recA-dependent and recA-independent N-ethyl-N-nitrosourea mutagenesis at a plasmid-encoded herpes simplex virus thymidine kinase gene in Escherichia coli

We have compared isogenic recA13/recA⁺ Escherichia coli K-12 strains for the induction by N-ethylN-nitrosourea (ENU) of forward mutations at a plasmid-encoded herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. Treatment of plasmid-bearing bacteria with ENU resulted in a dose-dependent increase in the mutant frequencies of the chromosomal udk locus and of the plasmid HSV-tk locus in both recA13 and recA⁺ strains. Although the recA13 strain was considerably more sensitive to the cytotoxic effects of ENU treatment than was the recA⁺ strain, the ENU-induced mutation frequency at both loci was greater for therecA⁺ strain than for the recA13 strain. When plasmid DNA modified by in vitro reaction with ENU was used to transform recA13, recA⁺, and UV pre-irradiated recA⁺ strains, an increase in the HSV-tk mutant frequency was observed in all 3 cases. The induction of mutations in recA13 and recA⁺ strains followed a similar dose-response, while the ENU-induced HSV-tk mutant frequency was significantly greater for UV pre-irradiated recA⁺ bacteria. These results indicate that fixation of ENU-induced premutagenic lesions can occur by both recA-independent pathways.