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) prod uced tRNA suppressor mutations (G:C to A:T) but only ENU produced a signifi cant number of backmutations (A:T to G:C, A:T to T:A and A:T to C:G). Furth er, the ENU-induced transversions were absent in a UmuC-defective strain. T his suggested that transition mutations could result from alkylation of gua nine or thymine at the O-6- and O-4-positions, respectively, but that trans versions might result from alkylation of thymine at the O-2-position. To te st 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 E NU and the resulting mutations were analyzed. With either MNU or ENU, the f requency of GlnV(o) suppressors was reduced about 70-fold in the Ogt-overex pressing cells, suggesting that Ogt eliminated O-6-alkylguanine. Similarly, GlnU(o) suppressor frequencies were substantially reduced. In contrast, th e reduction in frequency for the backmutations was slight, only about 2.5-f old 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 frequ ency of transversions, in comparison, was essentially unaltered. These resu lts implicate O-2-alkylthymine as a likely candidate for transversion mutag enesis induced by ENU. (C) 2001 Elsevier Science B.V. All rights reserved.