Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain

Abstract

We have previously isolated seven mutants of Escherichia coli which replicate their DNA with increased fidelity. These mutants were isolated as suppressors of the elevated mutability of a mismatch-repair-defective mutL strain. Each mutant was shown to contain a single amino acid substitution in the dnaE gene product, the ␣ (i.e., polymerase) subunit of DNA polymerase III holoenzyme responsible for replicating the E. coli chromosome. The mechanism(s) by which these antimutators exert their effect is of interest. Here, we have examined the effects of the antimutator alleles in a mutD5 mutator strain. This strain carries a mutation in the dnaQ gene, which results in defective exonucleolytic proofreading. Our results show that dnaE mutations also confer a strong antimutator phenotype in this background, the effects being generally much greater than those observed previously in the mutL background. The results suggest that the dnaE antimutator alleles can exert their effect independently of exonucleolytic proofreading activity. The large magnitude of the antimutator effects in the mutD5 background can be ascribed, at least in part, to the (additional) restoration of DNA mismatch repair, which is generally impaired in mutD5 strains because of error saturation. The high mutability of mutD5 strains was exploited to isolate a strong new dnaE antimutator allele on the basis of its ability to suppress the high reversion rate of an A ⅐ T3T ⅐ A transversion in this background. A model suggesting how the dnaE antimutator alleles might exert their effects in proofreading-proficient and -deficient backgrounds is presented.Mutations in DNA may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying a DNA template strand. It is important to understand the factors which contribute to the accuracy of this process. In general, three discrete steps to ensure fidelity have been identified (7,11). In the first, the base selection step, the polymerase strongly favors the insertion of correct nucleotides over incorrect ones. In the second step, fidelity is improved by a polymerase-associated 3Ј35Ј exonuclease, whose joint action with the DNA polymerase allows for the preferential removal of incorrectly inserted nucleotides (proofreading). Third, following replication, mismatches resulting from replication errors are subject to correction by postreplicative mismatch repair (for review, see reference 25), further enhancing overall replication fidelity.In the bacterium Escherichia coli, DNA polymerase III holoenzyme is responsible for most of the chromosomal replication and is the major determinant of fidelity (15,22,40). The polymerase III holoenzyme is a large asymmetric dimer containing some 10 distinct subunits (18 total). From these, the ␣, ε, and subunits bind together tightly to form the polymerase III core. The ␣ subunit encoded by the dnaE gene contains the 5Ј 3 3Ј polymerase activity. The ε subunit encoded by the dnaQ gene contains the 3Ј35Ј exonuclease (proofreading) activity. The role of is as ...

Isolation of a dnaE mutation which enhances RecA‐independent homologous recombination in the Escherichia coli chromosome

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Hypermutability and error catastrophe due to defects in ribonucleotide reductase

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