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

Forward mutations induced by 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea (a) in the lacl 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%) GC→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. © 1995 Wiley‐ Liss, Inc.