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• W2076333887 abstract "DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3′-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases. DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3′-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases. Dpo4 is strongly inhibited and highly mutagenic during bypass of m1G and m3C lesions In the Dpo4 extension complexes, m1G and m3C remain positioned within the DNA helix The 3′-terminal A, G, T, and C primer bases opposite the lesions are mispositioned Mechanisms of methylation damage recognition by repair demethylases are discussed Alkylation damage to DNA is induced by naturally occurring endogenous agents (De Bont and van Larebeke, 2004De Bont R. van Larebeke N. Endogenous DNA damage in humans: a review of quantitative data.Mutagenesis. 2004; 19: 169-185Crossref PubMed Scopus (691) Google Scholar, Sedgwick, 1997Sedgwick B. Nitrosated peptides and polyamines as endogenous mutagens in O6-alkylguanine-DNA alkyltransferase deficient cells.Carcinogenesis. 1997; 18: 1561-1567Crossref PubMed Scopus (67) Google Scholar), as well as by many environmental carcinogens (Hecht, 1999Hecht S.S. DNA adduct formation from tobacco-specific N-nitrosamines.Mutat. Res. 1999; 424: 127-142Crossref PubMed Scopus (319) Google Scholar, Singer and Grunberger, 1983Singer B. Grunberger D. Molecular Biology of Mutagens and Carcinogens: Intrinsic Properties of Nucleic Acids. Plenum Press, New York1983Crossref Google Scholar) and by cancer chemotherapeutics (Allan and Travis, 2005Allan J.M. Travis L.B. Mechanisms of therapy-related carcinogenesis.Nat. Rev. Cancer. 2005; 5: 943-955Crossref PubMed Scopus (221) Google Scholar, Park et al., 2010Park D.M. Sathornsumetee S. Rich J.N. Medical oncology: treatment and management of malignant gliomas.Nat. Rev. Clin. Oncol. 2010; 7: 75-77Crossref PubMed Scopus (21) Google Scholar). Secondary cancers that arise as a consequence of chemotherapy with alkylating agents are not unusual and are associated with mutation accumulation, genome instability and defects in DNA repair (Allan and Travis, 2005Allan J.M. Travis L.B. Mechanisms of therapy-related carcinogenesis.Nat. Rev. Cancer. 2005; 5: 943-955Crossref PubMed Scopus (221) Google Scholar). Among the diverse lesions produced by the SN2 type alkylating agents are methylation at the N1-position of adenine (m1A) and guanine (m1G), and at the N3-positions of cytosine (m3C) and thymine (m3T) (Figure 1A ) (Sedgwick, 2004Sedgwick B. Repairing DNA-methylation damage.Nat. Rev. Mol. Cell Biol. 2004; 5: 148-157Crossref PubMed Scopus (285) Google Scholar, Shrivastav et al., 2010Shrivastav N. Li D. Essigmann J.M. Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation.Carcinogenesis. 2010; 31: 59-70Crossref PubMed Scopus (213) Google Scholar). The addition of a methyl group to the endocyclic N-atoms that are normally involved in Watson-Crick base pairing is cytotoxic and blocks DNA replication in Escherichia coli in the absence of the AlkB repair protein (Delaney and Essigmann, 2004Delaney J.C. Essigmann J.M. Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli.Proc. Natl. Acad. Sci. USA. 2004; 101: 14051-14056Crossref PubMed Scopus (201) Google Scholar). This result suggests that the polymerase responsible for replicating genomic DNA, presumably the B-family Pol III, in E. coli, would be drastically impeded by the m1A, m1G, m3C, and m3T lesions. In vitro data indicate that the A-family gap-filling E. coli Pol I Klenow fragment is severally hindered by the m1A (Larson et al., 1985Larson K. Sahm J. Shenkar R. Strauss B. Methylation-induced blocks to in vitro DNA replication.Mutat. Res. 1985; 150: 77-84Crossref PubMed Scopus (193) Google Scholar), m3C (Boiteux and Laval, 1982Boiteux S. Laval J. Mutagenesis by alkylating agents: coding properties for DNA polymerase of poly (dC) template containing 3-methylcytosine.Biochimie. 