ROLE OF G-]T TRANSVERSIONS IN THE MUTAGENICITY OF ALKYLPEROXYL RADICALS - INDUCTION OF ALKALI-LABILE SITES IN BACTERIOPHAGE-M13MP19

The mutagenicity of peroxyl radicals, ubiquitous products of lipid per oxidation, was assessed using an in vitro M13 forward mutational assay . Single-stranded M13mp19 plasmids were incubated with a range of conc entrations of the azo initiator 2,2'-azobis(2-amidinopropane) hydrochl oride, and then transfected into competent, SOS-induced Escherichia co li JM105 cells. Incubation with peroxyl radicals produced a concentrat ion-dependent decrease in phage survival, with a 500 mu M concentratio n of the azo initiator reducing the transfection efficiency by more th an 90% while inducing a corresponding B-fold increase in lacZ(alpha), mutation frequencies. Peroxyl radical-induced mutagenesis was complete ly prevented by the peroxyl radical scavenger Trolox. Automated DNA se quence analysis of the lacZ(alpha), gene of 100 peroxyl radical-induce d mutants revealed that the most frequent sequence changes were base p air substitutions (92/95), with G-->T transversions predominating (73/ 92). Alkaline treatment prior to transfection diminished the mutagenic ity of damaged plasmids to a level resembling that of unmodified DNA. While abasic sites might account for the sensitivity to alkaline cleav age, the possibility that unidentified nonabasic alkaline-labile lesio ns also contribute to peroxyl radical mutagenesis cannot be excluded. Collectively, these findings raise the possibility that DNA damage cau sed by a major class of endogenous radicals contributes to one of the most common spontaneous mutational events, the G-->T transversion.