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Rad51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。Rad51旁系同源物在一个关键点起作用，即Rad51-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Rad51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Rad51旁系同源物在yRad51-ssDNA丝状体组装中的功能特化。突触前Rad51-ssDNA丝在其组装和拆卸之间存在亚稳态平衡。我们将确定Rad51旁系同源物在间隙与DSB断裂处Rad51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RAD51 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RAD51旁系同源物如何影响hRAD51-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。 公共卫生相关性：许多抗肿瘤治疗方式直接或间接诱导DNA双链断裂或缺口，同源重组是DNA损伤修复的中心途径，其主要功能是修复这种DNA损伤。拟议的工作集中在一类了解甚少的同源重组蛋白，在组装的RAD 51丝的功能的RAD 51旁系同源物。预期的结果将导致对重组DNA修复机制的更好理解，这对于理解旁系同源物中的突变如何促进癌症易感性至关重要，并且对于设计生物学方法以提高基于DNA损伤的抗肿瘤治疗的功效至关重要。

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. PUBLIC HEALTH RELEVANCE: Many modalities of anti-tumor therapy induce directly or indirectly DNA double-stranded breaks or gaps, and homologous recombination is a central DNA damage repair pathway, whose primary function is to repair such DNA damage. The proposed work focuses on a poorly understood class of homologous recombination proteins, the RAD51 paralogs that function in the assembly of the RAD51 filament. The expected results will lead to an improved mechanistic understanding of recombinational DNA repair, which is essential for understanding how mutations in the paralogs contribute to cancer predisposition and fundamental for designing biological approaches to improve the efficacy of DNA damage-based anti-tumor therapy.