SPONTANEOUS MUTATION IN ESCHERICHIA-COLI CONTAINING THE DNAE911 DNA-POLYMERASE ANTIMUTATOR ALLELE

We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants have a mutation in t he dnaE gene, encoding the cu subunit of DNA polymerase III. They were isolated in a mismatch-repair-defective mutL background, in which mut ations can be considered to represent uncorrected DNA replication erro rs. In the present study we analyze the effect of one of these alleles , dnaE911, an spontaneous mutagenesis in a mismatch-repair-proficient background. In this background, spontaneous mutations may be the sum o f uncorrected replication errors and mutations resulting from other pa thways. Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation. The data show that dnaE911 decreases the level of Rifr, lad and galK mutations in this background by 1.5-2-fold. DNA sequencing o f 748 forward mutants in the lad gene reveals that this effect has a c lear specificity. Transversions are decreased by similar to 3-fold, wh ereas transitions, frameshifts, deletions and duplications remain esse ntially unchanged. Among the tranversions, A.T --> T.A are affected mo st strongly (similar to 6-fold). In addition to this effect on transve rsions within the lacI gene, one previously recognized A.T --> G.C bas e-pair substitution hotspot in the lac operator is also reduced (simil ar to 5-fold). The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.