and Hanaoka, F. (2006) UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota Mol. Cell. Biol. 26, 7696– 7706
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      22
      UV-B radiation induces epithelial tumors in mice lacking DNA polymerase η and mesenchymal tumors in mice deficient for DNA polymerase
      Ohkumo, Tsuyoshi; Kondo, Yuji; Yokoi, Masayuki; Tsukamoto, Tetsuya; Yamada, Ayumi; Sugimoto, Taiki; Kanao, Rie; Higashi, Yujiro; Kondoh, Hisato; Tatematsu, Masae; Masutani, Chikahide; Hanaoka, Fumio
      Molecular and Cellular Biology (2006), 26 (20), 7696-7706CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)
      DNA polymerase η (Pol η) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol η is involved in the somatic hypermutation of Ig genes, but the cancer predisposition of Polh-/- mice has not been examd. until very recently. Another translesion synthesis polymerase, Pol ι, a Pol η paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol ι is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol ι deficiency does not influence the UV sensitivity of mouse fibroblasts irresp. of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histol. diagnosis after chronic treatment with UV light reveals that Pol ι deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not obsd. in Polh-/- mice. These results suggest the involvement of the Pol η and Pol ι proteins in UV-induced skin carcinogenesis.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFCgurrF&md5=2199a5c9d1becb57bcbcb69b42d4d514
    23. 23
      Iguchi, M., Osanai, M., Hayashi, Y., Koentgen, F., and Lee, G. H. (2014) The error-prone DNA polymerase iota provides quantitative resistance to lung tumorigenesis and mutagenesis in mice Oncogene 33, 3612– 3617
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      23
      The error-prone DNA polymerase ι provides quantitative resistance to lung tumorigenesis and mutagenesis in mice
      Iguchi, M.; Osanai, M.; Hayashi, Y.; Koentgen, F.; Lee, G.-H.
      Oncogene (2014), 33 (27), 3612-3617CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)
      Opposite undamaged nucleotide T, DNA polymerase ι (Polι) preferentially incorporates G rather than A, violating the Watson-Crick rule. Although the actual biol. role of Polι remains enigmatic, we have identified its coding gene as a candidate for pulmonary adenoma resistance 2 (Par2), a mouse quant. trait locus modulating chem. induced lung tumor susceptibility. Notably, the most tumor-sensitive Par2 allele possessed by the 129X1/SvJ mouse is assocd. with a loss-of-function mutation in Polι. To det. whether the nonfunctional Polι is responsible for the 129X1/SvJ-specific Par2 phenotype, we knocked out Polι in a C57BL/6J mouse carrying a less tumor-sensitive Par2 allele. Disruption of the C57BL/6J Polι conferred 129X1/SvJ-like sensitivity on the C57BL/6J Par2 locus and increased the in vivo mutation frequency in the lung, providing definitive proof that Polι causes the Par2 effect and inhibits tumorigenesis and mutagenesis, despite its extreme replication infidelity.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1ylsbbM&md5=b8fe6b0bba17659a2f15e6bbc137e025
    24. 24
      Yang, J., Chen, Z., Liu, Y., Hickey, R. J., and Malkas, L. H. (2004) Altered DNA polymerase iota expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis Cancer Res. 64, 5597– 5607
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      24
      Altered DNA polymerase ι expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis
      Yang, Jin; Chen, Zhiwen; Liu, Yang; Hickey, Robert J.; Malkas, Linda H.
      Cancer Research (2004), 64 (16), 5597-5607CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      The recently discovered human enzyme DNA polymerase ι (pol ι) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol ι in DNA lesion bypass, there is no in vivo evidence to confirm this action. It has been reported here that pol ι expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. Author also demonstrated that UV treatment of breast cancer cells addnl. increases pol ι expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol ι expression is an early event after UV-mediated DNA damage. That pol ι may play a role in the higher mutation frequencies obsd. in breast cancer cells was suggested when a redn. in mutation frequency was found after pol ι was immunodepleted from nuclear exts. of the cells. Anal. of the UV-induced mutation spectra revealed that >90% were point mutations. The anal. also demonstrated a decreased C → T nucleotide transition and an increased C → A transversion rate. Overall, the data strongly suggest that pol ι may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmslalurw%253D&md5=6e57151073b48272a3d2a3cbeea31bcf
    25. 25
      Zhou, J., Zhang, S., Xie, L., Liu, P., Xie, F., Wu, J., Cao, J., and Ding, W. Q. (2012) Overexpression of DNA polymerase iota (Poliota) in esophageal squamous cell carcinoma Cancer Sci. 103, 1574– 1579
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      25
      Overexpression of DNApolymerase iota (Polι) in esophageal squamous cell carcinoma
      Zhou, Jundong; Zhang, Shuyu; Xie, Ling; Liu, Pengfei; Xie, Fang; Wu, Jinchang; Cao, Jianping; Ding, Wei-Qun
      Cancer Science (2012), 103 (8), 1574-1579CODEN: CSACCM; ISSN:1349-7006. (Wiley-Blackwell)
      The present study investigated the transcriptional regulation of low-fidelity translesion DNA synthesis (TLS) polymerases in human esophageal carcinoma. Significantly higher mRNA expression of polymerase zeta (Polξ), RAD18,polymerase iota (Polι), and polymerase kappa (Polκ) was found in esophageal carcinomas. The increased expression of Polι in tumor samples was further confirmed by immunohistochem. The promoter of POLI that encodes Polι was found to be hypomethylated, although the overexpression of this gene was unlikely to be assocd. with methylation in tumors. We further identified Sp1 and Oct-1 binding sites present in the POLI promoter. We obsd. that the binding affinity of Sp1 to the POLI promoter was significantly increased in cancerous tissues and that Sp1 activated POLI gene transcription in cultured cell lines. The present study demonstrates overexpression of the TLS genes in esophageal carcinoma and identifies a key role for Sp1 in upregulating POLI gene expression. (Cancer Sci 2012; 103: 1574-1579).
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtV2isbfP&md5=2556a4bfa7f27c03fd1e6bd94761a598
    26. 26
      Albertella, M. R., Lau, A., and O’Connor, M. J. (2005) The overexpression of specialized DNA polymerases in cancer DNA Repair 4, 583– 593
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      26
      The overexpression of specialized DNA polymerases in cancer
      Albertella, Mark R.; Lau, Alan; O'Connor, Mark J.
