Kinetic Analysis of the Bypass of a Bulky DNA Lesion Catalyzed by Human Y-Family DNA Polymerases

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• 作者：Shanen M. Sherrer ; Laura E. Sanman ; Cynthia X. Xia ; Eric R. Bolin ; Chanchal K. Malik ; Georgia Efthimiopoulos ; Ashis K. Basu ; Zucai Suo
• 刊名：Chemical Research in Toxicology
• 出版年：2012
• 出版时间：March 19, 2012
• 年：2012
• 卷：25
• 期：3
• 页码：730-740
• 全文大小：421K
• 年卷期：v.25,no.3(March 19, 2012)
• ISSN：1520-5010

文摘

1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG^AP). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPol魏), iota (hPol喂), eta (hPol畏), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dG^AP on a synthetic DNA template but that hRev1 was stopped after lesion bypass. The time required to bypass 50% of the dG^AP sites (t₅₀^bypass) encountered by hPol畏, hPol魏, and hPol喂 was determined to be 2.5 s, 4.1 s, and 106.5 s, respectively. The efficiency order of catalyzing translesion synthesis of dG^AP (hPol畏 > hPol魏 > hPol喂 hRev1) is the same as the order for these human Y-family enzymes to elongate undamaged DNA. Although hPol畏 bypassed dG^AP efficiently, replication by both hPol魏 and hPol喂 was strongly stalled at the lesion site and at a site immediately downstream from dG^AP. By employing presteady state kinetic methods, a kinetic basis was established for polymerase pausing at these DNA template sites. Besides efficiency of bypass, the fidelity of those low-fidelity polymerases at these pause sites was also significantly decreased. Thus, if the translesion DNA synthesis of dG^APin vivo is catalyzed by a human Y-family DNA polymerase, e.g., hPol畏, the process is certainly mutagenic.