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 O(6)- and O(4)-positions, respectively, but that transversions might result from alkylation of thymine at the O(2)-position. To test this idea, the gene encoding O(6)-alkylguanine-DNA methyltransferase (ogt) was recombined into a plasmid to overexpress the cellular levels of this enzyme. Ogt protein can de-alkylate O(6)-alkylguanine and O(4)-alkylthymine, but not O(2)-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 O(6)-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 O(4)-alkylthymine. The frequency of transversions, in comparison, was essentially unaltered. These results implicate O(2)-alkylthymine as a likely candidate for transversion mutagenesis induced by ENU.