DNA Replication Checkpoint in Fission Yeast

裂殖酵母中的 DNA 复制检查点

• 批准号: 10557924
• 负责人: Yongjie Xu
• 金额: $ 37.5万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10557924
• 关键词: Affect; Antineoplastic Agents; Area; Biochemical; Cell Death; Cells; Chemotherapy-Oncologic Procedure; Collection; Complex; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Defect; Eukaryota; Fission Yeast; Genetic; Genome Stability; Genomic Instability; Goals; Human; In Vitro; Knowledge; Malignant Neoplasms; Mammalian Cell; Methods; Molecular; Monitor; Mutation; Pathway interactions; Phosphotransferases; Process; Proteins; Public Health; Publishing; Research; S phase; Signal Pathway; Signal Transduction; Stress; Study models; Work; anti-cancer; disorder prevention; forward genetics; helicase; improved; in vivo; mutant; prevent; programs; protein purification; reconstitution; replication stress; response; sensor; tumorigenesis

PROJECT SUMMARY/ABSTRACT . DNA replication checkpoint is a cell signaling pathway operating in all eukaryotes that monitors normal S phase progression and in response to perturbed DNA replication, activates cellular responses to prevent irreversible replication fork arrest, genomic instability, and cell death. The checkpoint senses the perturbed replication, maintains the genomic stability under stress and thus functions as an important anticancer barrier. Many anticancer drugs work by interfering with DNA replication and their efficacy is therefore influenced by the checkpoint status of cancer. Despite its importance in disease prevention and cancer chemotherapies, we still do not fully understand the checkpoint initiation process at the replication forks, nor do we know exactly how the checkpoint protects the fork functions under stress. As an established model for studying the cellular mechanisms that are conserved in humans, fission yeast offers several benefits for this research. The goal of this project is to investigate the newly screened checkpoint mutants in fission yeast with particular emphasis on two objectives: (1) understand the mechanistic underpinnings of checkpoint initiation at the perturbed forks, and (2) uncover the essential molecular details of the checkpoint-regulated fork protection. As a starting point, we have developed a combined approach of forward genetics and biochemical analysis and have identified several new mutants with various checkpoint initiation defects under replication stress. We have also screened a large collection of mutants that are defective in fork protection. Guided by our strong preliminary and published data, we will conduct in vivo and in vitro studies under the first objective to investigate how the checkpoint sensor kinase Rad3(ATR) signaling is affected by mutations in the Rad3-Rad26 complex, the RecQ helicase Rqh1, the Smc5/6 complex, and the RPA complex. Under the second objective, we will investigate how the activated checkpoint regulates DNA polymerase e on the leading strand and other yet-to-be identified targets for fork protection. The long-term goal of this research program is to provide a comprehensive understanding of the replication checkpoint that involves three primary areas of inquiry: First, by using our newly improved genetic method, replication proteins with conserved checkpoint functions will be identified. Second, reconstitution of the checkpoint pathway in vitro using purified proteins that can properly recapitulate the in vivo data we and others have obtained. Third, as we show in the studies on Rqh1, conservation of the checkpoint mechanisms in human cells will be evaluated. Overall, this research program will bring much improved clarity to the molecular mechanisms of the replication checkpoint in fission yeast as well as in mammalian cells. The proposed research is significant because of its relevance to genome instability, oncogenesis, and cancer chemotherapies.

项目摘要/摘要。 DNA复制检查点是在所有真核生物中运行的细胞信号通路 进展并响应扰动的DNA复制，激活细胞反应以防止不可逆 复制叉停滞，基因组不稳定性和细胞死亡。检查点感知扰动的复制， 在应力下保持基因组稳定性，从而充当重要的抗癌屏障。许多 抗癌药物通过干扰DNA复制而起作用，因此其功效受到 癌症的检查点状态。尽管它在预防疾病和癌症化学疗法方面具有重要意义，但我们仍然 请勿完全了解复制叉处的检查点启动过程，我们也不确切知道如何 检查点在压力下保护叉功能。作为研究细胞的既定模型 裂变酵母在人类中保守的机制为这项研究提供了一些好处。目标 该项目是为了调查裂变酵母中新筛选的检查点突变体，特别强调 两个目标：（1）了解扰动叉子的检查点启动的机械基础，以及 （2）揭示了检查点调节的叉子保护的基本分子细节。作为起点，我们 已经开发了远期遗传学和生化分析的联合方法，并确定了几个 在复制应力下具有各种检查点起始缺陷的新突变体。我们还筛选了一个大的 集合在叉子保护中有缺陷的突变体。在我们强大的初步和公开数据的指导下， 我们将在第一个目标下进行体内和体外研究，以研究检查点传感器 激酶RAD3（ATR）信号传导受Rad3-Rad26复合物中突变的影响，RECQ旋转酶RQH1， SMC5/6复合物和RPA复合物。在第二个目标下，我们将研究如何激活 检查点在领先链和其他尚未确定的叉子上调节DNA聚合酶E 保护。该研究计划的长期目标是提供对 复制检查点，涉及三个主要查询领域：首先，使用我们新的遗传 方法，将确定具有保守检查点功能的复制蛋白。其次，重组 使用纯化的蛋白质在体外进行检查点途径，该蛋白可以正确概括我们和其他人的体内数据 已经获得了。第三，正如我们在RQH1的研究中所显示的那样，人类检查点机制的保存 将评估细胞。总体而言，该研究计划将为分子带来很大的清晰度 裂变酵母以及哺乳动物细胞中复制检查点的机制。拟议的研究 由于与基因组不稳定性，肿瘤发生和癌症化学疗法的相关性而具有重要意义。