In Vitro Lesion Bypass Studies of O4-Alkylthymidines with Human DNA Polymerase η

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• 作者：Nicole L. Williams ; Pengcheng Wang ; Jiabin Wu ; Yinsheng Wang
• 刊名：Chemical Research in Toxicology
• 出版年：2016
• 出版时间：April 18, 2016
• 年：2016
• 卷：29
• 期：4
• 页码：669-675
• 全文大小：497K
• 年卷期：0
• ISSN：1520-5010

文摘

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O⁴-alkylthymidine (O⁴-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O⁴-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O⁴-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O⁴-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O⁴-iPrdT, O⁴-iBudT, and O⁴-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O⁴-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.