Functions of Rad51 Paralogs in Recombinational DNA Repair

描述（由申请人提供）：总体目标是确定和理解重组DNA修复的分子机制，这是保持基因组完整性的关键途径。该应用程序的重点是Rad51类似物，这是一类众所周知难以研究的蛋白质，其对重组的机制贡献仍有待确定。Rad51类似物在一个关键点上起作用，即Rad51- ssDNA丝的组装和维护，这是同源重组DNA进行高保真模板定向修复的重要中间体。在这个应用中，两位主要研究者（Heyer和Kowalczykowski）结合他们的专业知识，利用单分子生物物理学、集合生物化学和酵母遗传学，以全面的方法推进我们对Rad51在酵母和人类DNA修复中的平行功能的理解。具体目的是：(1)确定酵母Rad51同源物在yRad51-ssDNA丝组装中的功能专门化。突触前Rad51-ssDNA丝存在于其组装和拆卸之间的亚稳定平衡中。我们将确定Rad51类似物在Rad51丝形成与DSB断裂中的功能专门化，并确定特定抗重组酶的潜在选择性。(2)确定人类RAD51平行作用的功能后果。为了确定RAD51类似物的功能，我们将研究它们的生化行为。我们将确定它们的DNA结合特异性，它们刺激hRAD51促进的DNA链交换的能力，以及它们稳定hRAD51核蛋白丝的能力，使其免受内在和解旋酶的破坏（BLM, RECQL5， FANCJ和FBH1）；任何影响背后的机制都将被确定。我们还将研究与RAD51类似物相互作用的其他蛋白质是否对RAD51细丝的形成或稳定性起调节作用。(3)利用单分子分析确定人类RAD51相似物如何影响hRAD51-ssDNA细丝的动态行为。为了测试酵母研究中建立的范式，我们将测量人类RAD51相似物是否会改变hRAD51细丝组装或拆卸的速度。从原理上讲，类似物可以增加成核频率或提高丝的生长速率，也可以降低解离速率。这些测量将使用单分子荧光显微镜直接成像hRAD51灯丝在DNA单个分子上的动态。

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.