MUTANTS OF ESCHERICHIA-COLI WITH INCREASED FIDELITY OF DNA-REPLICATION

To improve our understanding of the role of DNA replication fidelity i n 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 dn aE and dnaQ genes, which encode, respectively, the DNA polymerase (alp ha subunit) and 3' exonucleolytic proofreading activity (epsilon subun it) of DNA polymerase III holoenzyme, the enzyme primarily responsible for replicating the bacterial chromosome. The mutated bacteria were s creened for antimutator phenotype in a strain defective in DNA mismatc h repair (mutL), using a papillation assay based on the reversion of t he galK2 mutation. In a mutL strain, mutations result primarily from D NA replication errors. Among 10,000 colonies, seven mutants were obtai ned whose level of papillation was reduced 5-30-fold. These mutants al so displayed decreased mutation frequencies for rifampicin or nalidixi c acid resistance as well as for other markers. Mapping by PI transduc tion and complementation showed each to reside in dnaE. These observat ions support the idea that the mutants represent antimutators which re plicate their DNA with increased fidelity. Mutation rates were reduced in both mutL and mutT backgrounds, but mutagenesis by ultraviolet lig ht was not significantly affected, suggesting that the antimutator eff ect may be largely restricted to normal DNA replication.