      DNA Repair (2005), 4 (5), 583-593CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)
      Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on noncanonical templates, such as undamaged DNA or on noncognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, the authors examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases β, λ, ι and κ, the authors also investigate the expression of the replicative polymerases α, δ and ε. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase β (pol β) was overexpressed at both the mRNA and protein level in approx. one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols λ, and ι were also overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol β. In contrast, pol κ was rarely overexpressed in tumors but was commonly underexpressed in many samples. Downregulation of pol β expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialized DNA polymerases, and particularly pol β, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitize tumors to chemotherapy.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXivFyru7s%253D&md5=c63ba33c74a51ba6193f37c730b59cb3
    27. 27
      Yuan, F., Xu, Z., Yang, M., Wei, Q., Zhang, Y., Yu, J., Zhi, Y., Liu, Y., Chen, Z., and Yang, J. (2013) Overexpressed DNA polymerase iota regulated by JNK/c-Jun contributes to hypermutagenesis in bladder cancer PLoS One 8, e69317
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      27
      Overexpressed DNA polymerase iota regulated by JNK/c-jun contributes to hypermutagenesis in bladder cancer
      Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin
      PLoS One (2013), 8 (7), e69317CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Human DNA polymerase iota (pol i) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol i may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol i and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation expt., EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol i in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol i, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, assocd. with elevated c-Jun activity, the overexpression of pol i is pos. correlated with the clin. tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol i-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol i by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol i or c-Jun in mutagenesis.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1GisLfK&md5=05f2e0ad776f497c2859b1eb4d20ac3e
    28. 28
      Luedeke, M., Linnert, C. M., Hofer, M. D., Surowy, H. M., Rinckleb, A. E., Hoegel, J., Kuefer, R., Rubin, M. A., Vogel, W., and Maier, C. (2009) Predisposition for TMPRSS2-ERG fusion in prostate cancer by variants in DNA repair genes Cancer Epidemiol. Biomarkers Prev. 18, 3030– 3035
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      28
      Predisposition for TMPRSS2-ERG Fusion in Prostate Cancer by Variants in DNA Repair Genes
      Luedeke, Manuel; Linnert, Carmen M.; Hofer, Matthias D.; Surowy, Harald M.; Rinckleb, Antje E.; Hoegel, Josef; Kuefer, Rainer; Rubin, Mark A.; Vogel, Walther; Maier, Christiane
      Cancer Epidemiology, Biomarkers & Prevention (2009), 18 (11), 3030-3035CODEN: CEBPE4; ISSN:1055-9965. (American Association for Cancer Research)
      The somatic fusion of TMPRSS2 to ETS oncogenes is a common event in prostate cancer (PCa). We hypothesized that defects in DNA repair may lead to an increase of chromosomal rearrangements and thus to the occurrence of ETS oncogene fusion. We have previously conducted a genome-wide linkage anal. in TMPRSS2-ERG fusion-pos. PCa families, revealing potential susceptibility loci on chromosomes 5q14, 9q21, 10q26, 11q24, 12q15, 13q12, 18q, and Xq27. In the present study, nine candidate genes from these regions were selected from the context of DNA repair and screened for mutations in TMPRSS2-ERG fusion-pos. families. Thirteen nonsynonymous variants, 5 of which had a minor allele frequency of <0.05, were genotyped in 210 familial cases, 47 of which with a known TMPRSS2-ERG status, 329 sporadic cases, and 512 controls. Significant assocn. of TMPRSS2-ERG fusion-pos. PCa was found with rare variants in the genes for POLI [variant F532S: P = 0.0011; odds ratios (OR), 4.62; 95% confidence interval (95% CI), 1.84-11.56] and ESCO1 (variant N191S: P = 0.0034; OR, 4.27; 95% CI, 1.62-11.28). Addnl. findings, regardless of TMPRSS2-ERG status, were the overrepresentation of a rare BRCA2 variant (V2728I: P = 0.03; OR, 6.16; 95% CI, 1.19-32.00) in familial PCa and of a common allele of RMI1 (variant N455S: P = 0.02; OR, 1.33; 95% CI, 1.04-1.70) in unselected PCa cases. The DNA repair genes POLI and ESCO1 are proposed as susceptibility genes for TMPRSS2-ERG fusion-pos. PCa that warrant further investigation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtl2kt7%252FE&md5=5086c48e52382132b2f59b86bec6464a
    29. 29
      Sakiyama, T., Kohno, T., Mimaki, S., Ohta, T., Yanagitani, N., Sobue, T., Kunitoh, H., Saito, R., Shimizu, K., Hirama, C., Kimura, J., Maeno, G., Hirose, H., Eguchi, T., Saito, D., Ohki, M., and Yokota, J. (2005) Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk Int. J. Cancer 114, 730– 737
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      29
      Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk
      Sakiyama, Tokuki; Kohno, Takashi; Mimaki, Sachiyo; Ohta, Tsutomu; Yanagitani, Noriko; Sobue, Tomotaka; Kunitoh, Hideo; Saito, Ryusei; Shimizu, Kimiko; Hirama, Chie; Kimura, Junko; Maeno, Go; Hirose, Hiroshi; Eguchi, Takashi; Saito, Daizo; Ohki, Misao; Yokota, Jun
      International Journal of Cancer (2005), 114 (5), 730-737CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
      Single nucleotide polymorphisms (SNPs) were searched for in 36 genes involved in diverse DNA repair pathways, and 50 nonsynonymous (assocd. with amino acid changes) SNPs identified were assessed for assocns. with lung cancer risk by a case-control study consisting of 752 adenocarcinoma cases, 250 squamous cell carcinoma cases and 685 controls. An SNP, Arg72Pro, of the TP53 gene encoding a DNA damage response protein showed the strongest assocn. with squamous cell carcinoma risk (OR Pro/Pro vs. Arg/Arg = 2.2), while 2 other SNPs, Phe257Ser of the REV1 gene encoding a translesion DNA polymerase and Ile658Val of the LIG4 gene encoding a DNA double-strand break repair protein, also showed assocns. (OR Ser/Ser vs. Phe/Phe = 2.0 and OR Ile/Val vs. Ile/Ile = 0.4, resp.). An SNP, Thr706Ala, in the POLI gene encoding another translesion DNA polymerase was assocd. with adenocarcinoma and squamous cell carcinoma risk, particularly in individuals of ages <61 years (OR Ala/Ala + Ala/Thr vs. Thr/Thr = 1.5 and 2.4, resp.). POLI is the human counterpart of PolI, a strong candidate for the Par2 (pulmonary adenoma resistance 2) gene responsible for adenoma/adenocarcinoma susceptibility in mice. The present results suggest that these 4 SNPs function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXitl2quro%253D&md5=e9444dac3ccb43ae6c758429d4895da4
