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

在人类中，同源重组(HR)执行关键功能，包括DNA修复、分离 同源染色体、遗传多样性的繁殖和端粒的维持。由人力资源部负责 为了修复由电离辐射(IR)和交联剂(CLA)引起的DNA双链断裂， 它们通常用于肿瘤治疗。HR功能障碍导致基因组不稳定导致癌症 以及各种染色体异常，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 HR酶机制分析的重构方法。 在DNA断裂时启动，HR促进对同源序列的搜索和随后的入侵 断裂的DNA末端形成同源的双链DNA，然后作为修复的模板。侵略者 产生一种交叉链结构，称为Holliday结(HJ)。HJ拥有非凡的能力 沿DNA轴的分支迁移(BM)。BM I)影响每种基因所贡献的遗传物质 父代，ii)导致HR中间体的分离，以及iii)通过他们的 回归。 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识别的马达蛋白，将能量消耗与机械运动结合起来， 促进了HJS的迁徙。此前，研究表明，hRad54可以刺激DNA链交换活动 HRad51是HR的关键蛋白。在这里，我们想了解hRad54，BM的这两个基本活动是如何 和hRad51的刺激是协调的(目标1)。布鲁姆综合征解旋酶(BLM)是另一种真核生物 促进HJ的BM的蛋白质。矛盾的是，hRad54和BLm突变体在 人类细胞。我们将研究这种差异的分子基础(目标2)。我们会鉴定出这种蛋白质 支持关键hRad54功能的区域：BM和hRad51刺激(目标3)。HJ的分辨率 仍然是人力资源最神秘的阶段。HRad54蛋白与MUS81/Eme1物理上相互作用，一个结构- 裂解HJS的特定内切酶。在这里，我们将确定此交互作用对 MUS81/EME1对HJ的决议(目标4)。

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).