Mechanisms of Double-strand Break Repair in Yeast

这项研究的长期目标是了解修复的细胞机制， 真核生物中的DMA双链断裂（DSB），使用芽殖酵母作为模型系统。的能力 通过HR修复DSB对于脊椎动物的细胞增殖和所有动物的基因组稳定性都是必不可少的。 特征的生物体。该过程的中心是Rad 51与ssDNA的复合物，其在细胞中具有活性。 同源性搜索和链交换阶段的反应。然而，不受控制的重组可能 对细胞有害，Rad 51细丝似乎是通过一些调节的共同目标。 蛋白质。Rad 52、Rad 54、Rad 55和Rad 57在Rad 51的形成和/或稳定中起作用 核蛋白丝，而Srs 2的功能是使丝不稳定。brca 2似乎有复杂的 通过促进核进入，将Rad 51靶向到DMA损伤位点， Rad 51灯丝的不稳定性。由于Rad 51灯丝动力学的核心作用， 在响应DMA损伤的重组中，重要的是要完全了解DNA的分子结构。 Rad 51/DNA复合物的形成和分解。这项建议的第一个目标是建立在我们以前的基础上。 rad 51功能获得性和改变的功能等位基因的研究，以进一步了解Rad 51细丝 与Rad 55、Rad 57和Srs 2相互作用。在原聚体界面上鉴定的残基的功能 还将确定Rad 51灯丝的晶体结构。该建议的第二个目的是 表征已知抑制因子，并识别rad 57的其他抑制因子，以确定Rad 57是否 仅在Rad 51细丝水平起作用，或在重组中具有额外的晚期作用。的 Rad 55和Rad 57在自发和诱导的染色体内和染色体间重组中的作用也将 被确定。最后一个目的包括体外研究以鉴定Rad 55的优选DNA底物：Rad 57 在存在或不存在Srs 2的情况下通过Rad 55：Rad 57结合和稳定Rad 51核蛋白。

The long-term objective of the proposed research is to understand the cellular mechanisms for the repair of DMA 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 DMA damage and destabilization of the Rad51 filament. Because of the central role of Rad51 filament dynamics for appropriate recombination in response to DMA 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.