Comparative Error-Free and Error-Prone Translesion Synthesis of N2-2′-Deoxyguanosine Adducts Formed by Mitomycin C and Its Metabolite, 2,7-Diaminomitosene, in Human Cells

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-adautts 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 eta, pol kappa, or pol zeta. For both adducts, the most significant reduction occurred with knockdown of pol kappa, which suggests that pol kappa. 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 eta and pol zeta reduced the MF of dG-N-2-MC and dG-N-2-2,7-DAM, whereas knockdown of pol kappa increased the MF of these adducts, This suggests that pol kappa predominantly carries out error-free TLS, whereas pol eta and pol zeta 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 eta, pol zeta, and Rev1 were simultaneously knocked down. This result strongly suggests that, unlike pol kappa, these three TLS polymerases cooperatively perform the error-prone TLS of these adducts.