MUTATIONAL SPECIFICITY OF 1-(2-CHLOROETHYL)-3-CYCLOHEXYL-1-NITROSOUREA IN THE ESCHERICHIA-COLI LAC1 GENE OF O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE-PROFICIENT AND ALKYLTRANSFERASE-DEFICIENT STRAINS

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso (CCNU) in the lad gene of Escherichia coli were recovered from bacteri a proficient (Ogt(+) Ada(+)) and deficient (Ogt(-) Ada(-)) in O-6-alky lguanine-DNA alkyltransferase activity. A CCNU dose of 1 mM was select ed for DNA sequence analysis. A total of 245 induced mutations were ch aracterized. The mutations were almost exclusively (95%) CG-->AT trans itions, indicating that CCNU-induced mutations arose in bacteria prima rily from misreplication of O-6-chloroethylguanine, in total agreement with results obtained for monofunctional alkylating agents. The distr ibution 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 b ase-pairs were on average 2.8 times more likely to mutate than those f lanked by G:C base-pairs on at least one side. This bias disappeared i n the Ogt(-) Ada(-) genetic background, thereby providing evidence tha t O-6-chloroethylated guanines adjacent to G:C base-pairs are better t argets for bacterial alkyltransferase than those not adjacent to G:C b ase-pairs. We recently reported a similar bias for ethyl methanesulfon ate, 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 maj or role in removing lesions responsible for GC-->AT transitions induce d by CCNU, influencing their ultimate distribution with respect to seq uence context. (C) 1995 Wiley-Liss, Inc.