Studies of the DNA replication checkpoint

描述(由申请人提供)：保持基因组的完整性对包括人类在内的所有生物体的健康和生存能力至关重要。可以理解的是，所有生物都有多种维持基因组稳定的机制。其中一个系统是复制检查点，它检测停滞或损坏的复制分叉。它通过阻止细胞分裂，稳定停滞不前的分叉，并确保一旦分叉进程的障碍消除后，完成DNA合成所需的足够复制蛋白质和核苷酸的供应，来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、发育、神经和免疫疾病以及癌症的易感性有关。因此，破译这个检查点是如何工作的，以及更广泛地说，当复制叉子失速、重新排列或坍塌时会发生什么，是一个有趣的生物学问题，也对改善人类健康具有广泛的影响。这项应用建议使用分裂酵母来研究真核细胞如何在DNA复制过程中保持基因组的完整性。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。第一个是鉴定和鉴定复制检查点激酶CDS1的底物。第二个目的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子，重点是结构特定的内切酶MUS81的功能。将使用多种技术：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去卷积显微镜，以及使用高密度寡核苷酸微阵列对定位于DNA损伤位置的蛋白质进行全基因组分析。这项研究中分析的所有蛋白质在人类中都有同源基因，其功能尚不完全清楚。因此，这项研究的发现将为人类基因组稳定机制的研究提供一个范例。

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.