    30. 30
      Ng, P. C. and Henikoff, S. (2001) Predicting deleterious amino acid substitutions Genome Res. 11, 863– 874
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      30
      Predicting deleterious amino acid substitutions
      Ng, Pauline C.; Henikoff, Steven
      Genome Research (2001), 11 (5), 863-874CODEN: GEREFS; ISSN:1088-9051. (Cold Spring Harbor Laboratory Press)
      Many missense substitutions are identified in single nucleotide polymorphism (SNP) data and large-scale random mutagenesis projects. Each amino acid substitution potentially affects protein function. We have constructed a tool that uses sequence homol. to predict whether a substitution affects protein function. SIFT, which sorts intolerant from tolerant substitutions, classifies substitutions as tolerated or deleterious. A higher proportion of substitutions predicted to be deleterious by SIFT gives an affected phenotype than substitutions predicted to be deleterious by substitution scoring matrixes in three test cases. Using SIFT before mutagenesis studies could reduce the no. of functional assays required and yield a higher proportion of affected phenotypes. SIFT may be used to identify plausible disease candidates among the SNPs that cause missense substitutions.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjs1Wmu7w%253D&md5=529639c792d6286a23b3f1b6e41f1147
    31. 31
      Ramensky, V., Bork, P., and Sunyaev, S. (2002) Human non-synonymous SNPs: server and survey Nucleic Acids Res. 30, 3894– 3900
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      31
      Human non-synonymous SNPs: server and survey
      Ramensky, Vasily; Bork, Peer; Sunyaev, Shamil
      Nucleic Acids Research (2002), 30 (17), 3894-3900CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Human single nucleotide polymorphisms (SNPs) represent the most frequent type of human population DNA variation. One of the main goals of SNP research is to understand the genetics of the human phenotype variation and esp. the genetic basis of human complex diseases. Non-synonymous coding SNPs (nsSNPs) comprise a group of SNPs that, together with SNPs in regulatory regions, are believed to have the highest impact on phenotype. Here we present a World Wide Web server to predict the effect of an nsSNP on protein structure and function. The prediction method enabled anal. of the publicly available SNP database HGVbase, which gave rise to a dataset of nsSNPs with predicted functionality. The dataset was further used to compare the effect of various structural and functional characteristics of amino acid substitutions responsible for phenotypic display of nsSNPs. We also studied the dependence of selective pressure on the structural and functional properties of proteins. We found that in our dataset the selection pressure against deleterious SNPs depends on the mol. function of the protein, although it is insensitive to several other protein features considered. The strongest selective pressure was detected for proteins involved in transcription regulation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xms1Klt7k%253D&md5=edc88439061ba8dc0d48665b707dc944
    32. 32
      Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S., and Sunyaev, S. R. (2010) A method and server for predicting damaging missense mutations Nat. Methods 7, 248– 249
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      32
      A method and server for predicting damaging missense mutations
      Adzhubei, Ivan A.; Schmidt, Steffen; Peshkin, Leonid; Ramensky, Vasily E.; Gerasimova, Anna; Bork, Peer; Kondrashov, Alexey S.; Sunyaev, Shamil R.
      Nature Methods (2010), 7 (4), 248-249CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      A method and corresponding software tool, PolyPhen-2, for predicting damaging effects of missense mutations is presented. HumDiv-trained PolyPhen-2 should be used to evaluate rare alleles at loci potentially involved in complex phenotypes, for dense mapping of regions identified by genome-wide assocn. studies and for anal. of natural selection from sequence data, in which even mildly deleterious alleles must be treated as damaging.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjvFKqu78%253D&md5=aafc33a76fbca3184c501aae083f6f75
    33. 33
      Choi, J.-Y. and Guengerich, F. P. (2004) Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase J. Biol. Chem. 279, 19217– 19229
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      33
      Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase
      Choi, Jeong-Yun; Guengerich, F. Peter
      Journal of Biological Chemistry (2004), 279 (18), 19217-19229CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease- (T7-). RT and T7- effectively bypassed N2-methyl(Me)G and readily extended primers but were strongly blocked by N2-ethyl(Et)G, N2-isobutylG, N2-benzylG, and N2-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7- showed a decrease of 103 in kcat/Km for dCTP incorporation opposite N2-MeG and a further large decrease opposite N2-EtG. Misincorporation frequency was increased 102-103-fold by a Me group and another ∼103-fold by an Et group. DATP was preferentially incorporated opposite bulky N2-alkylG mols. N2-MeG attenuated the pre-steady-state kinetic bursts with RT and T7-, and N2-EtG eliminated the bursts. Large elemental effects with thio-dCTP(αS) were obsd. with N2-EtG (6- and 72-fold decreases) but were much less with N2-MeG, indicating that the N2-Et group may affect the rate of the chem. step (phosphodiester bond formation). Similar values of Kd(dCTP) and Kd(DNA) and koff rates of DNA substrates from RT and T7- indicate that ground-state binding and dissocn. rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chem. step, demonstrating the role of bulk in DNA lesions.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjsVKmtbc%253D&md5=3b433b8a493242dd1bda212dce1a1808
    34. 34
      Boosalis, M. S., Petruska, J., and Goodman, M. F. (1987) DNA polymerase insertion fidelity: gel assay for site-specific kinetics J. Biol. Chem. 262, 14689– 14696
      [ PubMed], [ CAS], Google Scholar
      34
      DNA polymerase insertion fidelity. Gel assay for site-specific kinetics
      Boosalis, Michael S.; Petruska, John; Goodman, Myron F.
      Journal of Biological Chemistry (1987), 262 (30), 14689-96CODEN: JBCHA3; ISSN:0021-9258.
