MUTATION SPECTRA ANALYSIS SUGGESTS THAT 2-CHLOROETHYL)-N'-CYCLOHEXYL-N-NITROSOUREA-INDUCED LESIONS ARE SUBJECT TO TRANSCRIPTION-COUPLED REPAIR IN ESCHERICHIA-COLI

determine the influence of some bacterial DNA repair pathways on the m utagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-n itrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capac ities (uvr(+)ada(+)ogt(+), uvr(-)ada(+)ogt(+), and uvr(-)ada(-)ogt(-)) . Despite the differences in repair capacities, no statistically signi ficant difference in survival and mutability was observed among the te sted strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base -pair substitutions, most of which (more than 96%) were GC-->AT transi tions (the mutated G being almost exclusively preceded 5' by a purine) . Mutation distribution was not random. Position 160 (5'-G (G) under b ar T-3', nontranscribed (NT) strand) was a uvr(+)ada(+)og(+)-specific hot-spot. Position 123 (5'-G (G) under bar G-3', NT strand) was a comm on hot-spot but significantly more mutable in repair-proficient strain s than in repair-deficient strains. Conversely, position 168 (5'-G (G) under bar A-3; transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By app lying a computer program for comparison of mutational spectra, we foun d that the uvr(+) mutational spectrum was significantly different from those obtained in uvr(-) strains, whereas in the uvr(-) background, n o difference was observed between mutation spectra in ada(+)ogt(+) ver sus ada(-)ogt(-) strains. Our results are consistent with the hypothes is that O-6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC-->AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr(+) th an in uvr(-) strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli. (C) 1997 Wil ey-Liss, Inc.