Structure-functional studies of recombination/replication mediator proteins

重组/复制介体蛋白的结构功能研究

基本信息

批准号：
7821466
负责人：
SERGEY KOROLEV
金额：
$ 23.17万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7821466
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

项目摘要

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）：据信DMA损伤部位或其他屏障部位的复制中断是诱变，基因组重排和细胞中致死性的主要原因。 RECF途径是成功重新启动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重组酶载入DNA是必不可少的，这是触发SOS响应的事件，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很重要。这些蛋白质在所有生物体中均具有功能性。即使在细菌中，他们的活动机制也很熟悉，这是我们研究的长期目标。关于RECFOR活动的重要问题之一是，Recfors -Reciation/Recociation及其与DNA的相互作用的机制是什么？我们将通过对RECF和RECFOR复合物进行结构研究，并研究RECFOR反应的初始步骤的机制：RECF DNA结合和RECF对DNA募集RERS的初始步骤。以下目的，将研究以ATP驱动的二聚化在RECF活性中起着核心作用，并调节RECF与DNA和/或与RERS的相互作用的核心作用：1）RECF的晶体结构将在高分辨率上解决以揭示SMC域的特定特征和RECF特定域的新型结构； 2）将直接测量RECF和DNA结合后的ATP依赖性二聚化； RECF的结构引导的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）将使用野生型和突变蛋白研究RECF/RERM/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。我们还将试图将恢复复合物共结合，以研究其在原子分辨率水平上的相互作用。加上我们的协作研究，其中选定的突变体将在体外促进RECA核蛋白丝组装及其对紫外线辐照细胞的复制恢复的影响，并将提供重新组合/复制阶程的重要信息。将获得对RECF相互作用的新的详细机械理解在修复停滞的复制叉期间的RECF相互作用，从而促进了我们对复制重新启动和重组修复过程的理解。它将有助于制定对真核生物中类似过程的未来研究，在这些过程中，其故障与肿瘤发生高度相关。