Biochemistry 2010, 49, 2615–2626 2615 DOI: 10.1021/bi901516d 2 Structure of the 1,N -Etheno-20-deoxyguanosine Lesion in the 30-G(εdG)T-50 Sequence Opposite a One-Base Deletion† Ganesh Shanmugam,‡ Ivan D. Kozekov,‡ F. Peter Guengerich,§ Carmelo J. Rizzo,‡,§ and Michael P. Stone*,‡,§ ‡Department of Chemistry and §Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37235 Received August 28, 2009; Revised Manuscript Received November 19, 2009 ABSTRACT: The structure of the 1,N2-ethenodeoxyguanosine lesion (1,N2-εdG) has been characterized in 50-d(CGCATXGAATCC)-30 3 50-d(GGATTCATGCG)-30 (X = 1,N2-εdG), in which there is no dC opposite the lesion. This duplex (named the 1-BD duplex) models the product of translesion bypass of 1,N2-εdG by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) [Zang, H., Goodenough, A. K., Choi, J. Y., Irimia, A., Loukachevitch, L. V., Kozekov, I. D., Angel, K. C., Rizzo, C. J., Egli, M., and Guengerich, F. P. (2005) J. Biol. Chem. 280, 29750-29764], leading to a one-base deletion. The Tm of this duplex is 6 C higher than that of the duplex in which dC is present opposite the 1,N2-εdG lesion and 8 C higher than that of the unmodified 1-BD duplex. Analysis of NOEs between the 1,N2-εdG imidazole and deoxyribose H10 protons and between the 1,N2-εdG etheno H6 and H7 protons and DNA protons establishes that 1,N2-εdG adopts the anti conformation about the glycosyl bond and that the etheno moiety is accommodated within the helix. The resonances of the 1,N2-εdG H6 and H7 etheno protons shift upfield relative to the monomer 1,N2-εdG, attributed to ring current shielding, consistent with their intrahelical location. NMR data reveal that Watson-Crick base pairing is maintained at both the 50 and 30 neighbor base pairs. The structure of the 1-BD duplex has been refined using molecular dynamics calculations restrained by NMR-derived distance and dihedral angle restraints. The increased stability of the 1,N2-εdG lesion in the absence of the complementary dC correlates with the one-base deletion extension product observed during the bypass of the 1,N2-εdG lesion by the Dpo4 polymerase, suggesting that stabilization of this bulged intermediate may be significant with regard to the biological processing of the lesion. Ethenobases (1) are five-membered annelation products arising from the reactions of electrophiles derived from vinyl halides and other vinyl monomers, including chloroacetaldehyde, with dC, dA, and dG nucleotides in DNA (2-9). They also arise from endogenous exposure to lipid peroxidation products (10), parti- cularly 4,5-epoxy-2(E)-decanal (11, 12) and 4-hydroperoxynone- nal (13); related etheno adducts from 4-oxo-2(E)-nonenal (14, 15), 4-oxohexenal (16), and 9,12-dioxo-10(E)-dodecanoic acid (17) have also been characterized (15, 17). The 1,N2-ethenodeoxy- guanosine (1,N2-εdG)1 adduct (18) (Scheme 1) is one of two εdG lesions, the other being the N2,3-εdG adduct. One of our laboratories has elucidated the mechanism for formation of †This work was supported by National Institutes of Health (NIH) Grants P01 ES005355 (C.J.R., I.D.K., and M.P.S.) and R01 ES010375 (F.P.G.). Funding for the NMR spectrometers was supplied by Vanderbilt University, the Vanderbilt Center in Molecular Toxicology (P30 ES000267), and by NIH Grant RR005805. The Vanderbilt Ingram Cancer Center is supported by NIH Grant P30 CA068485. *To whom correspondence should be addressed. Phone: (615) 322-2589. Fax: (615) 322-7591. E-mail: michael.p.stone@vanderbilt.edu. 1Abbreviations: 1,N2-εdG, 3-(20-deoxy-β-D-erythro-pentofuranosyl)- 3,4-dihydro-9H-imidazo[1,2-a]purin-9-one or 1,N2-etheno-20-deoxy- guanosine; M1dG, 3-(20-deoxy-β-D-erythro-pentofuranosyl)pyrimido- [1,2-R]purin-10(3H)-one; PdG, 1,N2-propano-20-deoxyguanosine; 1-BD, one-base deletion; NOE, nuclear Overhauser enhancement; NOESY, two- dimensional NOE spectroscopy; COSY, correlation spectroscopy; DQF- COSY, double-quantum-filtered correlation spectroscopy; TPPI, time- proportional phase increment. A right superscript refers to the numerical position in the sequence starting from the 50 terminus of chain A and proceeding to the 30 terminus of chain A and then from the 50 terminus of chain B to the 30 terminus of chain B. 1,N2-εdG (19, 20). It has been detected in DNA treated with vinyl chloride metabolites (21) and β-carotene oxidation pro- ducts (22), isolated using immunohistochemistry techniques (23), and identified by mass spectrometry in liver DNA of rodents (24-27). The 1,Ν2-εdG lesion blocks the Watson-Crick hydrogen bonding face of dG and is mutagenic in Escherichia coli (28) and in mammalian (29) cells. The insertion of 1,N2-εdG into a 30-G(1,N2-εdG)TACT-50 template and its replication by the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) were reported by Zang et al. (30), who showed that dATP was preferentially incorporated. Crystallographic analyses (30) of either binary or ternary complexes with the Dpo4 polymerase led to the conclusion that this occurs via formation of a “type II” bypass structure at the active site of the polymerase, in which the incoming dATP pairs with the 50 neighboring T of the template strand, as opposed to the damaged 1,N2-εdG nucleotide (31). Further extension presumably led to the observed -1 frameshift event and the 50-CATGA-30 product (30). To improve our understanding of the structural basis of -1 frameshift events, we inserted 1,N2-εdG into the 12-mer 3 11-mer duplex [50-d(CGCATXGAATCC)-30 3 50-d(GGATTC_ATGCG)-30 (X = 1,N2-εdG)] containing a 30-G(1,N2-εdG)TAC-50 template sequence (underlined) . This will be termed the 1-BD duplex (Scheme 1). It models the situation following “type II” insertion of dATP in which the Dpo4 polymerase skips the damaged base in the 30-G(1,N2-εdG)TACT-50 template and instead utilizes the 50 neighbor thymine (underlined) to incorporate dATP (31), r 2010 American Chemical Society Published on Web 03/04/2010 pubs.acs.org/Biochemistry