BACTERIAL PROTEINS IN RECOMBINATIONAL DNA REPAIR

本提案的目标是提供对 大肠埃希菌五种蛋白的活性研究 重组DNA修复和同源基因重组。这些是 Ruva、RuvB、recf、reco和recR蛋白。每一个基因都有一些突变 相应的基因会导致某些基因的重组缺陷 背景。同样的突变使人对dna损伤剂敏感。 在另一种狂野的背景下。 RECF、O和R蛋白被认为在重组的早期起作用 流程。到目前为止已发现的一个功能是促进RecA蛋白 与单链断裂包被的单链DNA结合。每一个的氨基酸序列 这些蛋白质中有一个共同的核苷酸结合位点，尽管 目前还没有关于NTP水解的报道，关于NTP的水解也知之甚少 核苷酸在这些蛋白质可能具有的任何活动中的功能。我们 建议表征RECF，O， 和R蛋白的详细信息。我们还将研究这些措施的影响 体外蛋白质对RecA活性的影响。如果RecA与其形成混合细丝 这些蛋白质或它们的任何子集，我们将重点定义 这样修饰的细丝的活性。 Ruva和RuvB蛋白被认为有助于加工 由RecA蛋白产生的分支重组中间体。这些 蛋白质在体外促进DNA分支迁移，这可能有助于 重组过程中杂交DNA的快速生成。这些蛋白质 表现出有限的DNA解旋酶活性，并且RuvB蛋白可以水解三磷酸腺苷。 与RecA结合，它们促进了异质性区域的旁路 它们太长了，不能被RecA蛋白单独绕过。如果一道屏障 无法绕过，这些蛋白质促进有效逆转RecA- 介导的DNA链交换。这些蛋白质的一个功能可能是作用于 作为一种反重组系统，处理或扭转停滞 Holliday结构可能会导致潜在的有害 基因组内重组事件。我们对Ruva和B的研究将集中在 DNA结合和三磷酸腺苷水解酶的特性部分 阐明这些蛋白质在DNA上移位的机制。我们 还将研究Ruva和B处理停滞DNA的机制 由RecA生成的分支，作为所有Ruva和B操作的模型 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.