Bose Arindam, Surugihalli Chaitra, Pande Paritosh, Champeil Elise, Basu Ashis K
Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
Department of Science, John Jay College of Criminal Justice , New York, New York 10019, United States.
Chem Res Toxicol. 2016 May 16;29(5):933-9. doi: 10.1021/acs.chemrestox.6b00087. Epub 2016 Apr 27.
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-N(2) 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-N(2)-MC and dG-N(2)-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-N(2)-2,7-DAM is a stronger replication block than dG-N(2)-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-N(2) adducts. Analysis of the progeny showed that both adducts were mutagenic, and the mutation frequencies (MF) of dG-N(2)-MC and dG-N(2)-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-N(2)-MC and dG-N(2)-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-N(2)-MC and dG-N(2)-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.
丝裂霉素C（MC）是一种细胞毒性和致突变性抗肿瘤药物，在还原激活后可使DNA烷基化。2,7-二氨基丝裂霉素（2,7-DAM）是MC在肿瘤细胞中的主要代谢产物，它也能使DNA烷基化。MC形成七种DNA加合物，包括单加合物以及链间和链内交联，而2,7-DAM形成两种单加合物。在此，通过构建含有这些加合物的单链质粒并在人胚肾293T细胞中进行复制，比较了由MC和2,7-DAM形成的dG-N(2)加合物的生物学效应。与对照质粒相比，dG-N(2)-MC和dG-N(2)-2,7-DAM的跨损伤合成（TLS）效率分别为38±3%和27±3%。这表明两种加合物均会阻断DNA合成，并且dG-N(2)-2,7-DAM比dG-N(2)-MC是更强的复制阻断剂。在对pol η、pol κ或pol ζ进行小干扰RNA敲低后，每个加合体质粒的TLS均降低。对于两种加合物，pol κ敲低时TLS降低最为显著，这表明pol κ在这些dG-N(2)加合物的TLS中起主要作用。对后代的分析表明，两种加合物均具有致突变性，dG-N(2)-MC和dG-N(2)-2,7-DAM的突变频率（MF）分别为18±3%和10±1%。对于两种加合物，主要的突变类型是G→T颠换。pol η和pol ζ敲低降低了dG-N(2)-MC和dG-N(2)-2,7-DAM的MF，而pol κ敲低则增加了这些加合物的MF。这表明pol κ主要进行无错TLS，而pol η和pol ζ参与易错TLS。当pol η、pol ζ和Rev1同时被敲低时，dG-N(2)-MC和dG-N(2)-2,7-DAM构建体的MF分别最大降低了78%和80%。这一结果强烈表明，与pol κ不同，这三种TLS聚合酶协同进行这些加合物的易错TLS。
