RUI: Regulation of Bacillus subtilis Inducible DNA Repair response.

本研究的总体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调控机制。在之前的授权期间，我们纯化了枯草芽孢杆菌的SOS抑制因子，克隆了相应的基因，并鉴定了四个SOS操作子。我们发现抑制因子在体外失活需要RecA，由单链DNA和NTP激活，我们有证据表明，在这些条件下，抑制因子被磷酸化，但被切割。我们将在体外剖析抑制因子失活的分子细节，并通过以下方式确认其在体内的作用：(1)进行体外和体内动力学研究；(2)分离和鉴定失活机制缺陷突变体；(3)通过交联分析鉴定核苷酸结合域和操作符结合域；(4)研究抑制因子在体外和体内失活后构象的变化。我们之前的研究表明，在能力发展过程中，枯草芽孢杆菌RecA是由一种不同于SOS调控的机制诱导的，而其他SOS基因是由一种类似SOS的机制诱导的（Lovett等，1989）。在上一期研究中，我们发现能力特异性的RecA诱导并不伴随着SOS抑制因子失活，这表明在感态细胞中RecA的诱导是由于抑制因子的位移。我们将系统地测试三种可能发生位移的方式。我们最近发现，大肠杆菌Lex A阻断了能力特异性的RecA诱导，而不是sos样的诱导，这将被用作在能力发展过程中分离两种诱导途径的手段。这项研究将使我们深入了解细胞修复DNA损伤的重要机制。此外，它将为威廉姆斯学院的本科生提供有价值的研究培训。

The overall objective of this research is to elucidate the mechanisms involved in the regulation of B. subtilis SOS genes following DNA damage and during competence development. During the previous granting period we purified the B. subtilis SOS repressor, cloned the corresponding gene, and characterized four SOS operators. We showed that repressor inactivation in vitro requires RecA, activated with single-stranded DNA and NTP, and we have evidence suggesting that repressor is phosphorylated, but cleaved, under these conditions. We will dissect the molecular details of repressor inactivation in vitro and confirm its role in vivo by: (1) conducting kinetic studies in vitro and in vivo; (2) isolating and characterizing mutants defective in the inactivation mechanism; (3) identifying nucleotide binding and operator binding domains by cross-linking analysis ; and (4) studying repressor conformation changes upon inactivation in vitro and in vivo. We previously showed that during competence development B. subtilis RecA is induced by a mechanisms that is distinct from SOS regulation, whereas other SOS genes are induced by an SOS-like mechanism (Lovett, et al., 1989). During the last granting period we showed that competence-specific RecA induction is not accompanied by SOS repressor inactivation indicating the RecA induction in competent cells is due to repressor displacement. We will systematically test three possible ways in which displacement could occur. Our recent discovery that E. coli Lex A blocks competence-specific RecA induction, but not SOS-like induction will be exploited as a means to separate the two induction pathways during competence development. %%% This research will provide insight into an important mechanism by which cells repair damage to their DNA. In addition, it will provide valuable research training to undergraduate students at Williams College.