Title: The Spacious Active Site of a Y-Family DNA Polymerase Facilitates Promiscuous Nucleotide Incorporation Opposite a Bulky Carcinogen-DNA Adduct
Abstract: Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N2-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N2-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol κ. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N2-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N2-dG adduct, albeit to a lesser extent than unmodified guanine, and the Vmax values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N2-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion. Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N2-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N2-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol κ. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N2-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N2-dG adduct, albeit to a lesser extent than unmodified guanine, and the Vmax values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N2-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion. Members of the Y-family of DNA polymerases have been implicated in the bypass of DNA damage that poses blocks to high fidelity replicative A-family polymerases (1Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Crossref PubMed Google Scholar, 2Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 3Suzuki N. Ohashi E. Kolbanovskiy A. Geacintov N.E. Grollman A.P. Ohmori H. Shibutani S. Biochemistry. 2002; 41: 6100-6106Crossref PubMed Scopus (139) Google Scholar, 4Rechkoblit O. Zhang Y. Guo D. Wang Z. Amin S. Krzeminsky J. Louneva N. Geacintov N.E. J. Biol. Chem. 2002; 277: 30488-30494Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 5Bresson A. Fuchs R.P. EMBO J. 2002; 21: 3881-3887Crossref PubMed Scopus (87) Google Scholar, 6Wagner J. Etienne H. Janel-Bintz R. Fuchs R.P. DNA Repair (Amst). 2002; 1: 159-167Crossref PubMed Scopus (0) Google Scholar, 7Shen X. 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Nucleic Acids Res. 2001; 29: 4607-4616Crossref PubMed Google Scholar). In the present work, we have carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in the incorporation of different nucleotides opposite the bulky (+)-ta[BP]G adduct catalyzed by Dpo4. Our experimental results reveal that this enzyme is able to incorporate all four bases opposite the damaged guanine at similar rates, and the simulations provide molecular explanations for the accommodation of each incoming nucleotide opposite this bulky carcinogen-DNA adduct. Damaged Oligodeoxynucleotides and Primer Extension Template Construction—The oligodeoxynucleotides were synthesized using automated methods based on p