Homology Directed Repair

同源定向修复

• 批准号: 7992616
• 负责人: SCOTT W MORRICAL
• 金额: $ 20.25万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-03 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7992616
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Active Sites; Amino Acids; Base Excision Repairs; Binding; Biochemical; Biological Assay; Biological Process; Cells; Chromatin; Chromosomes; DNA; DNA Binding; DNA Polymerase beta; DNA Repair; DNA Repair Pathway; DNA-Directed DNA Polymerase; Defect; Diagnosis; Enzymes; Excision Repair; Family; Filament; Funding; Genome Stability; Genomic Instability; Germ Lines; Glean; Goals; Human; Knowledge; Lead; Link; Malignant Neoplasms; Methods; Modeling; Molecular; Mutagenesis; Mutation; Orthologous Gene; Pathway interactions; Phenotype; Play; Polymerase; Predisposition; Property; Proteins; Rad51 recombinase; Reaction; Recruitment Activity; Roentgen Rays; Role; Single-Stranded DNA; Sister; Site; Site-Directed Mutagenesis; Structure; Testing; Transferase; Tumor-Derived; Variant; Yeasts; base; cancer cell; cancer therapy; cell transformation; design; in vivo; interest; mutant; neoplastic cell; novel; presynaptic; programs; protein protein interaction; recombinational repair; repaired; structural biology; treatment strategy; tumor

Rad51 protein is the eukaryotic representative of the RecA/Rad51 fannlly of DNA strand transferase enzymes. Homologous DNA strand exchanges catalyzed by RadSI are critical for Homology-Dlrected DNA Repair (HDR) and therefore for genome stability. To promote HDR, RadSI must first assemble onto single stranded DNA In the form of a presynaptic filament. Filament assembly allosterically activates RadSI to catalyze ATP hydrolysis, to search for homology in a sister chromosome, and to perform DNA strand exchange reactions. There Is compelling evidence that defects in the assembly and activity of RadSI presynaptic filaments are linked to human cancer. The overall goal of Project 3 Is to understand how specific changes in the structure, function, and molecular interactions of RadSI can lead to genomic instability and cancer. The SPECIFIC AIMS of Project 3 are: (1) To test the hypothesis that key amino acid residues at the filament interface and in the ATPase active site of RadSI control the allosteric transitions that couple the ATPase catalytic cycle to DNA strand exchange. Using yeast RadSI as a model, the catalytic and allosteric mechanisms of RadSI will be probed using a combination of site-directed mutagenesis, biochemical and biophysical analyses, and structural biology methods. (2) To test the hypothesis that tumor-derived variants of human hRADSI protein have altered biochemical and/or regulatory properties. hRADSI variants identified in Project 1 will be characterized biochemically alongside wild-type hRADSI to identify any changes in DNA binding or catalytic properties, or in key protein-protein interactions. (3) To test the hypothesis that interactions between hRADSI and DNA polymerase beta (Pol-beta) help recruit hRADSI onto ssDNA generated as a result of abortive base excision repair (BER). hRADSI:Pol-beta interactions will be characterized biochemically and disrupted by mutagenesis to assess their importance for DNA repair functions. Interesting mutants from Aims 2-3 will be exported to Projects 1 and 4 for in vivo and chromatin studies. This project will provide rigorous models for the structure, function, and assembly of RadSI presynaptic filaments, and for potential cross-talk between HDR and BER pathways, in normal vs, tumor cells, which will be useful for predicting cancer susceptibility and for developing new cancer treatments.

Rad 51蛋白是DNA链转移酶RecA/Rad 51家族的真核生物代表蛋白 内切酶由RadSI催化的同源DNA链交换对于同源导向的DNA是至关重要的 修复（HDR），因此基因组稳定性。为了促进HDR，RadSI必须首先组装到单个 以突触前纤维的形式存在的DNA链。细丝组装变构激活RadSI， 催化ATP水解，在姐妹染色体中寻找同源性， 交换反应。有令人信服的证据表明，缺陷的组装和活动的RadSI 突触前纤维与人类癌症有关项目3的总体目标是了解如何具体 RadSI的结构、功能和分子相互作用的变化可导致基因组不稳定， 癌项目3的具体目标是：（1）检验在蛋白质中的关键氨基酸残基的假设， 丝界面和RadSI的ATP酶活性位点控制着与丝偶联的变构转换， ATP酶催化循环至DNA链交换。使用酵母RadSI作为模型，催化和变构 RadSI的机制将使用定点诱变，生物化学和 生物物理分析和结构生物学方法。(2)为了验证肿瘤衍生变异 人hRADSI蛋白的生物化学和/或调节特性改变。鉴定的hRADSI变体 在项目1中，将与野生型hRADSI一起进行生物化学表征，以确定DNA中的任何变化 结合或催化性质，或关键蛋白质-蛋白质相互作用。(3)为了验证这个假设， hRADSI和DNA聚合酶β（Pol-beta）之间的相互作用有助于将hRADSI募集到ssDNA上 由于碱基切除修复（BER）失败而产生。hRADSI：Pol-beta相互作用将 生物化学表征并通过诱变破坏，以评估其对DNA修复的重要性 功能协调发展的来自目标2-3的感兴趣的突变体将被输出到项目1和项目4，用于体内和染色质研究。 问题研究该项目将为RadSI的结构、功能和组装提供严格的模型 突触前纤维，以及HDR和BER通路之间的潜在串扰，在正常与肿瘤中 这将有助于预测癌症易感性和开发新的癌症治疗方法。