Comparative Error-Free and Error-Prone Translesion Synthesis of N(2)-2'-Deoxyguanosine Adducts Formed by Mitomycin C and Its Metabolite, 2,7-Diaminomitosene, in Human Cells.
Comparative Error-Free and Error-Prone Translesion Synthesis of N(2)-2'-Deoxyguanosine Adducts Formed by Mitomycin C and Its Metabolite, 2,7-Diaminomitosene, in Human Cells.
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N（2）-2'-脱氧鸟氨酸加合物的无误差和容易出现的转移合成由丝裂霉素C及其代谢物在人类细胞中形成的2,7-二氨基辛素。
DOI：
10.1021/acs.chemrestox.6b00087
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发表时间：
2016-05-16
影响因子：
4.1
通讯作者：
Basu AK
中科院分区：
文献类型：
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作者：
Bose A;Surugihalli C;Pande P;Champeil E;Basu AK
丝裂霉素C （Mitomycin C， MC）是一种细胞毒性和致突变性的抗肿瘤药物，通过还原激活使DNA烷基化。2,7-二氨基糖（2,7- dam）是肿瘤细胞中MC的主要代谢物，它也使DNA烷基化。MC形成7种DNA加合物，包括单加合物和链间和链内交联，而2,7- dam形成两种单加合物。本研究通过构建含有MC和2,7- dam加合物的单链质粒并在人胚胎肾293T细胞中复制，比较了MC和2,7- dam形成的dG-N2加合物的生物学效应。与对照质粒相比，dG-N2-MC和dG-N2-2、7-DAM的翻译合成效率分别为38±3%和27±3%。这表明这两种加合物都阻断DNA合成，并且dg - n2 -2,7- dam是比dG-N2-MC更强的复制阻断物。当siRNA敲低pol η、pol κ或pol ζ时，每个内合结构体的TLS都减少。对于这两种加合物，最显著的减少发生在pol κ的下调，这表明pol κ在这些dG-N2加合物的TLS中起主要作用。后代分析表明，两种加合物均具有致突变性，dG-N2-MC和dG-N2-2、7-DAM的突变频率（MF）分别为18±3%和10±1%。对于这两种加合物，主要的突变类型是G→T转化。敲低pol η和pol ζ降低了dG-N2-MC和dg - n2 -2,7- dam的MF，而敲低pol κ则增加了这些加合物的MF。这表明pol κ主要执行无错误的TLS，而pol η和pol ζ参与易出错的TLS。当pol η， pol ζ和Rev1同时被敲除时，dG-N2-MC和dG-N2-2,7-DAM的MF分别减少了78%和80%。这一结果强烈表明，与pol κ不同，这三种TLS聚合酶协同执行这些加合物容易出错的TLS。
Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates DNA upon reductive activation. 2,7-Diaminomitosene (2,7-DAM) is a major metabolite of MC in tumor cells, which also alkylates DNA. MC forms seven DNA adducts, including monoadducts and inter- and intrastrand cross-links, whereas 2,7-DAM forms two monoadducts. Herein, the biological effects of the dG-N2 adducts formed by MC and 2,7-DAM have been compared by constructing single-stranded plasmids containing these adducts and replicating them in human embryonic kidney 293T cells. Translesion synthesis (TLS) efficiencies of dG-N2-MC and dG-N2-2,7-DAM were 38 ± 3 and 27 ± 3%, respectively, compared to that of a control plasmid. This indicates that both adducts block DNA synthesis and that dG-N2-2,7-DAM is a stronger replication block than dG-N2-MC. TLS of each adducted construct was reduced upon siRNA knockdown of pol η, pol κ, or pol ζ. For both adducts, the most significant reduction occurred with knockdown of pol κ, which suggests that pol κ plays a major role in TLS of these dG-N2 adducts. Analysis of the progeny showed that both adducts were mutagenic, and the mutation frequencies (MF) of dG-N2-MC and dG-N2-2,7-DAM were 18 ± 3 and 10 ± 1%, respectively. For both adducts, the major type of mutation was G → T transversions. Knockdown of pol η and pol ζ reduced the MF of dG-N2-MC and dG-N2-2,7-DAM, whereas knockdown of pol κ increased the MF of these adducts. This suggests that pol κ predominantly carries out error-free TLS, whereas pol η and pol ζ are involved in error-prone TLS. The largest reduction in MF by 78 and 80%, respectively, for dG-N2-MC and dG-N2-2,7-DAM constructs occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down. This result strongly suggests that, unlike pol κ, these three TLS polymerases cooperatively perform the error-prone TLS of these adducts.