Studies of the DNA Replication Checkpoint

描述(由申请人提供)：中断DNA复制的内在和外在因素使复制真核生物基因组的挑战性任务变得更加困难。为了应对这些困难，细胞有一个DNA复制监测系统，可以感知停滞的复制叉子并指导各种反应。这些反应之一是S-M检查站，它推迟了有丝分裂(M)的开始，而DNA合成(S)正在进行。其他反应控制DNA复制、修复和重组。这些响应统称为复制检查点。本申请建议使用裂殖酵母裂殖酵母研究复制检查点。分裂酵母在发现和分析检查点机制方面发挥了开创性的作用，这些机制在包括人类在内的所有真核生物中都是保守的。这些研究将集中在蛋白激酶CDS1上，这是检查点的主要效应者。一个目的是了解CDS1是如何被调控的。这些研究将集中在MRc1上，这是一种蛋白质，然后将复制检查点信号从传感器激酶Rad3引导到CDS1。第二个目标是了解停滞的复制分叉是如何稳定的。将对参与这一过程的蛋白质进行鉴定和分析。第三个目标是确定Cds1靶标的特征。这些研究将集中在MUS81和Eme1，这是一种裂解X-结构DNA的Holliday连接解析酶的组成部分。所有这些基因在人类中都有确认或推定的同源基因。众所周知，其中一些同源物对维持基因组完整性和预防癌症非常重要，但它们如何完成这一任务在很大程度上尚不清楚。预计对分裂酵母中复制检查点的研究将为理解人类复制检查点控制提供一个有价值的框架。

DESCRIPTION (provided by applicant): The challenging task of replicating a eukaryotic genome is made more difficult by intrinsic and extrinsic agents that interrupt DNA replication. To cope with these difficulties, cells have a DNA replication monitoring system that senses stalled replication forks and direct various responses. One of these responses is the S-M checkpoint that delays the onset of mitosis (M) while DNA synthesis (S) is underway. Other responses control DNA replication, repair and recombination. These responses are collectively termed the replication checkpoint. This application proposes to use the fission yeast Schizosaccharomyces pombe to investigate the replication checkpoint. Fission yeast has played a pioneering role in the discovery and analysis of checkpoint mechanisms that are conserved amongst all eukaryotes, including humans. The studies will focus on he protein kinase Cds1, a major effector of the checkpoint. One aim is to understand how Cds1 is regulated. These studies will concentrate on Mrc1, a protein then channels the replication checkpoint signal from the sensor kinase Rad3 to Cds1. The second aim is to understand how stalled replication forks are stabilized. Proteins involved in this process I will be identified and analyzed. The third aim is to characterize Cds1 targets. These studies will focus on Mus81 and Eme1, components of a Holliday junction resolvase that cleaves X-structured DNA. All of these genes have confirmed or presumptive homologs in humans. Some of these homologs are known to be important for maintaining genome integrity and preventing cancer, but how they accomplish this task is largely unknown. It is anticipated that studies of the replication checkpoint in fission yeast will provide a valuable framework for understanding replication checkpoint control in humans.