Ethyl-nitrosourea-induced mutagenesis in Escherichia coli: Effects of neighboring base and DNA methyltransferase

In order to better understand mutagenesis induced by alkylating chemicals, this study investigated (1) the influence of different neighboring base contexts on the production of base substitutions generated by N-ethyl-N-nitrosourea (ENU), and (2) the role of O2-alkylT in the production of transversion mutations by ENU. A set of strains having all possible bases neighboring an ochre (TAA) nonsense defect in the tyrA gene of Escherichia coli were employed and backmutations of the defect were induced by two separate doses of ENU. Base substitution mutations were investigated by direct sequencing and colony-hybridization methods. Studies revealed that (1) mutations occurring at 5′-PuT sites were produced better, on average, than mutations involving 5′-PyT sites, and 5′ -TT sites contributed the least to the formation of mutations, (2) the order of preference for transitions was 5′-GT > 5 ′-AT, 5′-CT > 5′-TT, and (3) A:T to C:G transversions at the first position of the defect (i.e. GAA mutations) were produced best at 5′-AT sites, while A:T to T:A transversions occurring at the third position (TAT mutations) occurred more often at 5′-GT sites. These two transversion mutations occur in opposite strands during DNA replication; the GAA mutation results from lagging strand synthesis while the TAT mutation results from leading strand replication. Since the ENU-induced transversions were absent in a UmuC-defective strain, the results suggested that a mutation preference site should be determined by combination of factors which include preferential production of ethylated bases, preferential repair (including leading vs lagging strand differences) and type of mutation (transition or transversion).; For the second part of this study, a gene encoding O6-alkylguanine-DNA methyltransferase (ogt) was recombined onto a plasmid and introduced into E. coli to overexpress the level of this enzyme. Ogt protein can dealkylate O6-alkylguanine and O 4-alkylthymine, but not O2-alkylthymine. Cells with Ogt protein overexpressed were exposed to different concentrations of N-methyl-N-nitrosourea (MNU) or ENU and the resulting mutations were analyzed. The frequency of tRNA suppressor mutations (G:C to A:T) were significantly reduced, but the reduction in the frequency of backmutations was slight. However, DNA sequence analysis of the backmutations showed that only A:T to G:C transitions were affected by Ogt overexpression. The frequency of transversions, in comparison, was essentially unaltered. These results implicate O2-alkylthymine as a likely candidate for transversion mutagenesis induced by ENU.