Mechanisms of Double-strand Break Repair in Yeast

描述(申请人提供)：拟议研究的长期目标是了解真核生物中DNA双链断裂(DSB)修复的细胞机制，使用发芽酵母作为模型系统。HR修复双链断裂的能力对于脊椎动物的细胞增殖和所有特定生物的基因组稳定性都是必不可少的。这一过程的中心是RAD51与单链DNA的复合物，它在反应的同源搜索和链交换阶段活跃。然而，不受调控的重组很可能对细胞有害，而RAD51丝似乎是许多蛋白质调控的共同目标。Rad52、Rad54、Rad55和Rad57在RAD51核蛋白细丝的形成和/或稳定中起作用，而Srs2则作用于破坏该微丝的稳定。BRCA2似乎通过促进核进入，将RAD51靶向DNA损伤和RAD51丝不稳定的部位，在RAD51的调控中发挥着复杂的作用。由于RAD51丝动力学在DNA损伤反应中的适当重组中起着核心作用，因此全面了解RAD51/DNA复合体的形成和拆解是很重要的。这个建议的第一个目的是建立在我们之前对RAD51功能获得和改变的功能等位基因的研究基础上，以进一步了解RAD51细丝如何与Rad55、Rad57和Srs2相互作用。还将确定在RAD51细丝晶体结构中在原型界面确定的残基的功能。该提案的第二个目的是表征已知的抑制子，并确定rad57的其他抑制子，以确定Rad57是否仅在RAD51细丝水平上发挥作用，或者在重组中具有额外的晚期作用。RAD55和RAD57在自发和诱导、染色体内和染色体间重组中的作用也将被确定。最后一个目标包括在体外研究确定RAD55：RAD57与RAD51核蛋白结合的首选DNA底物，以及在存在或不存在Srs2的情况下由Rad55：RAD57稳定RAD51核蛋白。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to understand the cellular mechanisms for the repair of DNA double-strand breaks (DSBs) in eukaryotes, using budding yeast as a model system. The ability to repair DSBs by HR is essential for cell proliferation in vertebrates and for genome stability in all characterized organisms. Central to this process is the Rad51 complex with ssDNA, which is active in the homology search and strand exchange phases of the reaction. However, unregulated recombination is likely to be detrimental to cells and the Rad51 filament appears to be a common target for regulation by a number of proteins. Rad52, Rad54, Rad55 and Rad57 function in formation and/or stabilization of the Rad51 nucleoprotein filament, whereas Srs2 functions to destabilize the filament. Brca2 appears to have complex roles in the regulation of Rad51 by promoting nuclear entry, targeting Rad51 to sites of DNA damage and destabilization of the Rad51 filament. Because of the central role of Rad51 filament dynamics for appropriate recombination in response to DNA damage, it is important to have a complete understanding of the formation and disassembly of the Rad51/DNA complex. The first aim of this proposal builds on our previous studies of rad51 gain-of-function and altered function alleles to further understand how the Rad51 filament interacts with Rad55, Rad57 and Srs2. The function of residues identified at the protomer interface in the crystal structure of the Rad51 filament will also be determined. The second aim of the proposal is to characterize known suppressors, and identify additional suppressors of rad57, to determine whether Rad57 functions exclusively at the level of the Rad51 filament, or has additional late roles in recombination. The roles of Rad55 and Rad57 in spontaneous and induced, intra and inter-chromosomal recombination will also be determined. The last aim includes in vitro studies to identify preferred DNA substrates for Rad55:Rad57 binding and stabilization of the Rad51 nucleoprotein by Rad55:Rad57 in the presence or absence of Srs2.