The Novel Role of Branch Migration Protein Rad54 in Maintaining Genome Stability

描述(申请人提供)：同源重组是负责修复双链DNA断裂和在经历减数分裂的细胞中在同源染色体之间交换遗传物质的关键过程。我们的目标是了解人类同源重组的机制。同源重组的机制包括在两条同源染色体之间形成一种称为Holliday连接的交叉链结构。Holliday结具有一个显著的性质，这种结构可以在一个称为分支迁移的过程中沿着DNA轴自由移动。分支迁移在同源重组和DNA修复中的中心作用直到最近才得到充分认识。分支迁移驱动1)同源重组中间体的分解，2)DNA损伤部位丢失的信息的恢复，以及3)停滞的DNA复制叉子的重新启动。在人类细胞中，分支迁移是由RecQ解旋酶催化的，包括BLM和WRN。BLM和WRN基因的突变会导致高度的基因组不稳定，从而导致称为Bloom‘s和Werner’s综合征的过早衰老障碍。最近的遗传分析表明，Rad54在真核生物的同源重组中也起着重要的作用。双RAD54BLM基因敲除显示染色体不稳定程度显著增加，这表明这两种蛋白质之间存在功能冗余。事实上，我们的实验室最近发现，Rad54也可以促进Holliday连接的分支迁移。我们将在一系列专门设计的生化分析中使用纯化的人类蛋白来研究Rad54和BLM的分支迁移活性。该建议的目的是了解RAD51如何影响Rad54和BLm在同源重组中的功能，并研究Rad54在分支迁移过程中发挥作用的非典型解旋酶活性。这些分析将与Rad54的突变分析相结合，以确定哪些基序对其生物学活性至关重要。我们的目标是更好地了解这两种相似但不同的酶的分支迁移活动如何有助于维持人类细胞的基因组完整性。

DESCRIPTION (provided by applicant): Homologous recombination is a critically important process responsible for repairing double-strand DNA breaks and exchanging genetic material between homologous chromosomes in cells undergoing meiosis. Our goal is to understand the mechanisms of homologous recombination in humans. The mechanism of homologous recombination involves forming a cross-stranded structure between two homologous chromosomes called a Holliday junction. The Holliday junction possesses a remarkable property, in which this structure can freely translocate along the DNA axis in a process termed branch migration. The central role of branch migration in homologous recombination and DNA repair has only recently been fully appreciated. Branch migration drives 1) the resolution of homologous recombination intermediates, 2) the retrieval of information lost at sites of DNA damage, and 3) the restart of stalled DNA replication forks. In human cells, branch migration is catalyzed by RecQ helicases, including BLM and WRN. Mutations in the BLM and WRN genes cause a high degree of genome instability, which lead to premature aging disorders called Bloom's and Werner's Syndromes. Recent genetic analysis has demonstrated that Rad54 also plays an important role in homologous recombination of eukaryotes. Double RAD54 BLM knockouts show a dramatic increase in chromosomal instability over the level observed in either single knockout, indicating a functional redundancy between these two proteins. Indeed, our lab recently discovered that Rad54 can also promote branch migration of Holliday junctions. We will use purified human proteins in an array of biochemical assays specifically designed to investigate the branch migration activity of Rad54 and BLM. The goals of this proposal are to understand how Rad51 affects the function of Rad54 and BLM in homologous recombination and investigate the non-canonical helicase activity of Rad54 that functions during branch migration. These assays will be combined with a mutational analysis of Rad54 to determine which motifs are crucial to its biological activities. Our goal is to better understand how the branch migration activities of these two similar, but distinct enzymes contribute to the maintenance of genome integrity in human cells.