Homology Directed Repair

同源定向修复

基本信息

批准号：
8725060
负责人：
SCOTT W MORRICAL
金额：
$ 23.76万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-03 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8725060
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 Enzymes DNA Repair Pathway DNA-Directed DNA Polymerase Defect Diagnosis Enzymes 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

项目摘要

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.

Rad51蛋白是DNA链转移酶的RecA/Rad51的真核代表酶。由RADSI催化的同源DNA链交换对于同源性DNA至关重要修复（HDR），因此用于基因组稳定性。要促进HDR，Radsi必须首先组装到单个滞留的DNA以突触前细丝的形式。细丝组件变构将RADSI激活催化ATP水解，在姐妹染色体中寻找同源性并进行DNA链交换反应。有令人信服的证据表明RADSI的组装和活性缺陷突触前细丝与人类癌症有关。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会导致基因组不稳定性和癌症。项目3的具体目的是：（1）检验以下假设：细丝界面和在RADSI的ATPase活动位点控制的变构转变 ATPase催化循环到DNA链交换。使用酵母红色作为模型，催化和变构将使用位置定向诱变，生化和生物物理分析和结构生物学方法。（2）检验肿瘤衍生变体的假设人hradsi蛋白的生化和/或调节性质改变了。识别Hradsi变体在项目1中，将与野生型Hradsi一起在生化上进行表征，以识别DNA的任何变化结合或催化特性，或关键蛋白质蛋白质相互作用。（3）检验以下假设 Hradsi和DNA聚合酶β（POL-BETA）之间的相互作用有助于将Hradsi招募到ssDNA 由于堕胎基础切除修复（BER）而产生。 Hradsi：Pol-Beta互动将是在生化特征并被诱变而破坏以评估其对DNA修复的重要性功能。 AIM 2-3的有趣突变体将导出到体内和染色质的项目1和4 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细丝，以及HDR和BER途径之间的潜在串扰，在正常VS中，肿瘤细胞，这对于预测癌症的敏感性和开发新的癌症治疗非常有用。