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， 同源染色体、遗传多样性的繁殖和端粒的维持。HR负责 用于修复由电离辐射（IR）和交联剂（CLA）诱导的DNA双链断裂， 其通常用于肿瘤治疗。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，如唐氏综合征和其他综合征。我们的长期目标是 了解HR在人类细胞中的分子机制。我们将使用生物化学和体外 重组方法的酶机制的分析HR。 起始于DNA断裂，HR促进寻找同源序列和随后的入侵。 断裂的DNA以同源双链DNA结束，然后作为修复的模板。入侵 产生交叉链结构，称为霍利迪连接（HJ）。HJ拥有非凡的能力， 分支沿DNA轴沿着迁移。BM i）影响每个基因贡献的遗传物质的量。 亲本，ii）引起HR中间体的解离，和iii）通过其 回归分析 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识别的马达蛋白，将能量消耗与机械运动耦合， 并促进HJ的迁移。以前，已经表明hRad 54刺激DNA链交换活性， hRad 51是HR的一个关键蛋白。在这里，我们想了解hRad 54，BM 和hRad 51的刺激是协调的（目的1）。Bloom综合征解旋酶（BLM）是另一种真核生物 促进HJ的BM的蛋白质。巧合的是，hRad 54和BLM突变体在细胞中显示出不同的表型。 人体细胞我们将研究这种差异的分子基础（目标2）。我们将鉴定出 支持关键hRad 54功能的结构域：BM和hRad 51刺激（Aim 3）。HJ分辨率 hRad 54蛋白与Mus 81/Eme 1相互作用，后者是一种结构- 特异性内切核酸酶，其切割HJ。在这里，我们将确定这种相互作用对 通过Mus 81/Eme 1对HJ进行拆分（目的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).