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

Mol Carcinog. 1995 Dec;14(4):233-9. doi: 10.1002/mc.2940140403.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Base Sequence
  • DNA Repair / physiology
  • Escherichia coli / drug effects
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics*
  • Lac Operon / drug effects*
  • Lomustine / pharmacology*
  • Methyltransferases / deficiency*
  • Methyltransferases / metabolism*
  • Molecular Sequence Data
  • Mutation
  • O(6)-Methylguanine-DNA Methyltransferase
  • Repressor Proteins / drug effects
  • Repressor Proteins / genetics
  • Sensitivity and Specificity


  • Antineoplastic Agents
  • Repressor Proteins
  • Lomustine
  • Methyltransferases
  • O(6)-Methylguanine-DNA Methyltransferase