Structure-function of recombination/replication mediator

描述（由申请方提供）：认为DNA损伤或其他屏障部位的复制中断是细胞诱变、基因组重排和致死的主要原因。RecF途径是成功重启复制所必需的两个主要重组修复途径之一。RecF、-O和-R蛋白，也称为重组/复制介体，形成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，这是触发SOS反应的事件，并且对于维持停滞的复制叉的稳定性、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中都有功能对应物。即使在细菌中，它们的活性机制也知之甚少，这是我们研究的长期目标。关于RecFOR活性的一个重要的未回答的问题是RecFOR复合物的缔合/解离机制及其与DNA的相互作用。我们将通过对RecF和RecFOR复合物进行结构研究，并通过研究RecFOR反应的初始步骤的机制来解决这个问题：RecF DNA结合和RecR被RecF招募到DNA。本论文的主要目的是：1）解析RecF的晶体结构，揭示其SMC结构域的特征和RecF特异性结构域的新结构; 2）直接测量RecF单独和与DNA结合时的ATP依赖性二聚化;利用RecF的结构引导突变进一步研究SMC样二聚化在ATP水解和DNA结合中的作用;和3）RecF/RecR/DNA复合物的组装状态、其形成途径以及RecF ATP依赖性二聚化的作用将用野生型和突变蛋白质进行研究。我们还将尝试共结晶RecFOR复合物，以研究它们在原子分辨率水平上的相互作用。连同我们的合作研究，其中选定的突变体将被测试其能力，以促进RecA核蛋白丝组装在体外，并为他们在紫外线照射的细胞复制恢复的影响，拟议的研究将提供重大和新颖的信息重组/复制介质。一个新的详细的机械理解RecF与DNA和RecR的相互作用，在修复停滞的复制叉将获得从而推进我们的理解的复制重启和重组修复过程。这将有助于制定未来的研究类似的过程中，真核生物，他们的故障是高度相关的肿瘤发生。

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.