Repair of DNA breaks in Humans: the role of Rad54 protein

描述（由申请人提供）： 在人类中，同源重组（HR）执行关键功能，包括DNA修复，同源染色体的分离，遗传多样性的繁殖和端粒的维持。HR负责修复由电离辐射（IR）和交联剂（CLA）诱导的DNA双链断裂，这通常用于肿瘤治疗。HR的功能障碍导致基因组不稳定，导致癌症和各种染色体异常，如唐氏综合征和其他综合征。我们的长期目标是了解人类细胞中HR的分子机制。我们将使用生物化学和体外重建的方法来分析的酶机制的HR。启动在DNA断裂，HR促进寻找同源序列和随后的入侵到同源双链体DNA，然后作为修复的模板断裂的DNA末端。入侵产生交叉链结构，称为霍利迪连接（HJ）。HJ具有显著的沿着DNA轴分支迁移（BM）的能力。BM i）影响每个亲本贡献的遗传物质的量，ii）引起HR中间体的解离，和iii）通过它们的回归拯救停滞的复制叉。我们最近发现，hRad 54，一个重要的HR蛋白，催化一个ATP依赖的HJ BM。hRad 54是促进HJ识别、将能量消耗与机械运动耦合并促进HJ迁移的马达蛋白。以前，它表明，hRad 54刺激的DNA链交换活动的hRad 51，一个关键的蛋白质的HR。在这里，我们想了解这两个基本活动的hRad 54，BM和hRad 51的刺激，是如何协调的（目的1）。Bloom's syndrome helicase（BLM）是另一种促进HJ BM的真核蛋白。巧合的是，hRad 54和BLM突变体在人类细胞中显示出不同的表型。我们将研究这种差异的分子基础（目标2）。我们将鉴定支持关键hRad 54功能的蛋白质结构域：BM和hRad 51刺激（Aim 3）。HJ的分解仍然是HR中最神秘的阶段。hRad 54蛋白与结构特异性内切酶Mus 81/Eme 1物理相互作用，其切割HJ。在这里，我们将确定这种相互作用对Mus 81/Eme 1的HJ分辨率的影响（目的4）。 公共卫生相关性： 在人类中，同源重组系统执行关键功能，包括DNA修复、同源染色体分离、遗传多样性繁殖和端粒维持。同源重组负责修复由电离辐射和交联剂诱导的DNA双链断裂，其通常用于肿瘤治疗。同源重组机制的知识导致更有效的癌症治疗方法。

DESCRIPTION (provided by applicant): In humans, homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks induced by ionizing radiation (IR) and cross-linking agents (CLA), which are commonly used in tumor therapy. Malfunction of HR causes genome instability leading to cancer and various chromosomal abnormalities such as Down's and other syndromes. Our long-term goal is to understand the molecular mechanisms of HR in human cells. We will use biochemical and in vitro reconstitution approaches for analysis of the enzymatic machinery of HR. Initiated at DNA breaks, HR promotes a search for homologous sequences and subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for the repair. The invasion produces a cross-stranded structure, known as Holliday junction (HJ). HJ possesses a remarkable ability to branch migrate (BM) along the DNA axis. BM i) affects the amount of genetic material contributed by each parent, ii) causes dissociation of HR intermediates, and iii) rescues stalled replication forks through their regression. We discovered recently that hRad54, an important HR protein, catalyzes an ATP dependent BM of HJ. hRad54 is a motor protein that promotes HJ recognition, couples energy consumption with mechanical motion, and promotes migration of HJs. Previously, it was shown that hRad54 stimulates DNA strand exchange activity of hRad51, a key protein of HR. Here we want to understand how these two essential activities of hRad54, BM and stimulation of hRad51, are coordinated (Aim 1). Bloom's syndrome helicase (BLM) is another eukaryotic protein that promotes BM of HJ. Paradoxically, hRad54 and BLM mutants show dissimilar phenotypes in human cells. We will investigate the molecular basis for this difference (Aim 2). We will identify the protein domains which support critical hRad54 functions: BM and hRad51 stimulation (Aim 3). Resolution of HJ remains the most mysterious stage of HR. hRad54 protein physically interacts with Mus81/Eme1, a structure- specific endonuclease, which cleaves HJs. Here we will determine the effect of this interaction on the resolution of HJ by Mus81/Eme1 (Aim 4). PUBLIC HEALTH RELEVANCE: In humans, the system of homologous recombination performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. Homologous recombination is responsible for the repair of DNA double-strand breaks induced by ionizing radiation and cross-linking agents, which are commonly used in tumor therapy. Knowledge of the mechanisms of homologous recombination leads to more efficient approaches for cancer treatment.