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Article

Structure Determination of an Interstrand-Type Cis-Anti Cyclobutane Thymine Dimer Produced in High Yield by UVB Light in an Oligodeoxynucleotide at Acidic pH^†

Supporting Info

Dian G. T. Su, Jeffrey L.-F. Kao, Michael L. Gross and John-Stephen A. Taylor

Department of Chemistry, Washington University, St. Louis, Missouri 63130

J. Am. Chem. Soc., 2008, 130 (34), pp 11328–11337

DOI: 10.1021/ja8010836

Publication Date (Web): August 5, 2008

†

Abbreviations. (6−4), pyrimidine-(6−4)-pyrimidone; Ade, adenine; BIMP, bovine intestinal mucosa phosphodiesterase; BSP, bovine spleen phosphodiesterase; [c,a], cis,anti; [c,s], cis,syn; CPD, cyclobutane pyrimidine dimer; Cyt, cytosine; Dewar, pyrimidine-(6−4)-Dewar pyrimidone; ESI, electrospray ionization; Gua, guanine; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; NP1, nuclease P1; ODN, oligodeoxynucleotide; PDE, phosphodiesterase; Py, pyrimidine; Py=Py, pyrimidine dimer; RP-HPLC, reverse phase high performance liquid chromatography; SVP, snake venom phosphodiesterase; Thy, thymine; [t,a], trans,anti; [t,s], trans,syn.

Abstract

UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product pd(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3′-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an interstand type reaction. In addition to pH dependency, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this interstrand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.

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