Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in the Escherichia coli lacl gene of O6-alkylguanine-DNA alkyltransferase-proficient and -deficient strains.
Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the lacl gene of Escherichia coli were recovered from bacteria proficient (Ogt+ Ada+) and deficient (Ogt- Ada-) in O6-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 O6-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 O6-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 O6-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.
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, España.
J
J
Jurado
F
F
Ferrezuelo
C
C
Pueyo
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D004260:DNA Repair / Q000502:physiology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D007763:Lac Operon / Q000187:drug effects*; D008130:Lomustine / Q000494:pharmacology*; D008780:Methyltransferases / Q000172:deficiency* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012097:Repressor Proteins / Q000187:drug effects / Q000235:genetics; D012680:Sensitivity and Specificity
D000970:Antineoplastic Agents; D012097:Repressor Proteins; D008130:Lomustine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase