RUI: Regulation of Bacillus subtilis SOS Response

Lovett 9601398本研究的目的是阐明在DNA损伤后和感受态发育过程中诱导枯草芽孢杆菌SOS基因的机制。 以前的研究结果表明，诱导B。在感受态发育过程中，至少一个SOS基因（recA）的诱导似乎涉及阻遏物置换，但不涉及切割。我们将使用突变分析，蛋白质交联，NOR光谱和动力学分析的组合来研究RecA介导的DinR裂解发生在DNA损伤后的分子细节。我们还将测试的假设，在感受态细胞中的recA基因（也许其他SOS基因）的诱导是由于位移的DinR蛋白从SOS运营商的感受态转录因子，漫画。具体而言，我们将使用纯化蛋白质的定量羟基自由基足迹分析来检查ComK结合对DinR与recA操作子的结合亲和力的影响。为了建立这种结合分析的生物学相关性，我们将使用分级分离的感受态细胞的粗提物进行类似的研究，并研究ComK和DinR对recA启动子体外转录的影响。因为对B的完全了解。枯草杆菌SOS反应最终需要鉴定由DNA损伤和感受态发育诱导的SOS基因，我们将利用DinR蛋白的结合活性来寻找B。枯草杆菌基因组的其他SOS基因。 这项研究的结果将阐明细菌枯草芽孢杆菌中的DNA修复基因在DNA损伤处理（例如暴露于致癌物质）中被打开的方式;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两种蛋白质之间的相互作用，调节DNA修复基因的表达-激活剂和阻遏物。我们的研究结果有望提供从激活蛋白的激活导致的阻遏蛋白的破坏，导致在诱导DNA修复活性的事件序列的分子图片。由于破坏机制是一种明显广泛用于生物系统的生化策略，因此我们的研究结果也将为理解类似过程提供信息。此外，对B.枯草芽孢杆菌是特别重要的，因为在诱导型DNA修复的调节和专门进行遗传重组（即DNA从一个个体转移到另一个个体）的发育阶段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，在这个发育阶段某些DNA修复基因是如何被打开的。

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.