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

Abstract

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.

Scheme 1. Chemical Structures of MC, DMC, and 2,7-DAM and Their Major DNA Adducts

Not shown are the three epimers of adducts 1–3, which contain a β-linkage to dG at the C1 position of MC. Epimers of adducts 2 and 3 are formed at a high level by DMC.

Figure 1

Effect of siRNA knockdown of TLS polymerases on the extent of replicative bypass of dG-N²-MC (in 5′-CTAGTCG*TATCC-3′) and dG-N²-2,7-DAM (in 5′-CTAGTGG*TATCC-3′). Percent TLS in various polymerase knockdowns was measured using an internal control of unmodified plasmid in which a different 12-mer was ligated. The data represent the mean and standard deviation of results from two independent experiments. HEK 293T cells were treated with negative control (NC) siRNA (WT), whereas the other single, double, or triple polymerase knockdowns are indicated above the bar. TLS result from each knockdown experiment was considered statistically significant (p < 0.02) (except in pol η-knockdown cells) compared to that from HEK 293T cells treated with NC siRNA (WT). The p value of %TLS for each knockdown was calculated using a two-tailed, unpaired Student’s t test.

Figure 2

Mutational frequency of dG-N²-MC (in 5′-CTAGTCG*TATCC-3′) and dG-N²-2,7-DAM (in 5′-CTAGTGG*TATCC-3′) in HEK 293T cells cotransfected with NC siRNA (WT) or siRNA for single, double, or triple polymerase knockdowns (shown above the bar). The data represent the average of two independent experiments (shown in Table S2A–F).