Studies of the DNA replication checkpoint

描述（由申请人提供）：基因组完整性的保存对于所有生物体（包括人类）的健康和生存能力至关重要。可以理解的是，所有生物体都有多种维持基因组稳定性的机制。其中一个系统是复制检查点，它检测停止或损坏的复制分叉。它通过阻止细胞分裂，稳定停滞的分叉，以及确保一旦分叉进展的障碍被移除就有足够的复制蛋白和核苷酸来完成DNA合成，从而保护基因组的完整性。在人类中，损害复制检查点的突变与基因组不稳定性、发育、神经和免疫疾病以及癌症易感性有因果关系。因此，破译这个检查点是如何工作的，以及更普遍地说，当复制叉停止、重新排列或崩溃时会发生什么，是一个迷人的生物学问题，对改善人类健康也有广泛的意义。本申请提出使用裂殖酵母来研究真核细胞如何在DNA复制期间维持基因组完整性。这种生物体在发现和分析基因组稳定性机制方面发挥了核心作用。该提案有三个具体目标。第一个是识别和表征复制检查点激酶Cds1的底物。第二个目的是了解耐受复制应激所需的BRCT结构域蛋白的作用。第三个目标是确定细胞如何重新捕获断裂的复制叉，重点是结构特异性内切核酸酶Mus81的功能。将采用各种技术：酵母分子遗传学，染色质免疫沉淀，蛋白质相互作用屏幕涉及酵母双杂交和多维蛋白质鉴定技术，结构生物学，去卷积显微镜的荧光标记的蛋白质在活细胞中，和全基因组分析的蛋白质定位到网站的DNA损伤使用高密度寡核苷酸微阵列。在这个提议中分析的所有蛋白质在人类中都有直系同源物，其功能还不完全清楚。因此，这项研究的结果将作为研究人类基因组稳定性机制的范例。

DESCRIPTION (provided by applicant): Preservation of genome integrity is essential for the health and viability of all organisms, including humans. Understandably, all organisms have multiple mechanisms of maintaining genome stability. One of these systems is the replication checkpoint, which detects stalled or broken replication forks. It protects genome integrity by arresting cell division, by stabilizing the stalled forks, and by ensuring an adequate supply of replication proteins and nucleotides needed to complete DNA synthesis once the impediments to fork progression are removed. In humans, mutations that impair the replication checkpoint are causally associated with genomic instability, developmental, neurological and immunological diseases, and a predisposition for cancer. Deciphering how this checkpoint works, and more generally what happens when replication forks stall, rearrange or collapse, is therefore a fascinating biological problem that also has broad implications for improving human health. This application proposes to use fission yeast to study how eukaryotic cells maintain genome integrity during DNA replication. This organism has played a central role in the discovery and analyses of genome stability mechanisms. The proposal has three specific aims. The first is to identify and characterize substrates of the replication checkpoint kinase Cds1. The second aim is to understand the role of a BRCT domain protein that is needed to tolerate replication stress. The third aim is to determine how cells recapture broken replication forks with a focus on the function of the structure-specific endonuclease Mus81. A variety of techniques will be employed: yeast molecular genetics, chromatin immunoprecipitation, protein interaction screens involving yeast two-hybrid and multidimensional protein identification technology, structural biology, deconvolution microscopy of fluorescently tagged protein in live cells, and genome-wide analyses of proteins localized to site of DNA damage using high density oligonucleotide microarrays. All of the proteins analyzed in this proposal have orthologs in humans whose functions are incompletely understood. Therefore, the findings from this study will serve as a paradigm for the investigation of genome stability mechanisms in humans.