      A quant. assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase-α holoenzyme complex and M13 primer-template DNA. The Km and Vmax values are reported for correct insertion of adenine (A) and misinsertion of guanine (G), cytosine (C), and thymine (T) opposite a single template T site. The misinsertion frequencies were 2 × 10-4 for G-T and 5 × 10-5 for both C-T and T-T relative to normal A-T base pairs. The dissocn. const. of the polymerase-DNA-dNTP (deoxynucleotide triphosphate) complex, as measured by Km, played a dominant role in detg. the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 μM), the Km was 1100-fold greater for misinsertion of G opposite T (4.2 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 mM). These Km differences indicated that in the enzyme binding site the stability of A-T base pairs was 4.3 kcal/mol greater than T-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax were relatively small. There was only a 4-fold redn. in Vmax for insertion of G opposite T and an 8-fold redn. for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase-α appeared to be governed primarily by a Km discrimination mechanism.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlais7w%253D&md5=5870530d34c8ab0afe913d4251ab0999
    35. 35
      Song, I., Kim, E. J., Kim, I. H., Park, E. M., Lee, K. E., Shin, J. H., Guengerich, F. P., and Choi, J.-Y. (2014) Biochemical characterization of eight genetic variants of human DNA polymerase κ involved in error-free bypass across bulky N2-guanyl DNA adducts Chem. Res. Toxicol. 27, 919– 930
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      35
      Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky N2-Guanyl DNA Adducts
      Song, Insil; Kim, Eun-Jin; Kim, In-Hyeok; Park, Eun-Mi; Lee, Kyung Eun; Shin, Joo-Ho; Guengerich, F. Peter; Choi, Jeong-Yun
      Chemical Research in Toxicology (2014), 27 (5), 919-930CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky N2-guanyl DNA lesions induced by many carcinogens such as polycyclic arom. hydrocarbons. We analyzed the biochem. properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1-526) protein and the DNA template contg. an N2-CH2(9-anthracenyl)G (N2-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky N2-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtVWgsrg%253D&md5=3b892cadfb539736dddfdb191477c899
    36. 36
      Ketkar, A., Zafar, M. K., Maddukuri, L., Yamanaka, K., Banerjee, S., Egli, M., Choi, J.-Y., Lloyd, R. S., and Eoff, R. L. (2013) Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity Chem. Res. Toxicol. 26, 221– 232
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      36
      Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity
      Ketkar, Amit; Zafar, Maroof K.; Maddukuri, Leena; Yamanaka, Kinrin; Banerjee, Surajit; Egli, Martin; Choi, Jeong-Yun; Lloyd, R. Stephen; Eoff, Robert L.
      Chemical Research in Toxicology (2013), 26 (2), 221-232CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compds. identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC50 values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmvVGntg%253D%253D&md5=6bacbda93b04ec3e2bb56ba5086fb0cf
    37. 37
      Pence, M. G., Choi, J.-Y., Egli, M., and Guengerich, F. P. (2010) Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase ι J. Biol. Chem. 285, 40666– 40672
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      37
      Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι
      Pence, Matthew G.; Choi, Jeong-Yun; Egli, Martin; Guengerich, F. Peter
      Journal of Biological Chemistry (2010), 285 (52), 40666-40672CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      O6-Methylguanine (O6-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiol. ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was detd. for nucleoside triphosphate incorporation opposite O6-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this lab. using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O6-methylG. Misincorporation of dTTP opposite O6-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O6-methylG as the template base and incoming dCTP or dTTP were solved and showed that O6-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O6-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O6-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O6 atoms of O6-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O6-methylG by human pol ι, in contrast to the mispairing modes obsd. previously for O6-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFynsL3F&md5=e785844ad7d4e862280aee325acc4d69
    38. 38
      Kirouac, K. N. and Ling, H. (2011) Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota Proc. Natl. Acad. Sci. U.S.A. 108, 3210– 3215
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      38
      Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota
      Kirouac, Kevin N.; Ling, Hong
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (8), 3210-3215, S3210/1-S3210/3CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biol. role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA contg. an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equil. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the addnl. O8 atom of the lesion base, which influences nucleotide selection, enzymic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXislyjur0%253D&md5=b657fcab4ddbb7f765f3d1911e58b17d
    39. 39
      Manolio, T. A., Collins, F. S., Cox, N. J., Goldstein, D. B., Hindorff, L. A., Hunter, D. J., McCarthy, M. I., Ramos, E. M., Cardon, L. R., Chakravarti, A., Cho, J. H., Guttmacher, A. E., Kong, A., Kruglyak, L., Mardis, E., Rotimi, C. N., Slatkin, M., Valle, D., Whittemore, A. S., Boehnke, M., Clark, A. G., Eichler, E. E., Gibson, G., Haines, J. L., Mackay, T. F., McCarroll, S. A., and Visscher, P. M. (2009) Finding the missing heritability of complex diseases Nature 461, 747– 753
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      39
      Finding the missing heritability of complex diseases
      Manolio, Teri A.; Collins, Francis S.; Cox, Nancy J.; Goldstein, David B.; Hindorff, Lucia A.; Hunter, David J.; McCarthy, Mark I.; Ramos, Erin M.; Cardon, Lon R.; Chakravarti, Aravinda; Cho, Judy H.; Guttmacher, Alan E.; Kong, Augustine; Kruglyak, Leonid; Mardis, Elaine; Rotimi, Charles N.; Slatkin, Montgomery; Valle, David; Whittemore, Alice S.; Boehnke, Michael; Clark, Andrew G.; Eichler, Evan E.; Gibson, Greg; Haines, Jonathan L.; Mackay, Trudy F. C.; McCarroll, Steven A.; Visscher, Peter M.
      Nature (London, United Kingdom) (2009), 461 (7265), 747-753CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      A review. Genome-wide assocn. studies have identified hundreds of genetic variants assocd. with complex human diseases and traits, and have provided valuable insights into their genetic architecture. Most variants identified so far confer relatively small increments in risk, and explain only a small proportion of familial clustering, leading many to question how the remaining, missing heritability can be explained. Here we examine potential sources of missing heritability and propose research strategies, including and extending beyond current genome-wide assocn. approaches, to illuminate the genetics of complex diseases and enhance its potential to enable effective disease prevention or treatment.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1CisbrF&md5=dcdef1c2de0585e1a4d612589311aa19
    40. 40
      Nelson, M. R., Wegmann, D., Ehm, M. G., Kessner, D., St. Jean, P., Verzilli, C., Shen, J., Tang, Z., Bacanu, S. A., Fraser, D., Warren, L., Aponte, J., Zawistowski, M., Liu, X., Zhang, H., Zhang, Y., Li, J., Li, Y., Li, L., Woollard, P., Topp, S., Hall, M. D., Nangle, K., Wang, J., Abecasis, G., Cardon, L. R., Zollner, S., Whittaker, J. C., Chissoe, S. L., Novembre, J., and Mooser, V. (2012) An abundance of rare functional variants in 202 drug target genes sequenced in 14,002 people Science 337, 100– 104
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      40
      An Abundance of Rare Functional Variants in 202 Drug Target Genes Sequenced in 14,002 People
      Nelson, Matthew R.; Wegmann, Daniel; Ehm, Margaret G.; Kessner, Darren; St. Jean, Pamela; Verzilli, Claudio; Shen, Judong; Tang, Zhengzheng; Bacanu, Silviu-Alin; Fraser, Dana; Warren, Liling; Aponte, Jennifer; Zawistowski, Matthew; Liu, Xiao; Zhang, Hao; Zhang, Yong; Li, Jun; Li, Yun; Li, Li; Woollard, Peter; Topp, Simon; Hall, Matthew D.; Nangle, Keith; Wang, Jun; Abecasis, Goncalo; Cardon, Lon R.; Zoellner, Sebastian; Whittaker, John C.; Chissoe, Stephanie L.; Novembre, John; Mooser, Vincent
      Science (Washington, DC, United States) (2012), 337 (6090), 100-104CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Rare genetic variants contribute to complex disease risk; however, the abundance of rare variants in human populations remains unknown. We explored this spectrum of variation by sequencing 202 genes encoding drug targets in 14,002 individuals. We find rare variants are abundant (1 every 17 bases) and geog. localized, so that even with large sample sizes, rare variant catalogs will be largely incomplete. We used the obsd. patterns of variation to est. population growth parameters, the proportion of variants in a given frequency class that are putatively deleterious, and mutation rates for each gene. We conclude that because of rapid population growth and weak purifying selection, human populations harbor an abundance of rare variants, many of which are deleterious and have relevance to understanding disease risk.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpsFKltbg%253D&md5=b560cac006dd86b255383aec1469a4d3
    41. 41
      Zhu, Q., Ge, D., Maia, J. M., Zhu, M., Petrovski, S., Dickson, S. P., Heinzen, E. L., Shianna, K. V., and Goldstein, D. B. (2011) A genome-wide comparison of the functional properties of rare and common genetic variants in humans Am. J. Hum. Genet. 88, 458– 468
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      41
      A Genome-wide Comparison of the Functional Properties of Rare and Common Genetic Variants in Humans
      Zhu, Qian-Qn; Ge, Dong-Lang; Maia, Jessica M.; Zhu, Ming-Fu; Petrovski, Slave; Dickson, Samuel P.; Heinzen, Erin L.; Shianna, Kevin V.; Goldstein, David B.
