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)发挥着重要的功能，包括DNA修复、同源染色体分离、遗传多样性的传播和端粒的维持。HR负责修复由电离辐射(IR)和交联剂(CLA)引起的DNA双链断裂，这两种药物通常用于肿瘤治疗。HR功能障碍导致基因组不稳定，导致癌症和各种染色体异常，如唐氏症和其他综合征。我们的长期目标是了解人类细胞中HR的分子机制。我们将使用生化和体外重建的方法来分析HR的酶机制。在DNA断裂时，HR促进对同源序列的搜索，并随后将断裂的DNA末端侵入同源双链DNA，然后作为修复的模板。入侵产生了一种交叉链状结构，称为Holliday连接(HJ)。HJ具有沿DNA轴分支迁移(BM)能力。Bm i)影响每个亲本贡献的遗传物质的数量，ii)导致HR中间体的分离，iii)通过它们的回归拯救停滞不前的复制叉子。我们最近发现，hRad54是一种重要的HR蛋白，它催化HJ依赖于ATP的BM。HRad54是一种促进HJ识别，将能量消耗与机械运动结合起来，促进HJS迁移的马达蛋白。以往的研究表明，hRad54能刺激HR的关键蛋白hRad51的DNA链交换活性。这里我们想要了解hRad54的这两个基本活动，BM和刺激hRad51是如何协调的(目标1)。Bloom综合征解旋酶(BLM)是促进HJ骨髓形成的另一种真核蛋白。矛盾的是，hRad54和BLm突变体在人类细胞中表现出不同的表型。我们将研究这种差异的分子基础(目标2)。我们将确定支持hRad54关键功能的蛋白质结构域：BM和hRad51刺激(目标3)。HJ的解决仍然是HR最神秘的阶段。HRad54蛋白与MUS81/Eme1相互作用，MUS81/Eme1是一种切割HJS的结构特异性内切酶。在这里，我们将确定这种相互作用对MUS81/EME1(目标4)拆分HJ的影响。 公共卫生相关性： 在人类中，同源重组系统发挥着至关重要的功能，包括DNA修复、同源染色体分离、遗传多样性的传播和端粒的维持。同源重组负责修复由电离辐射和交联剂引起的DNA双链断裂，这是肿瘤治疗中常用的方法。对同源重组机制的了解为癌症治疗提供了更有效的方法。

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.