Bacterial Proteins Involved in DNA Repair

描述(由申请人提供)： 细菌中同源基因重组的主要功能是对受阻的复制叉子进行非诱变修复。实际上，起源于ORIC的每个复制分叉都会在某个时间点遇到DMA损坏，并且必须进行重组DNA修复。在DNA修复的主要途径中，这一过程可能是最复杂的，也是最不被了解的。GM52725支持的这项工作有三个目标，都是针对完全理解复制分叉修复路径，其中复制和重组系统紧密集成。 目前这项工作的重点是调节RecA蛋白功能的过程。第一个具体目标构成了努力的主要部分。本课程将探讨与RecA蛋白调控有关的蛋白质的基本生物化学，重点是RecFOR、RecX、DINL、RdgC、PsiB、UvrD和RecQ蛋白。RecA在多个层面上受到监管，这一努力旨在系统地探索这种监管。最近的研究概述了一个相互交织的调控系统，几乎调控RecA功能的每一个方面。另外两个目标代表了较小的努力投资，但允许整合和扩展目标1中获得的信息。第二个目标是重建一些分叉修复途径中的关键步骤，称为分叉回归。这项工作依赖于构建模仿失速叉子结构的新型DNA底物。这项工作将在很大程度上利用目标1中提供的信息。最终目标是在体外检查DNA聚合酶V对DNA损伤的诱变复制旁路。在这里，我们正在重新定义RecA在这一修复过程中的作用，并可能探索RecA调控的一个新方面。 这些实验将提供有关导致复制分叉重新激活的路径的关键部分的新信息。最终的目标是用纯酶完全重建这些途径。

DESCRIPTION (provided by applicant): The primary function of homologous genetic recombination in bacteria is the nonmutagenic repair of arrested replication forks. Virtually every replication fork originating at oriC encounters DMA damage at some point, and must undergo recombinational DNA repair. This process represents perhaps the most complex and certainly the least understood of the major pathways for DNA repair. The work supported by GM52725 has three goals, all directed at a complete understanding of replication fork repair pathways, where the replication and recombination systems are closely integrated. The work is currently focused on the processes that regulate the function of the RecA protein. The first specific aim constitutes the major portion of the effort. The fundamental biochemistry of proteins involved in the regulation of RecA protein will be explored, focusing on the RecFOR, RecX, Dinl, RdgC, PsiB, UvrD, and RecQ proteins. RecA is regulated on multiple levels, and this effort is designed to systematically explore that regulation. An interwoven system of regulatory activities modulating almost every aspect of RecA function has been outlined in recent studies. The other two aims represent a smaller investment in effort, but allow for an integration and expansion of the information obtained in aim 1. The second aim is to reconstitute a key step in some fork repair pathways, called fork regression. This effort relies on the construction of novel DNA substrates that mimic the structure of stalled forks. The effort will draw heavily on the information provided under aim 1. The final aim is to examine the mutagenic replicative bypass of DNA lesions by DNA polymerase V in vitro. Here we are redefining the role of RecA in this repair process and potentially exploring a novel aspect of RecA regulation. These experiments will provide new information about critical parts of the pathways leading to replication fork reactivation. The ultimate goal is a complete reconstitution of the pathways with pure enzymes.