1982; 64: 637-641Crossref PubMed Scopus (41) Google Scholar, Saffhill, 1984Saffhill R. Differences in the promutagenic nature of 3-methylcytosine as revealed by DNA and RNA polymerising enzymes.Carcinogenesis. 1984; 5: 691-693Crossref PubMed Scopus (19) Google Scholar), and m3T (Huff and Topal, 1987Huff A.C. Topal M.D. DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis.J. Biol. Chem. 1987; 262: 12843-12850Abstract Full Text PDF PubMed Google Scholar) lesions. Activation of the SOS-response in E. coli induces production of Pol IV and Pol V, the low-fidelity Y-family translesion synthesis (TLS) polymerases (Yang and Woodgate, 2007Yang W. Woodgate R. What a difference a decade makes: insights into translesion DNA synthesis.Proc. Natl. Acad. Sci. USA. 2007; 104: 15591-15598Crossref PubMed Scopus (309) Google Scholar), which increases bypass of the m1A, m3C, m1G, and m3T lesions in vivo by ∼3–4-fold (Delaney and Essigmann, 2004Delaney J.C. Essigmann J.M. Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli.Proc. Natl. Acad. Sci. USA. 2004; 101: 14051-14056Crossref PubMed Scopus (201) Google Scholar). The resulting progeny contain a staggering fraction of mutations: ∼70% in the case of the m3C- and m1G-modified templates and ∼53% in the case of the m3T; only m1A is weakly mutagenic generating ∼1% of errors (Delaney and Essigmann, 2004Delaney J.C. Essigmann J.M. Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli.Proc. Natl. Acad. Sci. USA. 2004; 101: 14051-14056Crossref PubMed Scopus (201) Google Scholar). In eukaryotes, as in E. coli, Y-family polymerases temporarily replace stalled high-fidelity polymerases with damage-bypass polymerases (McCulloch and Kunkel, 2008McCulloch S.D. Kunkel T.A. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases.Cell Res. 2008; 18: 148-161Crossref PubMed Scopus (378) Google Scholar, Waters et al., 2009Waters L.S. Minesinger B.K. Wiltrout M.E. D'Souza S. Woodruff R.V. Walker G.C. Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance.Microbiol. Mol. Biol. Rev. 2009; 73: 134-154Crossref PubMed Scopus (456) Google Scholar, Yang and Woodgate, 2007Yang W. Woodgate R. What a difference a decade makes: insights into translesion DNA synthesis.Proc. Natl. Acad. Sci. USA. 2007; 104: 15591-15598Crossref PubMed Scopus (309) Google Scholar). Currently, structure-function studies that address TLS polymerase-catalyzed bypass of blocking m1A, m3C, m1G, and m3T lesions are lacking. In contrast to high-fidelity B- and A-family polymerases that produce tight-fitting, solvent-excluding reaction-ready active sites on binding of a complementary dNTP (Johnson and Beese, 2004Johnson S.J. Beese L.S. Structures of mismatch replication errors observed in a DNA polymerase.Cell. 2004; 116: 803-816Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, Steitz and Yin, 2004Steitz T.A. Yin Y.W. Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 17-23Crossref PubMed Scopus (37) Google Scholar, Swan et al., 2009aSwan M.K. Johnson R.E. Prakash L. Prakash S. Aggarwal A.K. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase delta.Nat. Struct. Mol. Biol. 2009; 16: 979-986Crossref PubMed Scopus (193) Google Scholar), Y-family polymerases have more spacious and solvent-accessible active sites (Biertumpfel et al., 2010Biertumpfel C. Zhao Y. Kondo Y. Ramon-Maiques S. Gregory M. Lee J.Y. Masutani C. Lehmann A.R. Hanaoka F. Yang W. Structure and mechanism of human DNA polymerase eta.Nature. 2010; 465: 1044-1048Crossref PubMed Scopus (254) Google Scholar, Silverstein et al., 2010Silverstein T.D. Johnson R.E. Jain R. Prakash L. Prakash S. Aggarwal A.K. Structural basis for the suppression of skin cancers by DNA polymerase eta.Nature. 