      American Journal of Human Genetics (2011), 88 (4), 458-468CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
      One of the longest running debates in evolutionary biol. concerns the kind of genetic variation that is primarily responsible for phenotypic variation in species. Here, we address this question for humans specifically from the perspective of population allele frequency of variants across the complete genome, including both coding and noncoding regions. We establish simple criteria to assess the likelihood that variants are functional based on their genomic locations and then use whole-genome sequence data from 29 subjects of European origin to assess the relationship between the functional properties of variants and their population allele frequencies. We find that for all criteria used to assess the likelihood that a variant is functional, the rarer variants are significantly more likely to be functional than the more common variants. Strikingly, these patterns disappear when we focus on only those variants in which the major alleles are derived. These analyses indicate that the majority of the genetic variation in terms of phenotypic consequence may result from a mutation-selection balance, as opposed to balancing selection, and have direct relevance to the study of human disease.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXksFKlsb8%253D&md5=d62d08de43a29231292e1dcf98b381c3
    42. 42
      Ng, P. C. and Henikoff, S. (2006) Predicting the effects of amino acid substitutions on protein function Annu. Rev. Genomics Hum. Genet. 7, 61– 80
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      42
      Predicting the effects of amino acid substitutions on protein function
      Ng, Pauline C.; Henikoff, Steven
      Annual Review of Genomics and Human Genetics (2006), 7 (), 61-80CODEN: ARGHC4; ISSN:1527-8204. (Annual Reviews Inc.)
      A review. Nonsynonymous single nucleotide polymorphisms (nsSNPs) are coding variants that introduce amino acid changes in their corresponding proteins. Because nsSNPs can affect protein function, they are believed to have the largest impact on human health compared with SNPs in other regions of the genome. Therefore, it is important to distinguish those nsSNPs that affect protein function from those that are functionally neutral. Here, the authors provide an overview of amino acid substitution (AAS) prediction methods, which use sequence and/or structure to predict the effect of an AAS on protein function. Most methods predict approx. 25-30% of human nsSNPs to neg. affect protein function, and such nsSNPs tend to be rare in the population. The authors discuss the utility of AAS prediction methods for Mendelian and complex diseases as well as their broader applications for understanding protein function.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1WgsrzJ&md5=713c14f441a4cdde1e5e6cf125d5bef9
    43. 43
      Pence, M. G., Blans, P., Zink, C. N., Hollis, T., Fishbein, J. C., and Perrino, F. W. (2009) Lesion bypass of N2-ethylguanine by human DNA polymerase ι J. Biol. Chem. 284, 1732– 1740
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      43
      Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι
      Pence, Matthew G.; Blans, Patrick; Zink, Charles N.; Hollis, Thomas; Fishbein, James C.; Perrino, Fred W.
      Journal of Biological Chemistry (2009), 284 (3), 1732-1740CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Nucleotide incorporation and extension opposite N2-ethylguanine (N2-ethyl-Gua) by DNA polymerase ι (DNA pol ι) was measured and structures of the DNA polymerase ι-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase ι opposite N2-ethyl-Gua was detd. by steady state kinetic anal. with Mg2+ or Mn2+ as the activating metal. DNA polymerase ι incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase ι opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase ι extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase ι catalyzed reaction is well suited for N2-ethyl-Gua bypass. The structure of DNA polymerase ι with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the Et adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase ι complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase ι polymerase-assocd. domain to accommodate the adduct providing direct evidence that DNA polymerase ι efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtFOhtg%253D%253D&md5=cf9d56e62d17d269f4cc1075a93c1a56
    44. 44
      Nair, D. T., Johnson, R. E., Prakash, L., Prakash, S., and Aggarwal, A. K. (2006) An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site Structure 14, 749– 755
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      44
      An Incoming Nucleotide Imposes an anti to syn Conformational Change on the Templating Purine in the Human DNA Polymerase-ι Active Site
      Nair, Deepak T.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.
      Structure (Cambridge, MA, United States) (2006), 14 (4), 749-755CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      Substrate-induced conformational change of the protein is the linchpin of enzymic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallog. anal. of hPolι binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPolι active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjs1Gksrk%253D&md5=863b779888753f74965fc93fba1265cf
    45. 45
      Nair, D. T., Johnson, R. E., Prakash, L., Prakash, S., and Aggarwal, A. K. (2009) DNA synthesis across an abasic lesion by human DNA polymerase iota Structure 17, 530– 537
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      45
      DNA Synthesis across an Abasic Lesion by Human DNA Polymerase ι
      Nair, Deepak T.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.
