Structure-functional studies of recombination/replication mediator proteins

描述（由申请人提供）：在DMA损伤或其他屏障处的复制中断被认为是细胞突变、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重组修复的两个主要途径之一。RecF、-O和-R蛋白，也被称为重组/复制介质，构成了RecF途径的核心。它们对于将RecA重组酶装载到DNA上（触发SOS反应的事件）至关重要，并且对于维持停滞的复制分叉的稳定性，解决异常的DNA结构以及从中断点恢复复制非常重要。这些蛋白质在所有生物体中都有相应的功能。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解问题是RecFOR复合物的结合/解离及其与DNA的相互作用的机制。我们将在我们的提案中通过对RecF和RecFOR复合物进行结构研究，并通过研究RecFOR反应初始步骤的机制：RecF DNA结合和RecF向DNA募集RecR来解决这个问题。关于atp驱动的二聚化在RecF活性中起核心作用并调节RecF与DNA和/或RecR相互作用的假设将在以下方面进行研究：1)高分辨率解析RecF的晶体结构，揭示SMC结构域的具体特征和RecF特定结构域的新结构；2) RecF单独和DNA结合后的atp依赖性二聚化将被直接测量；利用RecF的结构导向诱变技术，进一步研究smc样二聚化在ATP水解和DNA结合中的作用；3) RecF/RecR/DNA复合物的组装状态、形成途径以及RecF atp依赖性二聚化的作用将在野生型和突变型蛋白中进行研究。我们还将尝试共结晶RecFOR配合物，以研究它们在原子分辨率水平上的相互作用。在我们的合作研究中，我们将测试选定的突变体在体外促进RecA核蛋白丝组装的能力，以及它们对紫外线照射细胞中复制恢复的影响。我们提出的研究将为重组/复制介质提供重要的新信息。在修复停滞的复制叉过程中，将获得RecF与DNA和RecR相互作用的新的详细机制理解，从而促进我们对复制重启和重组修复过程的理解。这将有助于制定未来真核生物中类似过程的研究，其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.