2010; 465: 1039-1043Crossref PubMed Scopus (119) Google Scholar, Yang and Woodgate, 2007Yang W. Woodgate R. What a difference a decade makes: insights into translesion DNA synthesis.Proc. Natl. Acad. Sci. USA. 2007; 104: 15591-15598Crossref PubMed Scopus (309) Google Scholar). Furthermore, Y-family polymerases do not have an exonuclease domain. Additionally, they do not check the minor groove edge of the template/primer to proofread mismatches and they select for the correct dNTP guided predominantly by base pairing complementarity (Yang and Woodgate, 2007Yang W. Woodgate R. What a difference a decade makes: insights into translesion DNA synthesis.Proc. Natl. Acad. Sci. USA. 2007; 104: 15591-15598Crossref PubMed Scopus (309) Google Scholar). These structural and functional features enable Y-family polymerases to bypass a variety of DNA lesions and at the same time cause a higher error rate on undamaged DNA templates (Broyde et al., 2008Broyde S. Wang L. Rechkoblit O. Geacintov N.E. Patel D.J. Lesion processing: high-fidelity versus lesion-bypass DNA polymerases.Trends Biochem. Sci. 2008; 33: 209-219Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, Waters et al., 2009Waters L.S. Minesinger B.K. Wiltrout M.E. D'Souza S. Woodruff R.V. Walker G.C. Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance.Microbiol. Mol. Biol. Rev. 2009; 73: 134-154Crossref PubMed Scopus (456) Google Scholar, Yang and Woodgate, 2007Yang W. Woodgate R. What a difference a decade makes: insights into translesion DNA synthesis.Proc. Natl. Acad. Sci. USA. 2007; 104: 15591-15598Crossref PubMed Scopus (309) Google Scholar). E. coli AlkB is an iron(II)-and 2-oxoglutarate-dependent repair protein that directly reverses alkylation damage on the endocyclic N-atoms of DNA and RNA bases by oxidative demethylation (Falnes et al., 2002Falnes P.O. Johansen R.F. Seeberg E. AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli.Nature. 2002; 419: 178-182Crossref PubMed Scopus (503) Google Scholar, Trewick et al., 2002Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage.Nature. 2002; 419: 174-178Crossref PubMed Scopus (633) Google Scholar). AlkB proficiently removes methyl groups from m1A and m3C adducts (Sedgwick et al., 2007Sedgwick B. Bates P.A. Paik J. Jacobs S.C. Lindahl T. Repair of alkylated DNA: recent advances.DNA Repair (Amst.). 2007; 6: 429-442Crossref PubMed Scopus (244) Google Scholar, Shrivastav et al., 2010Shrivastav N. Li D. Essigmann J.M. Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation.Carcinogenesis. 2010; 31: 59-70Crossref PubMed Scopus (213) Google Scholar) predominantly in single-stranded DNA, but it is markedly less efficient on the more relatively minor m1G and m3T lesions (Delaney and Essigmann, 2004Delaney J.C. Essigmann J.M. Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli.Proc. Natl. Acad. Sci. USA. 2004; 101: 14051-14056Crossref PubMed Scopus (201) Google Scholar, Falnes et al., 2002Falnes P.O. Johansen R.F. Seeberg E. AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli.Nature. 2002; 419: 178-182Crossref PubMed Scopus (503) Google Scholar, Trewick et al., 2002Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage.Nature. 2002; 419: 174-178Crossref PubMed Scopus (633) Google Scholar, Yu and Hunt, 2009Yu B. Hunt J.F. Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB.Proc. Natl. Acad. Sci. USA. 2009; 106: 14315-14320Crossref PubMed Scopus (72) Google Scholar). The human genome encodes at least nine AlkB family members, ABH1 through ABH8 (Aravind and Koonin, 2001Aravind L. Koonin E.V. The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases.Genome Biol. 2001; 2 (research0007.1–research0007.8)Crossref Google Scholar, Sedgwick et al., 2007Sedgwick B. Bates P.A. Paik J. Jacobs S.C. Lindahl T. Repair of alkylated DNA: recent advances.DNA Repair (Amst.). 