Structure-functional studies of recombination/replication mediator proteins

描述(由申请人提供)：DNA损伤或其他屏障部位的复制中断被认为是细胞突变、基因组重排和致死性的主要原因。RECF途径是成功重启复制所必需的两条主要的重组修复途径之一。RECF、-O和-R蛋白，也称为重组/复制介体，构成了RECF途径的核心。它们对于将RecA重组酶装载到DNA上是必不可少的，这一事件触发了SOS反应，并且对于维持停滞的复制叉子的稳定性、解决异常的DNA结构以及从中断点恢复复制是重要的。这些蛋白质在所有生物体中都有功能上的对应。即使在细菌中，它们的活动机制也知之甚少，这是我们研究的长期目标。关于RecFOR活性的一个重要问题是，RecFOR复合体的结合/解离机制以及它们与DNA的相互作用是什么。我们将在我们的提案中通过对RECF和RecFOR复合体的结构研究，以及通过研究RecFOR反应的初始步骤：RECF与DNA结合和RECF将RECR招募到DNA来解决这个问题。人们将从以下几个方面研究在RECF活性中起核心作用并调节RECF与DNA和/或RECR相互作用的假说：1)高分辨率求解RECF的晶体结构，揭示SMC结构域的特征和RECF特异区的新结构；2)直接测量RECF单独依赖于ATP的二聚化和DNA结合的二聚化；利用RECF的结构导向突变，进一步研究SMC样二聚体在ATP水解酶和DNA结合中的作用；3)利用野生型和突变型蛋白质研究RECF/RECR/DNA复合体的组装状态、形成途径以及RECF依赖于ATP的二聚化作用。我们还将尝试共结晶RecFOR络合物，以研究它们在原子分辨率水平上的相互作用。与我们的合作研究一起，我们将测试选定的突变体在体外促进RecA核蛋白细丝组装的能力，以及它们对紫外线照射细胞复制恢复的影响，拟议的研究将提供关于重组/复制介体的重要和新的信息。在修复停滞的复制叉过程中，我们将对RECF与DNA和RECR的相互作用有一个新的详细的机制理解，从而促进我们对复制重新启动和重组修复过程的理解。这将有助于形成未来对真核生物中类似过程的研究，在真核生物中，它们的故障与肿瘤发生高度相关。

DESCRIPTION (provided by applicant): Replication interruptions at the sites of DMA damage or other barriers are believed to be a primary cause of mutagenesis, genomic rearrangements, and lethality in cells. The RecF pathway is one of the two major pathways of recombinational repair essential for successful restart of replication. The RecF, -O and -R proteins, also known as recombination/replication mediators, form the core of the RecF pathway. They are essential for the loading of RecA recombinase onto DNA, the event which triggers the SOS response, and is important to maintain the stability of stalled replication forks, to resolve aberrant DNA structures, and to resume replication from the point of disruption. These proteins have functional counterparts in all organisms. The mechanism of their activity even in bacteria is poorly understood, and is a long term goal of our research. One of the important unanswered questions about RecFOR activities is what are the mechanisms of association/dissociation of RecFOR complexes and their interactions with DNA. We will address this question in our proposal by performing structural studies of RecF and RecFOR complexes, and by studying the mechanism of the initial steps of RecFOR reaction: RecF DNA binding and the recruitment of RecR by RecF to DNA. The hypotheses that the ATP-driven dimerization plays a central role in RecF activity and regulates RecF interactions with DNA and/or with RecR will be investigated in the following aims: 1) the crystal structure of RecF will be solved at high resolution to reveal specific features of the SMC domain and the novel structure of RecF specific domain; 2) ATP-dependent dimerization of RecF alone and upon DNA binding will be directly measured; structure-guided mutagenesis of RecF will be utilized to further study the role of SMC-like dimerization in ATP hydrolysis and DNA binding; and 3) the assembly state of the RecF/RecR/DNA complex, the pathway of its formation, and the role of the RecF ATP-dependent dimerization will be studied with wild type and mutant proteins. We also will attempt to co-crystallize RecFOR complexes to study their interactions at the atomic resolution level. Together with our collaborative studies, in which selected mutants will be tested for their ability to facilitate RecA nucleoprotein filaments assembly in vitro and for their effect on the replication recovery in UV irradiated cells, the proposed research will provide significant and novel information on recombination/replication mediators. A new detailed mechanistic understanding of RecF interactions with DNA and RecR during the repair of stalled replication forks will be obtained thus advancing our understanding of the replication restart and recombination repair processes. It will help formulate future studies of similar processes in eukaryotes where their malfunctions are highly associated with tumorigenesis.