Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in the Escherichia coli lac1 gene of O-6-alkylguanine-DNA alkyltransferase-proficient and -deficient strains

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso (CCNU) in the lad gene of Escherichia coli were recovered from bacteria proficient (Ogt(+) Ada(+)) and deficient (Ogt(-) Ada(-)) in O-6-alkylguanine-DNA alkyltransferase activity. A CCNU dose of 1 mM was selected for DNA sequence analysis. A total of 245 induced mutations were characterized. The mutations were almost exclusively (95%) CG-->AT transitions, indicating that CCNU-induced mutations arose in bacteria primarily from misreplication of O-6-chloroethylguanine, in total agreement with results obtained for monofunctional alkylating agents. The distribution of CCNU-induced GC-->AT mutations was significantly altered by the presence of DNA alkyltransferase activity (P = 0.01). In the Ogt(+) Ada(+) mutational spectrum, guanines flanked on both sides by A:T base-pairs were on average 2.8 times more likely to mutate than those flanked by G:C base-pairs on at least one side. This bias disappeared in the Ogt(-) Ada(-) genetic background, thereby providing evidence that O-6-chloroethylated guanines adjacent to G:C base-pairs are better targets for bacterial alkyltransferase than those not adjacent to G:C base-pairs. We recently reported a similar bias for ethyl methanesulfonate, strengthening the idea that CCNU is acting as a simple ethylating compound. In summary, this paper presents for the first time evidence that DNA repair by O-6-alkylguanine-DNA alkyltransferases plays a major role in removing lesions responsible for GC-->AT transitions induced by CCNU, influencing their ultimate distribution with respect to sequence context. (C) 1995 Wiley-Liss, Inc.