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

描述（由申请人提供）：同源重组是一个非常重要的过程，负责修复双链DNA断裂并在减数分裂细胞中同源染色体之间交换遗传物质。我们的目标是了解人类同源重组的机制。同源重组的机制涉及在两个同源染色体之间形成交叉链结构，称为霍利迪连接。霍利迪连接具有显着的性质，其中该结构可以在称为分支迁移的过程中沿着DNA轴自由移位。分支迁移在同源重组和DNA修复中的中心作用直到最近才被充分认识。分支迁移驱动1）同源重组中间体的解析，2）在DNA损伤位点丢失的信息的恢复，和3）停滞的DNA复制叉的重新启动。在人类细胞中，分支迁移由RecQ解旋酶催化，包括BLM和WRN。BLM和WRN基因的突变会导致基因组高度不稳定，从而导致称为Bloom's和Werner's Syndrome的过早衰老疾病。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作用。双RAD54 BLM敲除显示染色体不稳定性的急剧增加超过在任一单敲除中观察到的水平，表明这两种蛋白质之间的功能冗余。事实上，我们的实验室最近发现，Rad54也可以促进霍利迪连接的分支迁移。我们将在一系列专门设计用于研究Rad54和BLM的分支迁移活性的生化测定中使用纯化的人蛋白质。本研究的目的是了解Rad51如何影响Rad54和BLM在同源重组中的功能，并研究Rad54在分支迁移过程中的非经典解旋酶活性。这些试验将与Rad54的突变分析相结合，以确定哪些基序对其生物活性至关重要。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中基因组的完整性。

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.