The major mitomycin C-DNA monoadduct is cytotoxic but not mutagenic in Escherichia coli

To determine the mutagenic and genotoxic properties of the major guanine N2-adduct formed by the antitumor drug mitomycin C, we have synthesized a decanucleotide, d(TTACG[MC]TATCT), containing the adduct, which was inserted into a gapped bacteriophage M13 genome. Analysis of the constructed genome indicated that 41% ligation of the adducted 10-mer occurred on both sides of the gap, whereas the control 10-mer ligated with 34% efficiency. After transfection of the adducted single-stranded M13 DNA into Escherichia coli, the adduct was found to be highly genotoxic. Viability of the adducted genome in a repair-competent strain was only 7%, which increased to 12% and 15% upon induction of SOS by irradiating the cells with 254-nm light at 20 and 50 J/m2, respectively. Even lower viability of 2%, 4.6%, and 0.2% was observed in uvrA, uvrB, and uvrC strains, respectively, which increased up to 10-fold with SOS. An examination of the surviving phage populations revealed that the adduct was not detectably mutagenic. No mutants from the repair-proficient strain were detected after analysis of more than 2500 progeny phage. Only 0.2% of the survivors were mutants in the uvrA strain. It is uncertain, however, if they were induced by the adduct, since all the mutants showed untargeted mutations. We conclude that the major guanine N2-adduct formed by mitomycin C is cytotoxic but not appreciably mutagenic in E. coli.