The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to 
transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly 
restoring these alkylated DNA lesions to guanine and thymine. In addition to 
being exquisitely sensitive to the mutagenic effects of methylating agents, E. 
coli ada ogt null mutants display a higher spontaneous mutation rate than the 
wild type. Here, we determined which base substitution mutations are elevated in 
the MTase-deficient cells by monitoring the reversion of six mutated lacZ 
alleles that revert via each of the six possible base substitution mutations. 
During exponential growth, the spontaneous rate of G:C to A:T transitions and 
G:C to C:G transversions was elevated about fourfold in ada ogt double mutant 
versus wild-type E. coli. Furthermore, compared with the wild type, stationary 
populations of the MTase-deficient E. coli (under lactose selection) displayed 
increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) 
and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, 
and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated 
spontaneous mutation rates for any base substitution event, and the cloned ada 
and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt 
MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can 
repair the endogenously produced DNA lesions responsible for each of the five 
base substitution events that are elevated in MTase-deficient cells. Simple 
methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions 
in these strains but did not significantly induce G:C to C:G, A:T to C:G, and 
A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak 
methylating agent) is not the only metabolite responsible for endogenous DNA 
alkylation and that at least some of the endogenous metabolites that cause 
O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.