Mechanisms of Double-strand Break Repair in Yeast

描述（由申请人提供）：拟议研究的长期目标是了解真核生物DNA双链断裂（DSBs）修复的细胞机制，使用出芽酵母作为模型系统。通过HR修复dsb的能力对于脊椎动物的细胞增殖和所有特征生物的基因组稳定至关重要。这个过程的核心是Rad51与ssDNA的复合物，它在反应的同源性搜索和链交换阶段是活跃的。然而，不受调节的重组可能对细胞有害，Rad51丝似乎是许多蛋白质调节的共同靶标。Rad52, Rad54， Rad55和Rad57的作用是形成和/或稳定Rad51核蛋白丝，而Srs2的作用是破坏丝的稳定性。Brca2似乎在Rad51的调控中具有复杂的作用，通过促进核进入，将Rad51靶向到DNA损伤位点和Rad51丝的不稳定。由于Rad51丝动力学在DNA损伤响应中适当重组的核心作用，因此全面了解Rad51/DNA复合物的形成和分解非常重要。本提案的第一个目标建立在我们之前对rad51功能获得和改变功能等位基因的研究之上，以进一步了解rad51丝如何与Rad55， Rad57和Srs2相互作用。在Rad51灯丝的晶体结构中，在原相界面上发现的残基的功能也将被确定。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丝的水平上起作用，还是在重组中具有额外的后期作用。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。最后一个目标包括体外研究，以确定Rad55:Rad57结合的首选DNA底物，以及在Srs2存在或不存在的情况下Rad55:Rad57对Rad51核蛋白的稳定性。

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.