Homology-directed Repair of DNA Breaks in Humans

描述(申请人提供)：细胞持续暴露在内源性和外源性元素中，这些元素会导致脱氧核糖核酸双链断裂S，如果去除不当，可能会导致死亡或严重的染色体异常。同源重组(HR)是消除DSB的主要途径。有令人信服的证据表明，人类需要同源定向的DNA修复来避免癌症。 酵母中的遗传分析导致了HR所需的RAD52上位性基因组的鉴定。随后的克隆、生化和遗传学研究表明，在真核生物进化过程中，RAD52组基因和蛋白质的结构和功能得到了显著的保护。对RAD51编码产物的生化分析发现，它具有同源DNA配对和链交换活性，可以连接重组染色体。RAD51编码产物是RAD52组的关键成员。RAD51的重组酶活性受多层调控。本研究项目将利用多种分子工具描述由人RAD51蛋白介导的同源DNA配对和链交换反应，并确定各种人重组因子在调节hRad51重组酶活性中的作用。具体地说，我们将(1)研究ATP结合和水解如何调节hRad51与其DNA底物的亲和力和hRad51突触前细丝动力学，以及(2)定义hRad54和Rad54B蛋白的生化特性，剖析这些因素在HR中的多方面作用，并鉴定和表征包含这些因素的复合体。这一结果应该有助于阐明人类细胞中同源重组机制的机制基础，并将对癌症病因学和预防具有重要意义。

DESCRIPTION (provided by applicant): Cells are continually exposed to endogenous and exogenous elements that induce DNA double-strand breaks (DSB)s, which, if not properly removed, can cause death or gross chromosome aberrations. Homologous recombination (HR) is a major pathway for the elimination of DSBs. There is compelling evidence that homology-directed DNA repair is needed for cancer avoidance in humans. Genetic analyses in yeast have led to the identification of the RAD52 epistasis group of genes needed for HR. Subsequent cloning, biochemical, and genetic studies have shown remarkable conservation of the structure and function of the RAD52 group genes and proteins during eukaryotic evolution. Biochemical analyses of the RAD51-encoded product, a key member of the RAD52 group, have uncovered in it a homologous DNA pairing and strand exchange activity that can serve to link recombining chromosomes. The recombinase activity of Rad51 is subject to multiple layers of control. This research project will utilize a variety of molecular tools to delineate the homologous DNA pairing and strand exchange reaction mediated by human Rad51 protein and define the role of various human recombination factors in regulating the hRad51 recombinase activity. Specifically, we will (1) examine how ATP binding and hydrolysis modulate the affinity of hRad51 for its DNA substrates and hRad51 presynaptic filament dynamics, and (2) define the biochemical properties of the hRad54 and Rad54B proteins, dissect the multifaceted role of these factors in HR, and identify and characterize complexes that contain these factors. The results should shed some light on the mechanistic underpinnings of the homologous recombination machinery in human cells and will have important implications for cancer etiology and prevention.