2007; 6: 429-442Crossref PubMed Scopus (244) Google Scholar) and the fat mass- and obesity-associated FTO protein (Gerken et al., 2007Gerken T. Girard C.A. Tung Y.C. Webby C.J. Saudek V. Hewitson K.S. Yeo G.S. McDonough M.A. Cunliffe S. McNeill L.A. et al.The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.Science. 2007; 318: 1469-1472Crossref PubMed Scopus (1176) Google Scholar). ABH2 is recognized as a primary demethylase that protects genomic DNA, because mice lacking ABH2 accumulate significant levels of m1A in their genome even in the absence of any exogenous methylating agent (Ringvoll et al., 2006Ringvoll J. Nordstrand L.M. Vagbo C.B. Talstad V. Reite K. Aas P.A. Lauritzen K.H. Liabakk N.B. Bjork A. Doughty R.W. et al.Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA.EMBO J. 2006; 25: 2189-2198Crossref PubMed Scopus (152) Google Scholar). Consistently, in vitro, ABH2 works preferentially on double-stranded DNA and repairs m1A, m3C, and m3T (Aas et al., 2003Aas P.A. Otterlei M. Falnes P.O. Vagbo C.B. Skorpen F. Akbari M. Sundheim O. Bjoras M. Slupphaug G. Seeberg E. Krokan H.E. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA.Nature. 2003; 421: 859-863Crossref PubMed Scopus (520) Google Scholar, Duncan et al., 2002Duncan T. Trewick S.C. Koivisto P. Bates P.A. Lindahl T. Sedgwick B. Reversal of DNA alkylation damage by two human dioxygenases.Proc. Natl. Acad. Sci. USA. 2002; 99: 16660-16665Crossref PubMed Scopus (313) Google Scholar, Falnes, 2004Falnes P.O. Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins.Nucleic Acids Res. 2004; 32: 6260-6267Crossref PubMed Scopus (85) Google Scholar, Ringvoll et al., 2006Ringvoll J. Nordstrand L.M. Vagbo C.B. Talstad V. Reite K. Aas P.A. Lauritzen K.H. Liabakk N.B. Bjork A. Doughty R.W. et al.Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA.EMBO J. 2006; 25: 2189-2198Crossref PubMed Scopus (152) Google Scholar); m1G-containing DNA was not evaluated. ABH1 (Westbye et al., 2008Westbye M.P. Feyzi E. Aas P.A. Vagbo C.B. Talstad V.A. Kavli B. Hagen L. Sundheim O. Akbari M. Liabakk N.B. et al.Human AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine in DNA and RNA.J. Biol. Chem. 2008; 283: 25046-25056Crossref PubMed Scopus (137) Google Scholar), ABH3 (Aas et al., 2003Aas P.A. Otterlei M. Falnes P.O. Vagbo C.B. Skorpen F. Akbari M. Sundheim O. Bjoras M. Slupphaug G. Seeberg E. Krokan H.E. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA.Nature. 2003; 421: 859-863Crossref PubMed Scopus (520) Google Scholar, Duncan et al., 2002Duncan T. Trewick S.C. Koivisto P. Bates P.A. Lindahl T. Sedgwick B. Reversal of DNA alkylation damage by two human dioxygenases.Proc. Natl. Acad. Sci. USA. 2002; 99: 16660-16665Crossref PubMed Scopus (313) Google Scholar, Falnes, 2004Falnes P.O. Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins.Nucleic Acids Res. 2004; 32: 6260-6267Crossref PubMed Scopus (85) Google Scholar, Ringvoll et al., 2006Ringvoll J. Nordstrand L.M. Vagbo C.B. Talstad V. Reite K. Aas P.A. Lauritzen K.H. Liabakk N.B. Bjork A. Doughty R.W. et al.Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA.EMBO J. 2006; 25: 2189-2198Crossref PubMed Scopus (152) Google Scholar), and FTO (Gerken et al., 2007Gerken T. Girard C.A. Tung Y.C. Webby C.J. Saudek V. Hewitson K.S. Yeo G.S. McDonough M.A. Cunliffe S. McNeill L.A. et al.The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.Science. 2007; 318: 1469-1472Crossref PubMed Scopus (1176) Google Scholar, Jia et al., 2008Jia G. Yang C.G. Yang S. Jian X. Yi C. Zhou Z. He C. Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO.FEBS Lett. 2008; 582: 3313-3319Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) are most efficient on ssDNA and ssRNA; however, their substrate specificities are different. The exact cellular roles of ABH1 and ABH3, as well as ABH4–7 remain elusive. Surprisingly, FTO is a key factor in energy homeostasis regulation (Fischer et al., 2009Fischer J. Koch L. Emmerling C. Vierkotten J. Peters T. Bruning J.C. Ruther U. Inactivation of the Fto gene protects from obesity.Nature. 2009; 458: 894-898Crossref PubMed Scopus (710) Google Scholar), and ABH8 is involved in methylation of tRNA and participates in regulation of the DNA-damage response pathway (Fu et al., 2010aFu D. Brophy J.A. Chan C.T. Atmore K.A. Begley U. Paules R.S. Dedon P.C. Begley T.J. Samson L.D. Human AlkB homolog ABH8 Is a tRNA methyltransferase required for wobble uridine modification and DNA damage survival.Mol. Cell. Biol. 2010; 30: 2449-2459Crossref PubMed Scopus (149) Google Scholar, Fu et al., 2010bFu Y. Dai Q. Zhang W. Ren J. Pan T. He C. The AlkB domain of mammalian ABH8 catalyzes hydroxylation of 5-methoxycarbonylmethyluridine at the wobble position of tRNA.Angew. Chem. Int. Ed. Engl. 2010; 49: 8885-8888Crossref PubMed Scopus (114) Google Scholar). The crystal structures of AlkB with short ssDNA (Yu et al., 2006Yu B. Edstrom W.C. Benach J. Hamuro Y. Weber P.C. Gibney B.R. Hunt J.F. Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB.Nature. 2006; 439: 879-884Crossref PubMed Scopus (190) Google Scholar) and AlkB and ABH2 (Yang et al., 2008Yang C.G. Yi C. Duguid E.M. Sullivan C.T. Jian X. Rice P.A. He C. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA.Nature. 2008; 452: 961-965Crossref PubMed Scopus (203) Google Scholar) with dsDNA, where both ss- and dsDNAs contained the m1A lesion, as well as ABH3 in the absence of DNA or RNA (Sundheim et al., 2006Sundheim O. Vagbo C.B. Bjoras M. Sousa M.M. Talstad V. Aas P.A. Drablos F. Krokan H.E. Tainer J.A. Slupphaug G. Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage.EMBO J. 2006; 25: 3389-3397Crossref PubMed Scopus (141) Google Scholar) and FTO with the m3C nucleobase (Han et al., 2010Han Z. Niu T. Chang J. Lei X. Zhao M. Wang Q. Cheng W. Wang J. Feng Y. Chai J. Crystal structure of the FTO protein reveals basis for its substrate specificity.Nature. 2010; 464: 1205-1209Crossref PubMed Scopus (288) Google Scholar) revealed strikingly similar overall folds of the catalytic domains. These proteins flip damaged bases and insert them into the active site for repair (Han et al., 2010Han Z. Niu T. Chang J. Lei X. Zhao M. Wang Q. Cheng W. Wang J. Feng Y. Chai J. Crystal structure of the FTO protein reveals basis for its substrate specificity.Nature. 2010; 464: 1205-1209Crossref PubMed Scopus (288) Google Scholar, Sedgwick et al., 2007Sedgwick B. Bates P.A. Paik J. Jacobs S.C. Lindahl T. Repair of alkylated DNA: recent advances.DNA Repair (Amst.). 2007; 6: 429-442Crossref PubMed Scopus (244) Google Scholar, Sundheim et al., 2006Sundheim O. Vagbo C.B. Bjoras M. Sousa M.M. Talstad V. Aas P.A. Drablos F. Krokan H.E. Tainer J.A. Slupphaug G. Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage.EMBO J. 2006; 25: 3389-3397Crossref PubMed Scopus (141) Google Scholar, Yang et al., 2008Yang C.G. Yi C. Duguid E.M. Sullivan C.T. Jian X. Rice P.A. He C. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA.Nature. 2008; 452: 961-965Crossref PubMed Scopus (203) Google Scholar, Yu et al., 2006Yu B. Edstrom W.C. Benach J. Hamuro Y. Weber P.C. Gibney B.R. Hunt J.F. Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB.Nature. 