Structure-functional studies of recombination/replication mediator proteins

重组/复制介体蛋白的结构功能研究

• 批准号: 7931189
• 负责人: SERGEY KOROLEV
• 金额: $ 21.72万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931189
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Address; Architecture; Attenuated; Bacteria; Binding; Biological; Cells; Chromosomes; Collaborations; Complex; Crystallization; Crystallography; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Sequence Rearrangement; DNA Structure; Data; Dimerization; Dissociation; Escherichia coli; Eukaryota; Event; Filament; Future; Genetic Recombination; Genomics; Goals; In Vitro; Individual; Interruption; Maintenance; Maps; Measures; Mediating; Mediator of activation protein; Modeling; Mutagenesis; Mutate; Nucleoproteins; Organism; Pathway interactions; Play; Printing; Process; Property; Proteins; Reaction; Recovery; Research; Resolution; Role; SOS Response; Single-Stranded DNA; Site; Site-Directed Mutagenesis; Solutions; Structure; Testing; analog; design; dimer; foot; insight; intermolecular interaction; mutant; novel; protein complex; recombinase; recombinational repair; repaired; research study; success; tumorigenesis; ultraviolet irradiation

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.

描述（由申请人提供）：DMA 损伤或其他障碍位点的复制中断被认为是细胞诱变、基因组重排和致死的主要原因。 RecF 途径是重组修复的两条主要途径之一，对于复制的成功重启至关重要。 RecF、-O 和 -R 蛋白，也称为重组/复制介质，形成 RecF 途径的核心。它们对于将 RecA 重组酶装载到 DNA 上（触发 SOS 响应的事件）至关重要，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也很重要。这些蛋白质在所有生物体中都有功能对应物。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 活性尚未解答的重要问题之一是 RecFOR 复合物的缔合/解离机制及其与 DNA 的相互作用。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，并研究 RecFOR 反应初始步骤的机制来解决这个问题：RecF DNA 结合以及 RecF 将 RecR 招募到 DNA 上。 ATP 驱动的二聚化在 RecF 活性中发挥核心作用并调节 RecF 与 DNA 和/或 RecR 相互作用的假设将在以下目标中进行研究：1）将以高分辨率解析 RecF 的晶体结构以揭示特定的SMC结构域的特点和RecF特异性结构域的新颖结构； 2) 将直接测量 RecF 单独和 DNA 结合时的 ATP 依赖性二聚化； RecF 的结构引导诱变将用于进一步研究 SMC 样二聚化在 ATP 水解和 DNA 结合中的作用； 3) 将利用野生型和突变型蛋白研究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。我们还将尝试共结晶 RecFOR 复合物，以在原子分辨率水平上研究它们的相互作用。结合我们的合作研究（其中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），拟议的研究将提供有关重组/复制的重要且新颖的信息调解员。在修复停滞的复制叉过程中，我们将获得对 RecF 与 DNA 和 RecR 相互作用的新的详细机制理解，从而增进我们对复制重启和重组修复过程的理解。它将有助于未来对真核生物中类似过程的研究，这些过程的功能障碍与肿瘤发生高度相关。