Mutagenic DNA repair in Escherichia coli. XVII. Effect of temperature-sensitive DnaE proteins on the induction of streptomycin-resistant mutations by UV light

Abstract

In contrast to the dnaE486 mutation, which is nearly 'dead-stop', dnaE1026 allows DNA synthesis for some time at restrictive temperatures. When bacteria carrying the dnaE486 or dnaE1026 temperature-sensitive mutations were incubated at restrictive temperature after exposure to UV light they showed little or no fixation of mutations as determined by loss of photoreversibility and the mutation frequency fell progressively. These results confirm a role for DnaE protein (the alpha-subunit of DNA polymerase III holoenzyme) in UV mutagenesis. A derivative of dnaE1026 carrying the umuC122 allele which blocks normal UV mutagenesis showed induction of mutations when photoreversing light was given to UV-irradiated bacteria after a period of incubation at either permissive or restrictive temperature. The defective DnaE1026 protein is therefore able to carry out the misincorporations which have been postulated to be the first step in the mutagenic process. Its inability to give rise to mutations in umu+ bacteria may therefore be attributed to its inability to participate in a later step, perhaps because it is unable to interact with the UmuD, C gene products. In contrast, UV-irradiated dnaE486 umuC122 bacteria did not show mutagenesis when incubated at restrictive temperature before photoreversal, suggesting that the altered DnaE486 protein was not able to carry out the postulated misincorporation step at 43 degrees C. DNA polymerase III alpha-subunit therefore appears to be required for both the misincorporation and bypass steps in the two-step model for UV mutagenesis.