Activity of the Escherichia coli mutT mutator allele in an anaerobic environment.

Mutation frequencies for an Escherichia coli mutT strain were measured in both aerobic and anaerobic environments. When cells were grown in a rich medium (L broth), mutation frequencies were similar in both aerobic and anaerobic conditions. In contrast, when grown in a minimal medium, mutT anaerobic mutation frequencies were reduced dramatically compared with aerobic values, which were similar to L broth frequencies. L broth mutT cultures treated with a commercial enzyme complex that reduces free oxygen in the medium also showed strongly reduced anaerobic mutation frequencies. These results indicate that the biological role of the MutT protein is to prevent oxidative damage from becoming mutagenic.

The high mutator activity of the dnaQ49 allele of Escherichia coli is medium-dependent and results from both defective 3'-->5' proofreading and methyl-directed mismatch repair.

The Escherichia coli dnaQ49 mutator allele maps at the dnaQ locus, the structural gene for the epsilon subunit of the DNA polymerase III holoenzyme. Epsilon, when bound to the alpha subunit, provides the 3'-->5' exonuclease activity (proofreading) that removes 3' mismatched terminal nucleotides from the nascent DNA strand during replication. The temperature sensitive dnaQ49 allele lacks this catalytic activity which results in mutation frequencies 10(4)-10(5)-fold above wild-type values at 37 degrees C. At 30 degrees C dnaQ49 mutation frequencies are much lower but still higher than wild-type levels. We found that dnaQ49, like mutD5, another strong mutator allele of dnaQ, is medium-dependent with mutation frequencies ranging from 12 to nearly 1000-fold higher in rich media (L-broth) than in minimal media. In minimal media dnaQ49 retains modest mutator activity. In addition the base-pair substitution mutational spectrum of dnaQ49 was medium-dependent. Unlike mutD5 the addition of thymidine to minimal medium did not enhance dnaQ49 mutator activity. We also constructed dnaQ49mutL double mutator strains and compared mutator frequencies with single dnaQ49 and mutL strains. The mutL allele results in inactive methyl-directed mismatch repair. Double and single dnaQ49 mutators had similar mutation frequencies at 37 degrees C in L-broth suggesting that dnaQ49 strains are defective in mismatch repair as well as 3'-->5' exonuclease proofreading activity. In contrast in minimal media at 37 degrees C and in L-broth at 30 degrees C dnaQ49 mutL mutation frequencies were much higher than dnaQ49 values indicating the presence of active mismatch-repair activity in the latter strain. In addition at 37 degrees C dnaQ49mutL mutation frequencies were about 100-fold higher in L-broth than in minimal media. We conclude from this result that the rich media effect with dnaQ49 involves an actual increase in replication errors rather than a medium-dependent modulation of mismatch repair activity.

The interaction of the Escherichia coli mutD and mutT pathways in the prevention of A:T-->C:G transversions.

The Escherichia coli mutT mutator allele produces high frequencies of exclusively A:T-->C:G transversions. This is thought to be caused by a failure to prevent or remove A:G mispairs during DNA replication. The mutD5 mutator allele maps to the dnaQ locus which encodes the epsilon subunit of the DNA polymerase III holoenzyme. This subunit provides 3'-->5' exonuclease, proofreading, activity for removing mispaired nucleotides at the 3' end of the newly synthesized DNA strand. mutD5 has an altered epsilon resulting in reduced levels of proofreading and subsequent high mutation frequencies for all base-pair substitutions. We have analyzed the interaction between mutD5 and mutT-induced A:T-->C:G transversions by measuring reversion frequencies in mutD5 and mutT single mutator strains and mutD5mutT double mutator strains using the well-characterized trpA58 and trpA88 alleles. We find that the double mutator strains produce more A:T-->C:G substitutions than would be expected from simple additivity of the single mutator strains. We interpret this to mean that the two systems, at least in part, do act together to prevent the same mutational intermediate from producing A:T-->C:G transversions. It is estimated that over 90% of the mutT-induced A:G mispairs are corrected by proofreading at the trpA58 site while only about 30% are corrected at trpA88. Reversion frequencies in the mutD5mutT double mutator strains indicate A:G misincorporations occur about 100 x more frequently at trpA58 than at the trpA88 site. Using these and other data we also provide estimations of the fidelity contributions for mutT editing, proofreading and methyl-directed mismatch repair at the two trpA sites for both transversions and the transition that could be scored. In the case of A:T-->C:G transversions, both mutT editing and proofreading make major contributions in error reduction with mismatch repair playing a small or no role at all. For the A:T-->G:C transition, proofreading and mismatch repair were both important in preventing mutations while no contribution was observed for mutT editing.

Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'----5' exonuclease (proofreading) activity.

Escherichia coli strains carrying the temperature-dependent dnaQ49 allele are strong mutators at 37 degrees C. Since the dnaQ49 gene encodes the epsilon subunit of DNA polymerase III, it is thought that the large number of errors results in part from impaired proofreading activity during DNA replication. We have examined dnaQ49-induced reversion patterns of defined trpA alleles to determine the kinds of errors produced by dnaQ49 at 30 degrees C and 37 degrees C. We found that at 37 degrees C dnaQ49 produced all types of base-pair substitutions in addition to frameshifts with transitions generally occurring more frequently than transversions. This generalized mutator activity is very similar to that displayed in rich medium by mutD5, another mutator allele at the dnaQ locus. However, when dnaQ49 strains were cultured at 30 degrees C, not only were reversion frequencies much lower than at 37 degrees C, but in addition, the spectrum was altered. Transversions became proportionally more prevalent in the reversion spectra at the lower temperature. We suggest the possibility that at 37 degrees C dnaQ49 results in defective proofreading and methyl-directed postreplicative mismatch repair, while at 30 degrees C mismatch repair is fully and proofreading partially restored.