BACTERIAL PROTEINS IN RECOMBINATIONAL DNA REPAIR

本提案的目标是详细了解 来自大肠杆菌的五种蛋白质的活性， 重组DNA修复和同源遗传重组。这些是 RuvA、RuvB、RecF、RecO和RecR蛋白。每种基因的突变 相应的基因会导致某些遗传物质的重组缺陷， 背景相同的突变赋予对DNA损伤剂的敏感性 在野生型的背景下。 RecF、O和R蛋白被认为在重组的早期起作用。 流程.迄今为止确定的一个功能是促进RecA蛋白 与SSB包被的单链DNA结合。每者的氨基酸序列 这些蛋白质的特征是共有核苷酸结合位点，尽管 NTP水解尚未报道，并且关于NTP水解的情况知之甚少。 核苷酸在这些蛋白质可能具有的任何活性中的功能。我们 建议表征RecF，O， 和R蛋白的详细信息。我们还将研究这些因素的影响 蛋白质对RecA活性的影响如果RecA形成混合丝， 这些蛋白质或它们的任何子集，我们将专注于定义 如此修饰的细丝的活性。 RuvA和RuvB蛋白被认为有助于蛋白质的加工。 由RecA蛋白产生的分支重组中间体。这些 蛋白质促进体外DNA分支迁移，这可能有助于 在重组过程中杂交DNA的快速产生。的蛋白质 表现出有限的DNA解旋酶活性，RuvB蛋白水解ATP。 与RecA结合，它们促进绕过异质性区域， 太长了，RecA蛋白无法单独绕过。如果阻挡 不能绕过，这些蛋白质促进RecA的有效逆转- 介导的DNA链交换。这些蛋白质的功能之一可能是 作为一种反重组系统， 霍利迪结构，可能导致潜在的有害 基因组内重组事件。我们对RuvA和B的研究将集中在 部分关于DNA结合和ATP水解的表征， 阐明这些蛋白质在DNA上移位的机制。我们 我还将研究RuvA和B过程使DNA停滞的机制 由RecA生成的分支，作为RuvA和B动作的模型， 重组过程

The goals of this proposal is to provide a detailed understanding of the activities of five proteins from Escherichia coli that are involved in recombinational DNA repair and homologous genetic recombination. These are the RuvA, RuvB, RecF, RecO, and RecR proteins. Some mutations in each of the corresponding genes lead to recombination defects in certain genetic backgrounds. The same mutations confer sensitivity to DNA damaging agents in an otherwise wild type background. The RecF, O, and R proteins are believed to act early in recombinational processes. One function identified to date is to facilitate RecA protein binding to SSB-coated single-stranded DNA. The amino acid sequence of each of these proteins features a consensus nucleotide binding site, although NTP hydrolysis has not been reported and little is known about the function of nucleotides in any activity these proteins may have. We propose to characterize the DNA and NTP binding properties of the RecF, O, and R proteins in detail. We will also examine the effects of these proteins on RecA activities in vitro. If RecA forms mixed filaments with these proteins or any subset of them, we will focus on defining the activities of the filaments so modified. The RuvA and RuvB proteins are believed to contribute to the processing of branched recombination intermediates generated by RecA protein. These proteins promote DNA branch migration in vitro which could contribute to the rapid generation of hybrid DNA during recombination. The proteins exhibit a limited DNA helicase activity, and RuvB protein hydrolyzes ATP. In combination with RecA, they promote the bypass of regions of heterology that are too long to be bypassed by RecA protein alone. If a barrier cannot be bypassed, these proteins promote an efficient reversal of RecA- mediated DNA strand exchange. One function of these proteins may be to act as a kind of anti-recombination system, to process or reverse stalled Holliday structures that may result from potentially deleterious intragenomic recombination events. Our studies on RuvA and B will focus in part on the characterization of DNA binding and ATP hydrolysis to elucidate the mechanism by which these proteins translocate on the DNA. We will also examine the mechanism by which RuvA and B process stalled DNA branches generate by RecA, as a model for RuvA and B action in all recombination processes.