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.

The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra.

“…From the series of seven antimutators (FIJAL-KOWSKA, DUNN and SCHAAPER 1993), two (dnaE91 I and dnaE915) were selected for spectral analysis because they presented the most consistent antimutator effects when inspecting different mutational markers (FIJALKOWSKA, DUNN and SCHAAPER 1993). The spontaneous mutant frequencies obtained with these alleles in the mutL background are presented in Table 1.…”

“…Into this strain mutL::Tn5 was introduced by P1 transduction from donor strain ES1293 (SIEGEL et al 1982). The dnaE911 and dnaE915 antimutator alleles were then introduced by P1 transduction using their linkage with transposon rae502::TnlO as described (FIJALKOWSKA, DUNN and SCHAAPER 1993). Transductants carrying the antimutator allele were detected by backcrossing rae502::TnlO into strain NR9360 and testing for antimutator phenotype using the papillation assay as described (FIJALKOWSKA, DUNN and SCHAAPER 1993).…”

“…The dnaE911 and dnaE915 antimutator alleles were then introduced by P1 transduction using their linkage with transposon rae502::TnlO as described (FIJALKOWSKA, DUNN and SCHAAPER 1993). Transductants carrying the antimutator allele were detected by backcrossing rae502::TnlO into strain NR9360 and testing for antimutator phenotype using the papillation assay as described (FIJALKOWSKA, DUNN and SCHAAPER 1993). Finally, F'prolac (F'128-27), which provides the target for scoring lacl forward mutations and whose features have been described (SCHAAPER and DUNN 1991), was introduced by conjugation.…”

“…Because mutations in mismatch repair-deficient strains are largely transitions (SCHAAPER and DUNN 1987a), the antimutators may be assumed to reduce transition errors. Whether other mutations, such as transversions, are also affected was initially addressed by studying the effect in a mutT-defective strain (FIJALKOWSKA, DUNN and SCHAAPER 1993). mutT mutators produce exclusively A. T 3 C G transversions resulting from A .G mispairings during DNA replication (SCHAAPER and DUNN 1987b;AKIYAMA et al 1989;MAKI and SEKICUCHI 1992).…”

“…Our results showed that the antimutators were capable of reducing the mutT mutator activity, indicating that the antimutators were also capable of reducing this type of error. On the other hand, experiments in a recA730 background, where a mutator effect results from the constitutive induction of the SOS system (WITKIN et al 1982;WALKER 1984) and mutations are likely to be transversions as well (MILLER and LOW 1984;YATAGAI, HALLIDAY and GLICKMAN 1991), showed little or no susceptibility of this type of mutagenesis to the antimutators (FIJAL-KOWSKA, DUNN and SCHAAPER 1993). Thus, antimutators appear to have a defined specificity which could depend on both the type of mutation and the genetic background in which it is generated.…”