      Structure (Cambridge, MA, United States) (2009), 17 (4), 530-537CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      Summary: Abasic sites are among the most abundant DNA lesions formed in human cells, and they present a strong block to replication. DNA polymerase ι (Polι) is one of the few DNA Pols that does not follow the A-rule opposite an abasic site. We present here three structures of human Polι in complex with DNAs contg. an abasic lesion and dGTP, dTTP, or dATP as the incoming nucleotide. The structures reveal a mechanism of translesion synthesis across an abasic lesion that differs from that in other Pols. Both the abasic lesion and the incoming dNTPs are intrahelical and are closely apposed across a constricted active site cleft. The dNTPs partake in distinct networks of hydrogen bonds in the "void" opposite the lesion. These different patterns of hydrogen bonds, as well as stacking interactions, may underlie Polι's small preference for insertion of dGTP over other nucleotides opposite this common lesion.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVCms78%253D&md5=1bbf4b2b98af3f24bc0906ea71bd198b
    46. 46
      Johnson, R. E., Trincao, J., Aggarwal, A. K., Prakash, S., and Prakash, L. (2003) Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases Mol. Cell. Biol. 23, 3008– 3012
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      46
      Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases
      Johnson, Robert E.; Trincao, Jose; Aggarwal, Aneel K.; Prakash, Satya; Prakash, Louise
      Molecular and Cellular Biology (2003), 23 (8), 3008-3012CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)
      Although DNA polymerase η (Polη) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Polη of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equiv. to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, resp.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktV2ltL8%253D&md5=464585f4b76ef7860cd73a2bc3f87e89
    47. 47
      Ash, D. E. and Schramm, V. L. (1982) Determination of free and bound manganese(II) in hepatocytes from fed and fasted rats J. Biol. Chem. 257, 9261– 9264
      [ PubMed], [ CAS], Google Scholar
      47
      Determination of free and bound manganese(II) in hepatocytes from fed and fasted rats
      Ash, David E.; Schramm, Vern L.
      Journal of Biological Chemistry (1982), 257 (16), 9261-4CODEN: JBCHA3; ISSN:0021-9258.
      Free and total Mn(II) contents of hepatocytes from fed and fasted rats were detd. by EPR. The difference spectra of cumulative scans of cell samples and the mother medium allows a sensitivity of 0.1 μM free Mn(II). The free and total Mn(II) contents were 0.71 and 34.4 nmol/mL cell H2O in hepatocytes from fed animals and 0.25 and 35.9 nmol/mL cell H2O, resp., in hepatocytes from fasted animals. There was a decrease in free Mn(II) by a factor of 2.8 with no change in the total cell Mn(II) in response to fasting.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38Xlt1egsL0%253D&md5=f7d1d86f15a3578f7b4900cbc33544ea
    48. 48
      Markesbery, W. R., Ehmann, W. D., Alauddin, M., and Hossain, T. I. (1984) Brain trace element concentrations in aging Neurobiol. Aging 5, 19– 28
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      48
      Brain trace element concentrations in aging
      Markesbery, William R.; Ehmann, William D.; Alauddin, Mohammad; Hossain, T. I. M.
      Neurobiology of Aging (1984), 5 (1), 19-28CODEN: NEAGDO; ISSN:0197-4580.
      Trace element concns. are detd. in various human brain regions over the complete life span using instrumental neutron activation anal. Several different patterns of trace element alteration are obsd. with age. Brain Al, Cl, and Na concns. increase with advancing age, whereas K, P, and Rb decline. Ag, Co, Fe, Sb, and Sc concns. increase up to 40-79 age range then decline. Br, Se, and Zn remain relatively const. throughout adult life. Hg, Mn, and Cs show no consistent trend with age. In infant brains, Br and Cl increase and Al, Cr, Cs, Fe, Mn, P, Rb, Sc, Se, and Zn decrease compared to adults. The essential elements that remain within narrow concn. limits throughout adult life suggest the presence of an efficient homeostatic mechanism for their regulation in the brain, whereas those that are altered with age suggest modifications in control mechanisms or altered relationships with other elements. Increased concns. of nonessential elements may reflect accumulation from our environment, impaired removal, or altered balance with other elements.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXks12qu7c%253D&md5=f8fea6190665b0418d4b99d7a7186e16
    49. 49
      Versieck, J. (1985) Trace elements in human body fluids and tissues Crit. Rev. Clin. Lab. Sci. 22, 97– 184
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      49
      Trace elements in human body fluids and tissues
      Versieck, Jacques
      Critical Reviews in Clinical Laboratory Sciences (1985), 22 (2), 97-184CODEN: CRCLBH; ISSN:0590-8191.
      A review with 633 refs. on trace element anal. and nutrition and the contents of trace elements in human body fluids and tissues.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXkslSitrw%253D&md5=de4d5a49b8e32b042d854a372ad530b3
    50. 50
      Crossgrove, J. and Zheng, W. (2004) Manganese toxicity upon overexposure NMR Biomed. 17, 544– 553
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      50
      Manganese toxicity upon overexposure
      Crossgrove, Janelle; Zheng, Wei
      NMR in Biomedicine (2004), 17 (8), 544-553CODEN: NMRBEF; ISSN:0952-3480. (John Wiley & Sons Ltd.)
      A review on manganese toxicity upon overexposure. Manganese (Mn) is a required element and a metabolic byproduct of the contrast agent mangafodipir trisodium (MnDPDP). The Mn released from MnDPDP is initially sequestered by the liver for first-pass elimination, which allows an enhanced contrast for diagnostic imaging. The administration of i.v. Mn impacts its homeostatic balance in the human body and can lead to toxicity. Human Mn deficiency has been reported in patients on parenteral nutrition and in micronutrient studies. Mn toxicity has been reported through occupational (e.g. welder) and dietary overexposure and is evidenced primarily in the central nervous system, although lung, cardiac, liver, reproductive and fetal toxicity have been noted. Mn neurotoxicity results from an accumulation of the metal in brain tissue and results in a progressive disorder of the extrapyramidal system which is similar to Parkinson's disease. In order for Mn to distribute from blood into brain tissue, it must cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BCB). Brain import, with no evidence of export, would lead to brain Mn accumulation and neurotoxicity. The mechanism for the neurodegenerative damage specific to select brain regions is not clearly understood. Disturbances in iron homeostasis and the valence state of Mn have been implicated as key factors in contributing to Mn toxicity. Chelation therapy with EDTA and supplementation with levodopa are the current treatment options, which are mildly and transiently efficacious. In conclusion, repeated administration of Mn, or compds. that readily release Mn, may increase the risk of Mn-induced toxicity.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1Knsr0%253D&md5=6c3a61487421d5f40f0042b16071bc80
    51. 51
      Garcia-Rodriguez, N., Diaz de la Loza Mdel, C., Andreson, B., Monje-Casas, F., Rothstein, R., and Wellinger, R. E. (2012) Impaired manganese metabolism causes mitotic misregulation J. Biol. Chem. 287, 18717– 18729
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      51
      Impaired Manganese Metabolism Causes Mitotic Misregulation
      Garcia-Rodriguez, Nestor; Diaz de la Loza, Maria del Carmen; Andreson, Bethany; Monje-Casas, Fernando; Rothstein, Rodney; Wellinger, Ralf Erik
      Journal of Biological Chemistry (2012), 287 (22), 18717-18729CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn2+ acts as a cofactor for many enzymes and excess of Mn2+ is toxic. Alterations in Mn2+ homeostasis affect metabolic functions and mutations in the human Mn2+/Ca2+ transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn2+ on cell cycle progression and show that an excess of cytosolic Mn2+ alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn2+ pool requires a functional morphol. checkpoint to avoid the formation of polyploid cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsF2msrY%253D&md5=06a56a5f51b508be46aa5c04ca23c97a
    52. 52
      Wang, T. S., Eichler, D. C., and Korn, D. (1977) Effect of Mn2+ on the in vitro activity of human deoxyribonucleic acid polymerase beta Biochemistry 16, 4927– 4934
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      52
      Effect of manganese(2+) ions on the in vitro activity of human deoxyribonucleic acid polymerase β
      Wang, Teresa Shu-Fong; Eichler, Duane C.; Korn, David
      Biochemistry (1977), 16 (22), 4927-34CODEN: BICHAW; ISSN:0006-2960.
