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S唐氏综合症等。我们的长期目标是 了解人类HR蛋白的功能。我们将使用生化和体外重建的方法来 分析HR蛋白如何整合到HR机制中。 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)，这i)影响每个 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， 并促进HJ的进步。以前的研究表明，hRad54可以刺激DNA链交换 HR的关键蛋白hRad51的活性。在这里，我们想要了解这两项基本活动是如何 HRad54、BM和hRad51的刺激是协调的(目标1)。与人类免疫缺陷病毒54类似的布鲁姆S综合征 解旋酶促进HJ的BM。矛盾的是，hRad54和BLm突变体表现出不同的表型。我们会 研究这种差异的分子基础(目标2)。我们将确定支持的蛋白质结构域 关键hRad54功能：骨髓和hRad51刺激(目标3)。HJ的解决方案仍然是最神秘的 HR分期。HRad54蛋白与MUS81/Eme1，一种结构特异的内切酶相互作用， 割裂HJS。在这里，我们将描述这些相互作用及其对两种酶活性的影响 HRad54和MUS81/Eme1(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 functions of human HR proteins. We will use biochemical and in vitro reconstitution approaches to analyze how HR proteins are integrated within the HR machinery. 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, which 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 progression of the HJ. 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). Similar to hRad54, Bloom¿s syndrome helicase promotes BM of HJ. Paradoxically, hRad54 and BLM mutants show different phenotypes. 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 characterize these interactions and their effect on the enzymatic activities of both hRad54 and Mus81/Eme1 (Aim 4).