Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase 魏 Involved in Error-Free Bypass across Bulky N2-Guanyl DNA Adducts

详细信息    查看全文

• 作者：Insil Song ; Eun-Jin Kim ; In-Hyeok Kim ; Eun-Mi Park ; Kyung Eun Lee ; Joo-Ho Shin ; F. Peter Guengerich ; Jeong-Yun Choi
• 刊名：Chemical Research in Toxicology
• 出版年：2014
• 出版时间：May 19, 2014
• 年：2014
• 卷：27
• 期：5
• 页码：919-930
• 全文大小：415K
• 年卷期：v.27,no.5(May 19, 2014)
• ISSN：1520-5010

文摘

DNA polymerase (pol) 魏, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky N²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol 魏 variants positioned in the polymerase core domain, using the recombinant pol 魏 (residues 1鈥?26) protein and the DNA template containing an N²-CH₂(9-anthracenyl)G (N²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol 魏. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in k_cat/K_m for dCTP insertion opposite G and N²-AnthG compared to that of wild-type pol 魏. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in k_cat/K_m for next-base extension from C paired with N²-AnthG, compared to that of wild-type pol 魏. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol 魏 opposite bulky N²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol 魏 genetic variations translate into individual differences in susceptibility to genotoxic carcinogens.