Lovett 9601398 The objective of this research is to elucidate the mechanisms involved in the induction of Bacillus subtilis SOS genes following DNA damage and during competence development. Previous results indicate that induction of B. subtilis SOS genes by DNA damage follows the RecA-mediated cleavage of the SOS repressor (DinR) whereas during competence development the induction of at least one SOS gene (recA) appears to involve repressor displacement, but not cleavage. We will use a combination of mutational analysis, protein cross-linking, NOR spectroscopy, and kinetic analyses to investigate the molecular details of RecA-mediated DinR cleavage that occurs following DNA damage. We also will test the hypothesis that induction of the recA gene (and perhaps other SOS genes) in competent cells is due to the displacement of the DinR protein from the SOS operator by the competence transcription factor, comic. Specifically, we will examine the effect of ComK binding on the binding affinity of DinR to the recA operator using quantitative hydroxyl radical footprinting analysis with purified proteins. To establish the biological relevance of this binding analysis, we will conduct similar studies using crude extracts from fractionated competent cells and study the effects of ComK and DinR on transcription from the recA promoter in vitro. Since a complete understanding of the B. subtilis SOS response will ultimately require the characterization of over SOS genes that are induced by DNA damage and competence development, we will use the binding activity of the DinR protein to search the B. subtilis genome for other SOS genes. The results of this research will elucidate the way in which DNA repair genes in the bacteriumBacillus subtilis are turned on in response to DNA damaging treatments (e.g. exposure to carcinogenic agents); these results should have a significant impact on the understanding of similar DNA repair systems in other organisms. This work will focus primarily on the interactions that o ccur between the two proteins that regulate the expression of DNA repair genes-- an activator and a repressor. Our results are expected to provide molecular pictures of the sequence of events leading from the activation of the activator protein to the destruction of the repressor protein that results in the induction of DNA repair activity. Because the destruction mechanism is a biochemical strategy that is apparently used extensively in biological systems, our results will also inform the understanding of similar processes. In addition, the study of inducible DNA repair in B. subtilis is particularly important because there is a link between the regulation of inducible DNA repair and the regulation of a developmental stage that specializes in genetic recombination (i.e. the transfer of DNA from one individual to another). Our results will elucidate the way that certain DNA repair genes are turned on, in the absence of DNA damage, during this developmental stage.
