Mutagenic Spectrum Resulting from DNA Damage by Oxygen Radicals

Oxygen free radicals are highly reactive species that damage DNA and cause mutations. We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe²⁺. The Fe²⁺-treated DNA was transfected into competent Escherichia coli, and mutants within the nonessential lac Zα gene for β-galactosidase were identified by decreased α-complementation. The frequency of mutants obtained with 10 μM Fe²⁺ was 20- to 80-fold greater than that obtained with untreated DNA. Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS “error-prone” response. The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals. The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions. Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G → C transversions, followed by C → T transitions and G → T transversions. The clustering of mutations at distinct gene positions suggests that Fe²⁺/oxygen damage to DNA is nonrandom. This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.