RecA-mediated repair and mutagenesis in Escherichia coli: A genetic study of the molecular mechanism of SOS processing

Shi-Kau Liu, Purdue University

Abstract

In the absence of DNA-damaging agents SOS processing can be turned on by recA(Prt$\sp{\rm c}$) mutations which confer constitutive protease activity. The recA1202(Prt$\sp{\rm c}$) allele encodes a RecA protein with strong protease activity which efficiently mediates SOS processing at all times. As a result of the fully active SOS processing, spontaneous mutagenesis is increased dramatically in recA1202(Prt$\sp{\rm c}$) cells. The combination of the recA1202 mutation and a multicopy umuD$\sp+$C$\sp+$ plasmid further increases the spontaneous mutation frequency. In a study of the SOS mutagenesis in recA1202/pumuD$\sp+$C$\sp+$ cells, I found that the increase in spontaneous mutations was especially efficient in the recA1202 gene itself and in nearby regions. The cis-like mutagenic effect can be explained by the ease with which RecA1202(Prt$\sp{\rm c}$) is activated, which implies that there should be a relatively high concentration of activated RecA protein near the recA gene, where the protein is synthesized. The phenomenon observed in this study is a novel example of an overactive gene preferentially turning itself down by mutation. To further understand the spontaneous mutagenic process, I investigated the mutational specificity of this mutagenesis. Twenty-eight independently isolated recA mutants were characterized and sequenced. The analysis of the specificity of the mutations revealed that transversions and changes at AT sites were predominant. I also found that neighboring sequences play a role in the determination of the mutational sites. To account for the high mutation frequency observed in this system and the observed mutational specificity, an untargeted mutagenic mechanism was proposed. Intrigued by a previous study which showed that groE genes are involved in UV mutagenesis, I studied the role of the GroE heat shock proteins in Weigle reactivation and Weigle mutagenesis. I found that intact groE genes contribute to SOS repair; groE mutations reduced approximately 50% of the Weigle reactivation of UV-irradiated phage S13. The result also suggests that the inducibility of groE genes by UV plays a regulatory role in enhancing SOS repair after DNA damages occur. An unexpected phenomenon was observed in my study of the involvement of groE genes in Weigle mutagenesis. Most of the mutagenesis that accompanies the SOS repair of UV-irradiated phage S13 is eliminated when the groES or the groEL gene is defective. Therefore, SOS repair and SOS mutagenesis are separable; mutagenesis is not a necessary consequence of the so-called error-prone repair, but is additionally imposed on the repair process by GroE proteins.

Degree

Ph.D.

Advisors

Tessman, Purdue University.

Subject Area

Genetics|Microbiology|Molecular biology|Radiology

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