      Substitution of Mn2+ for Mg2+ has a profound effect on the kinetic parameters of the human DNA polymerase β reaction. At activated DNA concns. >100 μM in nucleotide, Mg2+ is the preferred cation, but at DNA concns. <100 μM, Mn2+ is preferred. With defined primer-template mols. at low concns. DNA synthesis can be detected only in the presence of Mn2+. The apparent Km for activated DNA with Mn2+ is <10 μM, whereas in reactions with Mg2+, the apparent Km is ∼300 μM. The effects of these divalent cations on the Km values for deoxynucleoside triphosphate substrates are qual. similar. Thus, under appropriate reaction conditions with DNA, Mn2+ may be as effective as Mg2+, strongly preferred or absolutely required for the demonstration of polymerase β activity. A new method was developed for the purifn. of DNA polymerase β from normal adult human liver.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXlvFKhurY%253D&md5=268fb32dc3f3eee6d5c70823905d95ee
    53. 53
      Blanca, G., Shevelev, I., Ramadan, K., Villani, G., Spadari, S., Hübscher, U., and Maga, G. (2003) Human DNA polymerase lambda diverged in evolution from DNA polymerase beta toward specific Mn(++) dependence: a kinetic and thermodynamic study Biochemistry 42, 7467– 7476
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      53
      Human DNA polymerase λ diverged in evolution from DNA polymerase β toward specific Mn++ dependence: A kinetic and thermodynamic study
      Blanca, Giuseppina; Shevelev, Igor; Ramadan, Kristijan; Villani, Giuseppe; Spadari, Silvio; Huebscher, Ulrich; Maga, Giovanni
      Biochemistry (2003), 42 (24), 7467-7476CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The recently discovered human DNA polymerase λ (DNA pol λ) has been implicated in translesion DNA synthesis across abasic sites. One remarkable feature of this enzyme is its preference for Mn2+ over Mg2+ as the activating metal ion, but the mol. basis for this preference is not known. Here, the authors present a kinetic and thermodn. anal. of the DNA polymerase reaction catalyzed by full length human DNA pol λ, showing that Mn2+ favors specifically the catalytic step of nucleotide incorporation. Besides acting as a poor coactivator for catalysis, Mg2+ appeared to bind also to an allosteric site, resulting in the inhibition of the synthetic activity of DNA pol λ and in an increased sensitivity to end product (pyrophosphate) inhibition. Comparison with the closely related enzyme human DNA pol β, as well as with other DNA synthesizing enzymes (mammalian DNA pol α and DNA pol δ, Escherichia coli DNA pol I, and HIV-1 reverse transcriptase) indicated that these features are unique to DNA pol λ. A deletion mutant of DNA pol λ, which contained the highly conserved catalytic core only representing the C-terminal half of the protein, showed biochem. properties comparable to the full length enzyme but clearly different from the close homolog DNA pol β, highlighting the existence of important differences between DNA pol λ and DNA pol β, despite a high degree of sequence similarity.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktFymtbo%253D&md5=2238f0022a80a84b197256221d3ee47d
    54. 54
      Dominguez, O., Ruiz, J. F., Lain de Lera, T., Garcia-Diaz, M., Gonzalez, M. A., Kirchhoff, T., Martinez, A. C., Bernad, A., and Blanco, L. (2000) DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells EMBO J. 19, 1731– 1742
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      54
      DNA polymerase mu (Pol μ), homologous to TdT, could act as a DNA mutator in eukaryotic cells
      Dominguez, Orlando; Ruiz, Jose F.; De Lera, Teresa Lain; Garcia-Diaz, Miguel; Gonzalez, Manuel A.; Kirchhoff, Tomas; Martinez-A, Carlos; Bernad, Antonio; Blanco, Luis
      EMBO Journal (2000), 19 (7), 1731-1742CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
      A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol μ), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol μ, overproduced in Escherichia coli in a sol. form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn2+ ions. Interestingly, unlike TdT, the catalytic efficiency of polymn. carried out by Pol μ was enhanced by the presence of a template strand. Using activating Mg2+ ions, template-enhanced polymn. was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn2+ ions, template-enhanced polymn. produced a random insertion of nucleotides. Northern-blotting and in situ anal. showed a preferential expression of Pol μ mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol μ, present in the databases, derived from germinal center B cells. Therefore, Pol μ is a good candidate to be the mutator polymerase responsible for somatic hypermutation of Ig genes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXislymtLc%253D&md5=11e1f0d206762d4f2301bc5d1673bd7f
    55. 55
      Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., and Kraut, J. (1996) Crystal structures of human DNA polymerase β complexed with DNA: implications for catalytic mechanism, processivity, and fidelity Biochemistry 35, 12742– 12761
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      55
      Crystal structures of human DNA polymerase β complexed with DNA: Implications for catalytic mechanism, processivity, and fidelity
      Pelletier, Huguette; Sawaya, Michael R.; Wolfle, William; Wilson, Samuel H.; Kraut, Joseph
      Biochemistry (1996), 35 (39), 12742-12761CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Mammalian DNA polymerase β (pol β) is a small (39 kDa) DNA gap-filling enzyme that comprises an N-terminal 8-kDa domain and a C-terminal 31-kDa domain. Here, crystal structures of human pol β complexed with blunt-ended segments of DNA showed that, although the crystals belonged to a different space group, the DNA was nevertheless bound in the pol β binding channel in the same way as the DNA in previously reported structures of rat pol β complexed with a template-primer and ddCTP. The 8-kDa domain was in 1 of 3 previously obsd. positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small no. of stable conformations. The thumb subdomain was in a more open position in the human pol β-DNA binary complex than in the rat pol β-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that was obsd. in pre-steady-state kinetic studies. Intermol. contacts between the DNA and the 8-kDa domain of a symmetry-related pol β mol. revealed a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addn. to a Lys-rich binding pocket that accommodated a 5'-phosphate end group, the 8-kDa domain also contained a newly discovered helix-hairpin-helix (HhH) motif that bound to DNA in the same way as did a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs was facilitated by a metal cation. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions obsd. in pol β may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol β was particularly striking in that all of the conserved residues were clustered in a short sequence segment, LPGVGXK, where LPGV corresponds to a type II β-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be crit. for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol β may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their resp. DNA lyase activities. A model for productive binding of pol β to a gapped-DNA substrate required a 90° bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlsVymu7s%253D&md5=9ad490433e93546c8f3bd5a23792f5d1
