INFLUENCE OF DNA-REPAIR BY ADA AND OGT ALKYLTRANSFERASES ON THE MUTATIONAL SPECIFICITY OF ALKYLATING-AGENTS

We investigated the influence of the alkyltransferases (ATases) encode d by the ada and ogt genes of Escherichia coli on the mutational speci ficity of alkylating agents. A new mutational assay for selection of s upF(-) mutations in shuttle-vector plasmids was used. Treating plasmid -bearing bacteria with N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitroso urea (ENU), and ethyl methanesulfonate (EMS) dramatically increased th e mutation frequency (from 33-fold to 789-fold). The vast majority of mutations (89-100%) were G:C-->A:T transitions. This type of mutation increased in ada(-) (MNU) or ogt(-) (ENU) bacteria, suggesting that re pair of O-6-methylguanine by ada ATase and repair of O-6-ethylguanine by ogt ATase contribute mainly to the decrease in G:C-->A:T transition s. The analysis of neighboring base sequences revealed an overabundanc e of G:C-->A:T transitions at 5'-GG sequences. The 5'-PuG bias increas ed in ATase-defective cells, suggesting that these sequences were not refractory to repair. G:C-->A:T transitions occurred preferentially in the untranscribed strand after in vivo exposure. That this strand spe cificity was detected even in bacteria devoid of ATase activity (ada(- ) ogt(-)) and not after in vitro mutagenesis suggests a bias for damag e induction rather than for DNA repair. Highly significant differences were found between the in vivo and in vitro incidences of G:C-->A:T s ubstitutions at the two major hotspots, positions 123 (5'-GGG-3'; anti sense strand) and 168 (5'-GGA-3'; sense strand). These results are exp lained by differences in the probability of formation of stem-loop str uctures in vivo and in vitro. (C) 1994 Wiley-Liss, Inc.