期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 291, 期 46, 页码 24304-24313出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.755462
关键词
DNA damage; DNA polymerase; enzyme kinetics; mass spectrometry (MS); X-ray crystallography
资金
- National Institutes of Health [R01 ES010375, R01 ES010546, P01 CA160032]
O-6-Methyl-2-deoxyguanosine (O-6-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the translesion Y-family human DNA polymerases (hpols), hpol is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol . Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O-6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O-6-MeG and 2-deoxythymidine-5-[(, )-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O-6- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol to insert both dC and dT opposite the O-6-MeG lesion with similar efficiencies. Comparison of the hpol active site configurations with either O-6-MeG:dC or O-6-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of approximate to 4, helps rationalize the more error-prone synthesis opposite the lesion by hpol eta.
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