Overexpression of Ogt reduces MNU and ENU induced transition, but not transversion, mutations in E. coli

Studies of alkylation-induced mutations in Escherichia coli FX-11 revealed that both N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) produced tRNA suppressor mutations (G:C to A:T) but only ENU produced a significant number of backmutations (A:T to G:C, A:T to T:A and A:T to C:G). Further, the ENU-induced transversions were absent in a UmuC-defective strain. This suggested that transition mutations could result from alkylation of guanine or thymine at the O⁶- and O⁴-positions, respectively, but that transversions might result from alkylation of thymine at the O²-position. To test this idea, the gene encoding O⁶-alkylguanine-DNA methyltransferase (ogt) was recombined into a plasmid to overexpress the cellular levels of this enzyme. Ogt protein can de-alkylate O⁶-alkylguanine and O⁴-alkylthymine, but not O²-alkylthymine. Cells harboring the plasmid (or a control plasmid lacking the ogt gene) were exposed to different concentrations of MNU or ENU and the resulting mutations were analyzed. With either MNU or ENU, the frequency of GlnV^o suppressors was reduced about 70-fold in the Ogt-overexpressing cells, suggesting that Ogt eliminated O⁶-alkylguanine. Similarly, GlnU^o suppressor frequencies were substantially reduced. In contrast, the reduction in frequency for the backmutations was slight, only about 2.5-fold with MNU and less than two-fold for ENU. However, DNA sequence analysis of the backmutations showed that only A:T to G:C transitions were affected by overexpression of Ogt, suggesting repair of O⁴-alkylthymine. The frequency of transversions, in comparison, was essentially unaltered. These results implicate O²-alkylthymine as a likely candidate for transversion mutagenesis induced by ENU.