Homology Directed Repair

RAD51蛋白是DNA链转移酶RecA/RAD51家族的真核代表 酵素。RADSI催化的同源DNA链交换是同源定向DNA的关键 修复(HDR)，从而保证基因组的稳定性。为了推广HDR，RadSI必须首先组装到Single上 以突触前细丝的形式搁浅的DNA。灯丝组件变构激活RadSI到 催化ATP水解，在姐妹染色体中寻找同源性，并执行DNA链 交换反应。有令人信服的证据表明，RADSI的组装和活性存在缺陷 突触前细丝与人类癌症有关。项目3的总体目标是了解具体到 RADSI结构、功能和分子相互作用的变化可导致基因组不稳定和 癌症。项目3的具体目标是：(1)检验假设关键氨基酸残基在 RADSI的细丝界面和ATPase活性部位控制着偶联 ATPase催化循环至DNA链交换。以酵母RadSI为模型，催化和变构 RADSI的机制将结合定点突变、生化和 生物物理分析和结构生物学方法。(2)检验肿瘤衍生变异体的假设 已经改变了人类hRADSI蛋白的生化和/或调节特性。发现hRADSI变异体 在项目1中，将与野生型hRADSI一起进行生化表征，以确定DNA的任何变化 结合或催化特性，或关键的蛋白质-蛋白质相互作用。(3)检验假设 HRADSI与DNA聚合酶β(POL-β)的相互作用有助于将hRADSI募集到单链DNA上 这是碱基切除修复(BER)流产的结果。HRADSI：POL-Beta交互将是 具有生化特征并被突变破坏，以评估它们对DNA修复的重要性 功能。来自AIMS 2-3的有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 学习。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，以及正常和肿瘤中HDR和BER通路之间的潜在串扰 细胞，这将有助于预测癌症易感性和开发新的癌症治疗方法。

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.