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.

项目总结/摘要。 DNA复制检查点是所有真核生物中运行的细胞信号传导途径，其监控正常的S期 疾病的进展和对干扰的DNA复制的反应，激活细胞反应，以防止不可逆的 复制叉停滞、基因组不稳定和细胞死亡。检查点检测到被干扰的复制， 在应激下保持基因组稳定性，因此作为重要的抗癌屏障发挥作用。许多 抗癌药物通过干扰DNA复制而起作用，因此它们的功效受到DNA复制的影响。 癌症的检查点状态。尽管它在疾病预防和癌症化疗中的重要性，我们仍然 不完全了解复制分叉处的检查点启动过程，也不确切知道 检查点在压力下保护分叉功能。作为研究细胞的一个既定模型， 虽然这些机制在人类中是保守的，但裂变酵母为这项研究提供了几个好处。的目标 本项目是研究新筛选的裂变酵母中的检查点突变体，特别强调 两个目标：（1）了解在扰动分叉处检查点启动的机械基础， (2)揭示检查点调节的分叉保护的基本分子细节。作为起点，我们 已经开发了正向遗传学和生物化学分析的组合方法，并且已经鉴定了几种 在复制应激下具有各种检查点起始缺陷的新突变体。我们还筛选了大量 在分叉保护方面有缺陷的突变体的集合。在我们强有力的初步和公布的数据的指导下， 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如何 激酶Rad 3（ATR）信号传导受Rad 3-Rad 26复合物、RecQ解旋酶Rqh 1、 Smc 5/6复合物和RPA复合物。在第二个目标下，我们将研究如何激活 检查点调节前导链上的DNA聚合酶e和其他尚未确定的fork靶点 保护这项研究计划的长期目标是提供一个全面的了解 复制检查点，涉及三个主要领域的调查：首先，通过使用我们新改进的遗传 方法，将鉴定具有保守检查点功能的复制蛋白。第二，重建 我们和其他人使用纯化的蛋白质在体外研究检查点通路， 已经获得。第三，正如我们在对Rqh 1的研究中所显示的，人类中检查点机制的保守性， 细胞将被评估。总的来说，这项研究计划将大大提高分子的清晰度， 裂变酵母以及哺乳动物细胞中复制检查点的机制。拟议研究 由于其与基因组不稳定性、肿瘤发生和癌症化疗相关，因此具有重要意义。