RUI Regulation of the Bacillus subtillis Inducible DNA Repair System

这项拟议研究的总体目标是鉴定和分离枯草芽孢杆菌SOS DNA修复系统的调控成分，并开始表征其调控的分子细节。到目前为止已有的证据表明，大肠杆菌SOS系统在枯草杆菌中可能是高度保守的。在大肠杆菌中，SOS DNA修复基因的诱导是由于RecA蛋白介导的SOS基因抑制物LexA蛋白的切割。似乎与枯草杆菌RecA蛋白类似的机制也存在于枯草杆菌中。该项目的具体目标是：(1)鉴定四个不同的枯草杆菌DNA损伤诱导基因的调控DNA序列；(2)纯化与这些序列结合的蛋白质(可能是LexA类似物)，并克隆相应的基因；以及(3)鉴定该蛋白与调控DNA序列以及与枯草杆菌RecA蛋白类似物的相互作用。这些方法将包括使用调控的DNA序列来(1)鉴定蛋白质并在DNA酶足迹分析中跟踪其纯化；(2)构建亲和柱以促进蛋白质的纯化；以及(3)探测编码蛋白质的基因的lambda gt11文库。目前，对诱导DNA修复的调控只对大肠杆菌有一定的了解。这项研究将是了解枯草杆菌SOS DNA修复系统调控的决定性一步，枯草杆菌是一种与大肠杆菌关系不密切的生物。

The overall objective of the proposed research is to identify and isolate the regulatory components of the Bacillus subtilis SOS DNA repair system and start to characterize the molecular details of its regulation. Evidence available to date suggests that the well-characterized Escherichia coli SOS system may be highly conserved in B. subtilis. In E. coli, induction of the SOS DNA repair genes is due to the RecA protein-mediated cleavage of the SOS gene repressor, LexA protein. It appears that an analogous mechanism, involving the B. subtilis RecA protein analogue, occurs in B. subtilis. The specific aims of this project are to (1) characterize the regulatory DNA sequences of four distinct B. subtilis DNA damage-inducible genes; (2) purify the protein (presumably a LexA analogue) that binds to these sequences and clone the corresponding gene; and (3) characterize the interactions of this protein with the regulatory DNA sequences and with the B. subtilis RecA protein analogue. The methods will include using the regulatory DNA sequences to (1) identify the protein and track its purification in DNase footprinting assays; (2) construct an affinity column to facilitate the protein purification; and (3) probe a lambda gt11 library for the gene that codes for the protein. At present, the regulation of inducible DNA repair is only understood for E. coli. This research will be a decisive step toward understanding the regulation of the SOS DNA repair system in B. subtilis, an organism that is not closely related to E. coli.