RUI: Regulation of Bacillus subtilis SOS Response

本研究的目的是阐明枯草芽孢杆菌SOS基因在DNA损伤后和能力发育过程中的诱导机制。先前的研究结果表明，DNA损伤诱导枯草芽孢杆菌SOS基因的诱导遵循recA介导的SOS抑制因子（DinR）的裂解，而在能力发育过程中，至少一个SOS基因（recA）的诱导似乎涉及抑制因子位移，而不是裂解。我们将结合突变分析、蛋白质交联、NOR光谱和动力学分析来研究DNA损伤后reca介导的DinR切割的分子细节。我们还将验证一个假设，即在受感细胞中诱导recA基因（可能还有其他SOS基因）是由于能力转录因子comic取代了SOS操作符中的DinR蛋白。具体来说，我们将使用纯化蛋白的定量羟基自由基足迹分析来研究ComK结合对DinR与recA算子结合亲和力的影响。为了建立这种结合分析的生物学相关性，我们将使用从分离的受感细胞中提取的粗提取物进行类似的研究，并在体外研究ComK和DinR对recA启动子转录的影响。由于对枯草芽孢杆菌SOS反应的全面了解最终将需要对DNA损伤和能力发展诱导的超过SOS基因进行表征，因此我们将使用DinR蛋白的结合活性来搜索枯草芽孢杆菌基因组中其他SOS基因。这项研究的结果将阐明枯草芽孢杆菌（bacillus subtilis）中的DNA修复基因在DNA损伤治疗（例如暴露于致癌物质）下是如何被激活的；这些结果将对理解其他生物体中类似的DNA修复系统产生重大影响。这项工作将主要集中在调节DNA修复基因表达的两种蛋白质之间发生的相互作用-激活因子和抑制因子。我们的结果有望提供从激活蛋白的激活到抑制蛋白的破坏导致DNA修复活性诱导的事件序列的分子图片。因为破坏机制是一种生物化学策略，显然在生物系统中广泛使用，我们的结果也将告知类似过程的理解。此外，枯草芽孢杆菌诱导DNA修复的研究尤其重要，因为诱导DNA修复的调节与专门进行基因重组的发育阶段（即DNA从一个个体转移到另一个个体）的调节之间存在联系。我们的研究结果将阐明在没有DNA损伤的情况下，某些DNA修复基因在这个发育阶段是如何被激活的。

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.