    56. 56
      Yang, W., Lee, J. Y., and Nowotny, M. (2006) Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity Mol. Cell 22, 5– 13
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      56
      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.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFWiurg%253D&md5=71303ca14fea285a44031d5c2eb5b1d3
    57. 57
      Yuan, B., You, C., Andersen, N., Jiang, Y., Moriya, M., O’Connor, T. R., and Wang, Y. (2011) The roles of DNA polymerases κ and ι in the error-free bypass of N2-carboxyalkyl-2′-deoxyguanosine lesions in mammalian cells J. Biol. Chem. 286, 17503– 17511
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      57
      The roles of DNA polymerases κ and ι in the error-free bypass of N2-carboxyalkyl-2'-deoxyguanosine lesions in mammalian cells
      Yuan, Bifeng; You, Changjun; Andersen, Nisana; Jiang, Yong; Moriya, Masaaki; O'Connor, Timothy R.; Wang, Yinsheng
      Journal of Biological Chemistry (2011), 286 (20), 17503-17511CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochem. studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N2-modified 2'-deoxyguanosine derivs. Herein, we used shuttle vector technol. and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) and N2-carboxymethyl-2'-deoxyguanosine (N2-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N2-CEdG lesions. In contrast, no mutation was found after the N2-CEdG- and N2-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N2-CEdG and N2-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtVGjtr4%253D&md5=f55b7b6d4a8bcb103e8b9d4c988c105f
    58. 58
      Kirouac, K. N. and Ling, H. (2011) Poli: Shining light on repair of oxidative DNA lesions and mutations Cell Cycle 10, 1520– 1521
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      58
      Polι: shining light on repair of oxidative DNA lesions and mutations
      Kirouac, Kevin N.; Ling, Hong
      Cell Cycle (2011), 10 (10), 1520-1521CODEN: CCEYAS; ISSN:1538-4101. (Landes Bioscience)
      A review on DNA polymerase ι and its role in repairing oxidative DNA lesions and mutations.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1ChtbnE&md5=0170b601e8847cd1878da5458289a821
    59. 59
      Petta, T. B., Nakajima, S., Zlatanou, A., Despras, E., Couve-Privat, S., Ishchenko, A., Sarasin, A., Yasui, A., and Kannouche, P. (2008) Human DNA polymerase iota protects cells against oxidative stress EMBO J. 27, 2883– 2895
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      59
      Human DNA polymerase iota protects cells against oxidative stress
      Petta, Tirzah Braz; Nakajima, Satoshi; Zlatanou, Anastasia; Despras, Emmanuelle; Couve-Privat, Sophie; Ishchenko, Alexander; Sarasin, Alain; Yasui, Akira; Kannouche, Patricia
      EMBO Journal (2008), 27 (21), 2883-2895CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)
      Human DNA polymerase iota (polι) is a unique member of the Y-family of specialized polymerases that displays a 5'deoxyribose phosphate (dRP) lyase activity. Although polι is well conserved in higher eukaryotes, its role in mammalian cells remains unclear. To investigate the biol. importance of polι in human cells, we generated fibroblasts stably downregulating polι (MRC5-polιKD) and examd. their response to several types of DNA-damaging agents. We show that cell lines downregulating polι exhibit hypersensitivity to DNA damage induced by hydrogen peroxide (H2O2) or menadione but not to ethylmethane sulfonate (EMS), UVC or UVA. Interestingly, exts. from cells downregulating polι show reduced base excision repair (BER) activity. In addn., polι binds to chromatin after treatment of cells with H2O2 and interacts with the BER factor XRCC1. Finally, green fluorescent protein-tagged polι accumulates at the sites of oxidative DNA damage in living cells. This recruitment is partially mediated by its dRP lyase domain and ubiquitin-binding domains. These data reveal a novel role of human polι in protecting cells from oxidative damage.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlalt7fE&md5=364752b1d8a33d4da7327f15c7f3b887
    60. 60
      Ren, J., Wen, L., Gao, X., Jin, C., Xue, Y., and Yao, X. (2009) DOG 1.0: illustrator of protein domain structures Cell Res. 19, 271– 273
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      60
      DOG 1.0: illustrator of protein domain structures
      Ren, Jian; Wen, Longping; Gao, Xinjiao; Jin, Changjiang; Xue, Yu; Yao, Xuebiao
      Cell Research (2009), 19 (2), 271-273CODEN: CREEB6; ISSN:1001-0602. (Nature Publishing Group)
      Software DOG (Domain Graph, version 1.0) to prep. publication-quality figures of protein domain structures is described. The DOG 1.0 software was written in JAVA 1.5 and packed with Install4j 4.0.8.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1ams7k%253D&md5=4f86a979e028e2c41b2275c215bd1052

  • Supporting Information

    Supporting Information

    ARTICLE SECTIONS
    Jump To

    Analysis of human pol ι (1–445) wild-type and variant proteins by SDS-polyacrylamide gel electrophoresis (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

    Terms & Conditions

    Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at http://pubs.acs.org/page/copyright/permissions.html.

PDF [ 5MB]

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

OOPS

You have to login with your ACS ID befor you can login with your Mendeley account.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect

This website uses cookies to improve your user experience. By continuing to use the site, you are accepting our use of cookies. Read the ACS privacy policy.

CONTINUE