Signaling Mechanism of the DNA Replication Checkpoint

DNA 复制检查点的信号传导机制

基本信息

批准号：
9695226
负责人：
Yongjie Xu
金额：
$ 25.9万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9695226
关键词：
Address Binding Biochemical Genetics Biological Models Cell Cycle Cell Death Cell Survival Clinical Cytokinesis DNA Damage DNA biosynthesis DNA replication fork DNA-Directed DNA Polymerase Data Defect Endogenous Factors Enzymes Eukaryota Exogenous Factors Fission Yeast Genetic Genetic Screening Genome Stability Genomic Instability Goals Human In Vitro Individual Knowledge Link Malignant Neoplasms Maps Methods Mission Modeling Molecular Mutation Outcome Pathway interactions Pharmaceutical Preparations Phosphorylation Phosphorylation Site Play Proteins Public Health Publications Publishing Research Rest Ribonucleotide Reductase Inhibitor Role Series Signal Pathway Signal Transduction Sterols Stress Surveys System Testing Therapeutic United States National Institutes of Health Work Yeasts base cell killing disability genetic approach genome integrity helicase hydroxyurea in vivo innovation insight mutant novel public health relevance replication stress response sensor tumorigenesis

项目摘要

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.

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配置的信号通路。它在保持压力下扰动复制叉的DNA合成功能方面起着关键作用。如果没有检查点未发现灾难性的叉子，则会发生灾难性塌陷，从而导致染色体DNA损伤甚至细胞死亡。途径中的缺陷与基因组不稳定性和癌症有关。然而，要求其重要性和强烈的研究工作，我们对受叉叉，叉稳定和细胞存活的检查点信号涉及的机制的理解仍然不完整。我们研究的长期目标是了解复制机制与检查点途径之间的分子相互作用，以使用S. Pombe作为模型系统，以进行适当的检查点信号传导和叉子保护。这里的目的是定义DNA聚合酶pol的重要检查点功能，复制性解旋酶CMG和固醇合成酶ERG11（人类中的CYP51）。 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 reduces inhibitor hydroxyurea.我们的假设是我们强大的初步数据和最新出版物的结果。拟议研究的理由是，了解这些基本酶的检查点功能将提供有关如何启动复制检查点信号传导以及如何稳定扰动叉以实现细胞存活的新见解，这是该领域中最突出的两个问题。在强大的初步数据的指导下，这些假设将通过追求三个具体目的来检验：（1）确定POL如何受到稳定的扰动叉子的检查点；（2）在压力下发现细胞存活的复制检查点的主要目标（）；（3）定义ERG11的功能在检查点信号传导和羟基脲诱导的细胞因子停滞。在前两个目标下，我们将在体外和体内进行研究，以研究POL和CMG的活性如何受CHEKCPORT的调节，以进行叉子保护和细胞存活。我们还将系统地分析裂变酵母中的所有复制蛋白，以识别可能单独或与已知靶标有用的主要检查点目标（）。在第三个目标下，将表征ERG11新近确定的ERG11功能。该方法具有创新性，因为它旨在在代表较高真核生物的模型系统中提供一种全面的分子机制，用于检查点信号传导。该提案很重要，因为预计它将垂直提高并扩展我们对在扰动叉子上如何生成检查点信号的理解以及如何保护叉叉。最终，这种知识将提高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被破坏的理解。