2006; 439: 879-884Crossref PubMed Scopus (190) Google Scholar). Thus far only ABH2 has been identified as a dsDNA repair protein; it interacts with both damaged and undamaged strands and employs an aromatic residue to intercalate into the duplex DNA and fill the gap resulting from the base flipping (Han et al., 2010Han Z. Niu T. Chang J. Lei X. Zhao M. Wang Q. Cheng W. Wang J. Feng Y. Chai J. Crystal structure of the FTO protein reveals basis for its substrate specificity.Nature. 2010; 464: 1205-1209Crossref PubMed Scopus (288) Google Scholar, Yang et al., 2008Yang C.G. Yi C. Duguid E.M. Sullivan C.T. Jian X. Rice P.A. He C. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA.Nature. 2008; 452: 961-965Crossref PubMed Scopus (203) Google Scholar). It is currently unclear how ABH2 identifies damaged bases opposite their cognate partners in the context of the vast amount of undamaged dsDNA. Moreover, no data are available on lesion recognition opposite incorrect base partners that were mistakenly incorporated by DNA polymerases during replication. The structures of the cognate m1A⋅T and m3C⋅G alignments within dsDNA duplexes have provided valuable information into a possible DNA scanning mechanism employed by ABH2 (Lu et al., 2010Lu L. Yi C. Jian X. Zheng G. He C. Structure determination of DNA methylation lesions N1-meA and N3-meC in duplex DNA using a cross-linked protein-DNA system.Nucleic Acids Res. 2010; 38: 4415-4425Crossref PubMed Scopus (40) Google Scholar), but much remains to be elucidated. The objectives of this work were to explore structural and biochemical features of Dpo4-catalyzed bypass of the m1G and m3C lesions. We demonstrate that this TLS polymerase, that is able to efficiently and in many cases accurately bypass a variety of DNA lesions ranging in size and chemical functionality (Bauer et al., 2007Bauer J. Xing G. Yagi H. Sayer J.M. Jerina D.M. Ling H. A structural gap in Dpo4 supports mutagenic bypass of a major benzo[a]pyrene dG adduct in DNA through template misalignment.Proc. Natl. Acad. Sci. USA. 2007; 104: 14905-14910Crossref PubMed Scopus (68) Google Scholar, Eoff et al., 2007Eoff R.L. Irimia A. Egli M. Guengerich F.P. Sulfolobus solfataricus DNA polymerase Dpo4 is partially inhibited by “wobble” pairing between O6-methylguanine and cytosine, but accurate bypass is preferred.J. Biol. Chem. 2007; 282: 1456-1467Crossref PubMed Scopus (75) Google Scholar, Ling et al., 2003Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Replication of a cis-syn thymine dimer at atomic resolution.Nature. 2003; 424: 1083-1087Crossref PubMed Scopus (202) Google Scholar, Rechkoblit et al., 2006Rechkoblit O. Malinina L. Cheng Y. Kuryavyi V. Broyde S. Geacintov N.E. Patel D.J. Stepwise translocation of Dpo4 polymerase during error-free bypass of an oxoG lesion.PLoS Biol. 2006; 4: e11Crossref PubMed Scopus (127) Google Scholar, Rechkoblit et al., 2010Rechkoblit O. Kolbanovskiy A. Malinina L. Geacintov N.E. Broyde S. Patel D.J. Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4.Nat. Struct. Mol. Biol. 2010; 17: 379-388Crossref PubMed Scopus (40) Google Scholar, Zhang et al., 2009Zhang H. Eoff R.L. Kozekov I.D. Rizzo C.J. Egli M. Guengerich F.P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translesion synthesis past bulky N2-alkylguanine adducts.J. Biol. Chem. 2009; 284: 3563-3576Crossref PubMed Scopus (56) Google Scholar), is strongly inhibited by m1G and m3C lesions. Furthermore, the extension products, which are nevertheless generated during bypass, are predominately mutagenic. The crystal structures of eight ternary Dpo4 complexes, with either correct or incorrect terminal 3′-partner bases opposite m1G and m3C lesions and cognate dNTP paired with a template base 5′ to the lesion site, reveal the basis of the observed blockage and mutagenicity. Moreover, the observed alignments of m1G- and m3C- lesions with C, T, A, and G partner bases, which reveal pronounced