Mutants of Escherichia coli with increased fidelity of DNA replication

Abstract

To improve our understanding of the role of DNA replication fidelity in mutagenesis, we undertook a search for Escherichia coli antimutator strains with increased fidelity of DNA replication. The region between 4 and 5 min of the E. coli chromosome was mutagenized using localized mutagenesis mediated by bacteriophage P1. This region contains the dnaE and dnaQ genes, which encode, respectively, the DNA polymerase (alpha subunit) and 3' exonucleolytic proofreading activity (epsilon subunit) of DNA polymerase III holoenzyme, the enzyme primarily responsible for replicating the bacterial chromosome. The mutated bacteria were screened for antimutator phenotype in a strain defective in DNA mismatch repair (mutL), using a papillation assay based on the reversion of the galK2 mutation. In a mutL strain, mutations result primarily from DNA replication errors. Among 10,000 colonies, seven mutants were obtained whose level of papillation was reduced 5-30-fold. These mutants also displayed decreased mutation frequencies for rifampicin or nalidixic acid resistance as well as for other markers. Mapping by P1 transduction and complementation showed each to reside in dnaE. These observations support the idea that the mutants represent antimutators which replicate their DNA with increased fidelity. Mutation rates were reduced in both mutL and mutT backgrounds, but mutagenesis by ultraviolet light was not significantly affected, suggesting that the antimutator effect may be largely restricted to normal DNA replication.