Signaling Mechanism of the DNA Replication Checkpoint

描述（由申请人提供）： DNA复制检查点是所有真核生物中高度保守的信号通路。它在维持受干扰的复制叉在应激下的DNA合成功能中起关键作用。如果检查点没有检测到，受干扰的分叉会发生灾难性的崩溃，导致染色体DNA损伤甚至细胞死亡。该途径的缺陷与基因组不稳定和癌症有关。然而，尽管它的重要性和密集的研究工作，我们的理解参与检查点信号的启动在pertubed叉，叉稳定，和细胞存活的机制仍然是不完整的。我们研究的长期目标是通过使用S. pombe作为模型系统。本文的目的是确定DNA聚合酶Pol β、复制解旋酶CMG和甾醇合成酶Erg11（人类Cyp51）的重要检查点功能。我们的中心假设是（1）激活的复制检查点靶向前导链上的Pol和CMG，以抑制应激下的分叉进程，从而保护受干扰的分叉免受灾难性崩溃，以及（2）Erg11可能作为核糖核苷酸还原酶抑制剂羟基脲诱导的应激的新传感器。我们的假设是我们强大的初步数据和最近的出版物的结果。这项研究的基本原理是，了解这些必需酶的检查点功能将为复制检查点信号是如何启动的以及受干扰的叉子如何稳定细胞存活提供新的见解，这是该领域最突出的两个问题。在强有力的初步数据的指导下，这些假设将通过追求三个具体目标进行测试：（1）确定Pol？如何被检查点调节以稳定扰动叉;（2）发现压力下细胞存活的复制检查点的主要靶标（）;（3）定义Erg11的功能。 在检查点信号传导和羟基脲诱导的胞质分裂阻滞中的作用。在前两个目标下，我们将进行体外和体内研究，以研究Pol和CMG的活性如何受到叉保护和细胞存活的检查点的调节。我们还将系统地分析裂变酵母中的所有复制蛋白，以确定可能单独或与已知靶标冗余工作的主要检查点靶标（）。在第三个目标下，Erg11在检查点信号传导和胞质分裂中的新鉴定的功能将被表征。该方法是创新的，因为它旨在为代表高等真核生物的模型系统中的检查点信号传导提供全面的分子机制。该提议是重要的，因为它有望垂直推进和扩展我们对检查点信号如何在扰动分叉处生成以及如何保护扰动分叉的理解。最终，这些知识将促进我们对基因组完整性如何维持以及如何在所有真核生物中破坏的理解。

DESCRIPTION (provided by applicant): Project Summary DNA replication checkpoint is a highly conserved signaling pathway in all eukaryotes. It plays a critical role in maintaining the DNA synthesis function of perturbed replication forks under stress. Perturbed forks, if undetected by the checkpoint, undergo catastrophic collapse resulting in chromosomal DNA damage or even cell death. Defects in the pathway are linked to genome instability and cancer. However, despite its importance and intense research efforts, our understanding of the mechanisms involved in the initiation of checkpoint signaling at the pertubed forks, fork stabilization, and cell survival remains incomplete. The long-term goal of our research is to understand the molecular interactions between the replication machinery and the checkpoint pathways for proper checkpoint signaling and fork protection by using S. pombe as the model system. The objective here is to define the important checkpoint functions of DNA polymerase Pol �, the replicative helicase CMG, and the sterol synthesis enzyme Erg11 (Cyp51 in humans). Our central hypotheses are (1) that the activated replication checkpoint targets Pol � and CMG on the leading strand to suppress the fork progression under stress and hence protect the perturbed forks against catastrophic collapse, and (2) that Erg11 may function as a new sensor of the stress induced by the ribonucleotide reductase inhibitor hydroxyurea. Our hypotheses are the results of our strong preliminary data and recent publications. The rationale for the proposed research is that understanding the checkpoint functions of these essential enzymes will provide novel insights into how the replication checkpoint signaling is initiated and how perturbed forks are stabilized for cell survival, the two most prominent questions in the field. Guided by strong preliminary data, these hypotheses will be tested by pursuing three specific aims: (1) determine how Pol � is regulated by the checkpoint for stabilization of perturbed forks; (2) discover the major target() of the replication checkpoint for cell survival under stress; and (3) define the functions of Erg11 in checkpoint signaling and hydroxyurea-induced cytokinesis arrest. Under the first two aims, we will conduct in vitro and in vivo studies to investigate how the activties of Pol � and CMG are regulated by the chekcpoint for fork protection and cell survival. We will also systematically analyze all replication proteins in fission yeast in order to identify the major checkpoint target() that may work alone or redundantly with the known targets. Under the third aim, the newly identified functions of Erg11 in checkpoint signaling and cytokinesis will be characterized. The approach is innovative, because it aims to provide a comprehensive molecular mechanism for checkpoint signaling in a model system representative of higher eukaryotes. The proposal is significant, because it is expected to vertically advance and expand our understanding of how checkpoint signaling is generated at perturbed forks and how perturbed forks are protected. Ultimately, such knowledge will advance our understanding of how genomic integrity is maintained and how it can be disrupted in all eukaryotes.