primer terminal nucleotide distortion, have important implications for the efficiency of recognition by ABH2 and, thus, the cytotoxicity and mutagenicity of these lesions. To obtain the crystals of the Dpo4 extension ternary complexes with correct as well as with misinserted 3′ terminal primer bases opposite the m1G and m3C lesions (Figure 1A) we used 5′-CTAAC[X∗]C-…3′ 19-mer templates, where [X∗] is m1G or m3C, and 2′,3′-dideoxy-Y terminated 13-mer primer strands, where Y is C, G, T, or A (Figure 1B). An incoming dGTP was added to pair with the template base C flanking the m1G or m3C on its 5′-side. The structures of the Dpo4 m1G-modified (designated m1G⋅C, m1G⋅G, m1G⋅T, m1G⋅A) and m3C-modified (m3C⋅G, m3C⋅A, m3C⋅C, m3C⋅T) ternary complexes were solved by the molecular replacement method. The crystal data, together with the data collection and refinement statistics are summarized in Table 1.Table 1Data Collection and Refinement Statisticsm1G⋅Cm1G⋅Tm1G⋅Am1G⋅Gm3C⋅Cm3C⋅Tm3C⋅Am3C⋅GSpace groupP1P1P21212P21P21P21P21P21Cell dimensions a, b, c (Å)52.2, 62.1, 91.552.9, 61.6, 92.793.2, 110.4, 51.952.4, 110.4, 102.352.9, 109.3, 100.752.9, 109.9, 100.352.9, 109.3, 100.852.3, 110.0, 101.5 α, β, γ (°)99.0, 103.8, 93.798.5, 103.8, 93.390.0, 90.0, 90.090.0, 101.3, 90.090.0, 101.1, 90.090.0, 101.0, 90.090.0, 101.1, 90.090.0, 101.4, 90.0Complexes per AU22122222Resolution range (Å)aValues in parentheses are for highest-resolution shell.20–2.25(2.31–2.25)20–1.89(1.93–1.89)20–2.80(2.91–2.80)20–3.20(3.31–3.20)20–2.50(2.57–2.50)20–2.80(2.90–2.80)20–2.70(2.79–2.70)20–2.80(2.89–2.80)Rmerge (%)10.7 (75.0)4.3 (45.0)6.6 (36.2)18.3 (39.4)7.9 (47.6)8.4 (61.7)8.7 (73.4)7.3 (35.3)I/σI12.2 (1.7)25.4 (2.1)17.9 (3.6)8.1 (2.0)18.5 (1.9)16.8 (2.1)14.3 (2.6)15.8 (2.2)Completeness (%)98.4 (96.8)95.9 (79.0)94.4 (83.0)96.6 (83.7)96.7 (75.6)96.0 (99.9)98.3 (95.8)98.6 (91.6)Redundancy3.6 (2.8)3.8 (2.8)4.6 (3.3)3.4 (1.9)4.2 (2.9)4.1 (3.9)3.7 (3.6)4.7 (3.5)Resolution range (Å)20–2.2520–1.8920–2.8020–3.2220–2.5020–2.8020–2.7020–2.80Reflections (n)48,43881,25112,44216,78335,52625,33628,87725,974Rfactor/Rfree19.4/23.519.7/23.021.7/26.422.3/29.420.4/23.822.6/26.620.4/24.720.8/25.8Atoms (n) Protein54805480274054805480548054805480 DNA1255123662711811089109610731096 Ligand (dGTP)6262316262626262 Ligand (HEPES)30—15—15—15— Ion (Ca2+)66366666 Water3266163635129646164 B-factors Protein26.4225.0534.647.337.828.333.128.3 DNA30.8827.1750.1778.651.546.837.441.0 Ligand (dGTP)15.67.6134.744.234.927.628.527.7 Ligand (HEPES)41.1—90.9—51.7—51.4— Ion (Ca2+)31.529.146.767.2652.747.247.644.9 Water32.735.828.626.236.529.327.629.0 Rmsd Bond length (Å)0.0080.0080.0090.0090.0090.0090.0080.009 Bond angles (°)1.421.371.581.441.461.521.441.47AU, asymmetric unit; rmsd, root-mean-square deviation. See also Figure S1.a Values in parentheses are for highest-resolution shell. Open table in a new tab AU, asymmetric unit; rmsd, root-mean-square deviation. See also Figure S1. The overall structure and conformation of the m1G⋅C extension ternary complex with correct primer C base opposite the lesion (Figure 1C) is similar (Cα root-mean-square deviation [rmsd] = 0.99 Å) to the type I unmodified ternary complex (Ling et al., 2001Ling H. Boudsocq F. Woodgate R. Yang W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication.Cell. 2001; 107: 91-102Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar) (see Figure S1 available online). The Dpo4 polymerase embraces the template/primer DNA by the palm, finger, and thumb domains, that are prese" @default.
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• W2076333887 title "Implications for Damage Recognition during Dpo4-Mediated Mutagenic Bypass of m1G and m3C Lesions" @default.
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