Regulation of DNA polymerase IV-dependent mutagenesis in Escherichia coli.

摘要

In their natural environments, bacteria are constantly assaulted by a variety of stresses. To survive these stresses, they have evolved coordinated stress responses to regulate the expression of genes whose products can help to alleviate the stressful situation. The general stress response in Escherichia coli is mediated by the sigma factor, RpoS. Sigma factors are subunits of RNA polymerase that direct it to transcribe genes that are appropriate for the particular condition. One RpoS-regulated gene is the specialized DNA polymerase, DNA Polymerase IV (Pol IV). Pol IV is error-prone, generating mutations at a much higher rate than achieved by normal replicative polymerases.;The cellular levels of Pol IV increase when the cells enter stationary phase because RpoS directs the transcription of dinB. Because RpoS affects both Pol IV-dependent and Pol IV-independent adaptive mutation, RpoS probably regulates another gene or genes in addition to dinB that affect adaptive mutation. Chapter two of this dissertation presents data from two genetic screens that were performed to identify an RpoS-regulated gene that affects adaptive mutation.;The data in Chapter three reveal that RpoS indirectly regulates Pol IV activity in normally growing cells via an RpoS-regulated factor. When Pol IV is overexpressed in growing cells, the rate of Pol IV-dependent spontaneous mutation increases. If the cells overexpressing Pol IV are missing RpoS, the rate of Pol IV-dependent mutagenesis is decreased, but the amount of Pol IV is not affected.;Chapter four describes microarray analyses comparing the transcriptional profiles of exponentially growing wildtype and rpoS-- strains. These microarrays did not reveal any intermediate factor(s) in the regulation of Pol IV.;SbcCD, a structural maintenance of chromosomes (SMC)-like protein affects Pol IV-dependent spontaneous mutation. In Chapter five, I show that when one or both genes in the sbcDC operon are deleted, the Pol IV-dependent spontaneous mutation rate decreased in cells that were wildtype for RpoS, but not in cells that were missing RpoS. Mutant alleles of sbcDC and recB, which encodes a double strand break repair protein, are epistatic for Pol IV-dependent spontaneous mutation as are mutant alleles of recB and rpoS. We conclude that RpoS, RecB, and SbcCD are all in the same pathway, and this pathway is required for most of the Pol IV-dependent mutations that occur in growing cells.