Studies of the DNA replication checkpoint

描述（由申请人提供）：保存基因组完整性对包括人类在内的所有生物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，用于检测停滞或损坏的复制分叉。它通过阻止细胞分裂，稳定停滞的分叉，并确保在分叉进展的障碍被消除后完成DNA合成所需的复制蛋白和核苷酸的充足供应来保护基因组的完整性。在人类中，破坏复制检查点的突变与基因组不稳定、发育、神经和免疫疾病以及患癌症的易感性有因果关系。因此，破译这个检查点是如何工作的，以及更普遍地说，当复制分叉停止、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影响。本应用建议使用裂变酵母来研究真核细胞在DNA复制过程中如何保持基因组完整性。这种生物在基因组稳定性机制的发现和分析中发挥了核心作用。该提案有三个具体目标。首先是鉴定和表征复制检查点激酶Cds1的底物。第二个目标是了解耐受复制压力所需的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，重点关注结构特异性内切酶Mus81的功能。将采用多种技术：酵母分子遗传学，染色质免疫沉淀，涉及酵母双杂交和多维蛋白质鉴定技术的蛋白质相互作用筛选，结构生物学，活细胞中荧光标记蛋白质的反卷积显微镜，以及使用高密度寡核苷酸微阵列对DNA损伤位点的蛋白质进行全基因组分析。本研究中分析的所有蛋白质在人类中都有同源物，其功能尚不完全清楚。因此，本研究的发现将作为人类基因组稳定机制研究的范例。

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.