Functions of Rad51 Paralogs in Recombinational DNA Repair

描述（由申请人提供）：总体目标是鉴定和理解重组DNA修复的分子机制，这是保持基因组完整性的关键途径。本申请集中于Rad 51旁系同源物，这是一类众所周知的难以研究的蛋白质，其对重组的机制贡献仍有待确定。Rad 51旁系同源物在一个关键点起作用，即Rad 51- ssDNA细丝的组装和维持，这是通过同源重组DNA进行高保真模板定向修复的必要中间体。在这个应用程序中，两个主要的研究人员（海耶和Kowalczykowski）联合收割机结合他们的专业知识，在一个全面的方法，以提高我们的理解Rad 51在酵母和人类利用单分子生物物理学，整体生物化学和酵母遗传学的DNA修复的parallel功能。具体目的是：（1）确定酵母Rad 51旁系同源物在yRad 51-ssDNA丝状体组装中的功能特化。突触前Rad 51-ssDNA丝在其组装和拆卸之间存在亚稳态平衡。我们将确定Rad 51旁系同源物在间隙与DSB断裂处Rad 51细丝形成中的功能特化，并确定对特定抗重组酶的潜在选择性。(2)确定人RAD 51 paradox作用的功能后果。为了确定RAD 51旁系同源物的功能，将检查它们的生化行为。我们将确定它们的DNA结合特异性，它们刺激由hRAD 51促进的DNA链交换的能力，以及它们稳定hRAD 51核蛋白丝免于内在和解旋酶（BLM，RECQL 5，FANCJ和FBH 1）分解的能力;将确定任何作用的机制。我们还将研究是否其他蛋白质，与RAD 51旁系同源物相互作用，功能调节RAD 51丝的形成或稳定性。(3)使用单分子分析来确定人RAD 51旁系同源物如何影响hRAD 51-ssDNA细丝的动态行为。测试在酵母研究中建立的范例，我们将测量人RAD 51旁系同源物是否改变hRAD 51细丝组装或拆卸的速率。旁系同源物原则上可以增加成核频率或细丝生长速率，或者它们可以降低解离速率。这些测量将使用通过单分子荧光显微镜对单个DNA分子上的hRAD 51细丝动力学的直接成像。

DESCRIPTION (provided by applicant): The general goal is to identify and understand the molecular mechanisms of recombinational DNA repair, a key pathway for preserving genomic integrity. This application focuses on the Rad51 paralogs, a class of proteins that has been notoriously difficult to study, and whose mechanistic contribution to recombination remains to be defined. The Rad51 paralogs act at a pivotal point, namely the assembly and maintenance of the Rad51- ssDNA filament, an essential intermediate for high-fidelity template-directed repair by homologous recombination DNA. In this application, two principal investigators (Heyer and Kowalczykowski) combine their expertise in a comprehensive approach to advance our understanding of Rad51 paralog function in DNA repair in yeast and humans utilizing single-molecule biophysics, ensemble biochemistry, and yeast genetics. The Specific Aims are: (1) Determine the functional specialization of yeast Rad51 paralogs in yRad51-ssDNA filament assembly. The presynaptic Rad51-ssDNA filament exists in a meta-stable balance between its assembly and disassembly. We will determine the functional specialization of the Rad51 paralogs in Rad51 filament formation at gaps versus DSB breaks and determine potential selectivity for specific anti-recombinases. (2) Determine functional consequences of human RAD51 paralog action. To ascertain the functions of the RAD51 paralogs, their biochemical behavior will be examined. We will determine their DNA binding specificity, their capacity to stimulate DNA strand exchange promoted by hRAD51, and their ability to stabilize hRAD51 nucleoprotein filaments from disassembly both intrinsically and by helicases (BLM, RECQL5, FANCJ, and FBH1); the mechanisms underlying any effect will be determined. We will also examine whether other proteins, which interact with the RAD51 paralogs, function to regulate the formation or stability of RAD51 filaments. (3) Use single-molecule analyses to determine how the human RAD51 paralogs affect the dynamic behavior of hRAD51-ssDNA filaments. Testing the paradigm established in the yeast studies, we will measure whether the human RAD51 paralogs alter the rate of hRAD51 filament assembly or disassembly. The paralogs could, in principle, increase the nucleation frequency or the filament growth rate, or they could decrease the dissociation rate. These measurements will use direct imaging by single-molecule fluorescence microscopy of hRAD51 filament dynamics on individual molecules of DNA.