Mutation spectra analysis suggests that N-(2-Chloroethyl)-N'-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli

Raffaella Iannone, Alberto Inga, Francisco L. Luque-Romero, Paola Menichini, Angelo Abbondandolo, Nieves Abril, Carmen Pueyo, Gilberto Fronza

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To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitros (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr+ada+ogt+, and uvr-ada-ogt-). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC→AT transitions (the mutated C being almost exclusively preceded 5' by a purine). Mutation distribution was not random. Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt+-specific hot-spot. Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains. Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr+ strains, whereas in the uvr+ background, no difference was observed between mutation spectra in ada+ogt+ versus ada+-ogt+ strains. Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of CC→AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli.

Original languageEnglish
Pages (from-to)39-45
Number of pages7
JournalMolecular Carcinogenesis
Issue number1
Publication statusPublished - May 1997



  • DNA repair
  • Hot spot
  • Mutation spectrum
  • N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea
  • supF

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Biology

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