Functions of Rad51 Paralogs in Recombinational DNA Repair

描述(由申请人提供)：总体目标是识别和理解重组DNA修复的分子机制，这是保持基因组完整性的关键途径。这一应用重点是RAD51 Paralog，这是一类出了名的难以研究的蛋白质，其对重组的机制贡献仍有待确定。RAD51类似物起着关键作用，即RAD51-ssDNA细丝的组装和维护，RAD51-ssDNA细丝是同源重组DNA高保真模板定向修复的重要中间产物。在这一应用中，两位主要研究人员(Heyer和Cotalczykowski)结合他们的专业知识，利用单分子生物物理、总体生物化学和酵母遗传学，促进了我们对RAD51在酵母和人类DNA修复中的Paralog功能的理解。具体目的是：(1)确定酵母RAD51蛋白在yRad51-ssDNA微丝组装中的功能专一性。突触前RAD51-ssDNA微丝在组装和拆解之间处于亚稳定平衡状态。我们将确定RAD51并列基因在RAD51细丝形成中的功能专化性与DSB断裂之间的关系，并确定特定抗重组酶的潜在选择性。(2)确定人类RAD51Paralog动作的功能后果。为了确定RAD51对虾的功能，将对它们的生化行为进行检测。我们将确定它们的DNA结合特异性，它们刺激hRAD51促进DNA链交换的能力，以及它们稳定hRAD51核蛋白细丝不被内在和解旋酶(BLM、RECQL5、FANCJ和FBH1)分解的能力；任何影响的机制都将被确定。我们还将检查是否有其他与RAD51并列蛋白相互作用的蛋白质调节RAD51细丝的形成或稳定性。(3)使用单分子分析来确定人类RAD51对hRAD51-ssDNA微丝动态行为的影响。测试酵母研究中建立的范式，我们将测量人类RAD51平行基因是否改变了hRAD51细丝组装或拆解的速度。PARALLOGS原则上可以提高成核频率或细丝生长速率，也可以降低解离速率。这些测量将使用单分子荧光显微镜对DNA单个分子上的hRAD51细丝动态进行直接成像。

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.