Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene

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• 作者：Srikanth Kotapati ; Susith Wickramaratne ; Amanda Esades ; Emily J. Boldry ; Danae Quirk Dorr ; Matthew G. Pence ; F. Peter Guengerich ; Natalia Y. Tretyakova
• 刊名：Chemical Research in Toxicology
• 出版年：2015
• 出版时间：July 20, 2015
• 年：2015
• 卷：28
• 期：7
• 页码：1496-1507
• 全文大小：573K
• ISSN：1520-5010

文摘

N⁶-(2-Hydroxy-3-buten-1-yl)-2鈥?deoxyadenosine (N⁶-HB-dA I) and N⁶,N⁶-(2,3-dihydroxybutan-1,4-diyl)-2鈥?deoxyadenosine (N⁶,N⁶-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N⁶ position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the N⁶-H atom of adenine is required for Watson鈥揅rick hydrogen bonding with thymine, N⁶-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N⁶-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N⁶-HB-dA I and (R,R)-N⁶,N⁶-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases 尾, 畏, 喂, and 魏. While (S)-N⁶-HB-dA I was readily bypassed by all four enzymes, only polymerases 畏 and 魏 were able to carry out DNA synthesis past (R,R)-N⁶,N⁶-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N⁶-HB-dA I. In contrast, hPol 尾 was completely blocked by (R,R)-N⁶,N⁶-DHB-dA, while hPol 畏 and 魏 inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (S)-N⁶-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N⁶,N⁶-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N⁶-HB-dA I lesions are not miscoding, but that exocyclic (R,R)-N⁶,N⁶-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson鈥揅rick pairs with dT.