Signaling Mechanism of the DNA Replication Checkpoint

描述(由申请人提供)： 项目摘要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子在应激状态下的DNA合成功能方面起着关键作用。受到干扰的叉子如果没有被检查站发现，就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努力，我们仍然不了解在受干扰的叉子上启动检查点信号、叉子稳定和细胞存活的机制。我们研究的长期目标是以S.pombe为模型系统，了解复制机制和检查点通路之间的分子相互作用，以获得适当的检查点信号和叉子保护。本研究的目的是确定DNA聚合酶Pol�、复制解旋酶Cmg和固醇合成酶Erg11(在人类中为Cyp51)的重要检查点功能。我们的中心假设是(1)激活的复制检查点针对前导链上的PolErg11和Cmg，以抑制应激下的叉子进展，从而保护受干扰的叉子免受灾难性的崩溃，以及(2)Erg11可能作为一种新的传感器来检测核糖核苷酸还原酶抑制剂羟基脲诱导的应激反应。我们的假设是我们强劲的初步数据和最近发表的文章的结果。拟议研究的基本原理是，了解这些关键酶的检查点功能将为如何启动复制检查点信号以及如何稳定受干扰的叉子以保证细胞生存提供新的见解，这是该领域最突出的两个问题。在强大的初步数据的指导下，这些假说将通过追求三个具体目标来检验：(1)确定Pol�是如何被干扰的叉子稳定的检查点调节的；(2)发现复制检查点的主要目标()在压力下细胞存活；以及(3)定义Erg11的功能 在检查点信号和羟基脲诱导的胞质分裂停滞中。在前两个目标下，我们将进行体外和体内研究，以探讨Pol�和CMG的活性如何受到检查点的调节，以实现分叉保护和细胞存活。我们还将系统地分析裂解酵母中的所有复制蛋白，以确定可能与已知目标单独或冗余地工作的主要检查点目标()。在第三个目标下，将对Erg11在检查点信号和胞质分裂中新发现的功能进行表征。这种方法是创新的，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提供一个全面的分子机制。这项提议意义重大，因为它有望垂直推进并扩大我们对受扰叉口如何产生检查点信号以及如何保护受扰叉口的理解。最终，这些知识将促进我们对如何维持基因组完整性以及如何在所有真核生物中破坏基因组完整性的理解。

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.