Mechanisms of replication termination in vertebrates

脊椎动物复制终止机制

• 批准号: 10225443
• 负责人: James M Dewar
• 金额: $ 39.25万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10225443
• 关键词: Address; Biochemical; Cell Cycle; Cell Shape; Cells; Chemotherapy-Oncologic Procedure; Chromosomes; Clinic; Clinical Trials; Complement; DNA; DNA Repair Pathway; DNA biosynthesis; DNA replication fork; Data Set; Daughter; Defect; Disease; Dissection; Etoposide; Event; Failure; Foundations; Genome Stability; Genomic Instability; Goals; Head; Health; Human; Malignant Neoplasms; Molecular Structure; Monitor; Mutation; Nerve Degeneration; Process; Proteins; Proteomics; Set protein; Site; Stress; Stretching; Topoisomerase II; Vertebrates; Work; Xenopus; biochemical model; cancer cell; chromosome missegregation; egg; event cycle; genomic locus; reconstitution; tumorigenesis

PROJECT SUMMARY DNA replication is carried out by macromolecular structures called ‘replication forks’, which are loaded at discrete sites called ‘origins’. DNA Replication termination (‘termination’) occurs when two replication forks from adjacent origins converge head-on upon the same stretch of DNA and ~60,000 termination events occur each cell cycle in a typical human cell. Termination involves completion of DNA synthesis, unlinking of daughter chromosomes, and unloading of replication proteins. Failure to terminate DNA replication leads to chromosome missegregation and/or activation of error-prone DNA repair pathways that elevate the mutation rate of DNA replication. Therefore, termination defects are expected to cause genomic instability, which is a hallmark of cancer cells. Furthermore, termination involves Topoisomerase II and p97, which are targeted during cancer chemotherapy. Overall, it is critical that we understand how termination works. However, termination is poorly-characterized, in large part because termination cannot be monitored in cells. This technical limitation arises because termination is asynchronous and not localized to specific genomic loci, owing to stochastic origin usage and variable replication fork rates. To overcome this limitation, I recently developed a biochemical approach to monitor synchronous, localized termination in Xenopus egg extracts. This approach allows for extensive mechanistic dissection of termination events and includes the full complement of proteins required for termination, including proteins that are critically important but not yet discovered. This approach led to a biochemical model for termination, which involves rapid convergence of replication forks in contrast to the stalling that was previously-thought to occur. I also generated a large proteomic dataset of potential regulators of termination. My lab will leverage these expertise to address three broad questions about replication termination: 1. How does topological stress influence termination? 2. How does termination regulate subsequent cell cycle events? 3. What is the full set of proteins required for replication termination? Overall, this work will identify new mechanisms and proteins involved in termination, and expand our view of termination to include new stages that occur before and after completion of DNA synthesis. This work provides a foundation for some of the long-term goals of my lab, which are to: 1. Understand the biochemical problems posed by termination and the solutions employed by cells 2. Determine how termination shapes the cell cycle and impacts genomic stability 3. Reconstitute the termination of DNA replication in vertebrates

项目摘要 DNA复制是通过称为“复制叉”的大分子结构进行的，该结构被加载在 离散网站称为“起源”。当两个复制叉时发生DNA复制终止（'终止'） 在相同的DNA和约60,000个终止事件中，从相邻的起源正面汇合 每个细胞周期在典型的人类细胞中。终止涉及完成DNA合成，未链接 女儿染色体和复制蛋白的卸载。未能终止DNA复制导致 染色体错误陈化和/或激活易错的DNA修复途径，以提升突变 DNA复制速率。因此，预期终止缺陷会导致基因组不稳定性，这是 癌细胞的标志。此外，终止涉及针对性的拓扑异构酶II和P97 在癌症化疗期间。总体而言，我们了解终止的工作原理至关重要。然而， 终止表征较差，在很大程度上是因为无法在细胞中监测终止。这 出现技术限制是因为终止是异步的，并且不局部到特定的基因组基因局， 由于随机起源的使用和可变复制叉率。为了克服这一限制，我最近 开发了一种生化方法，以监测异爪蟾卵提取物中的同步局部终止。 这种方法允许对终止事件进行广泛的机械解剖，并包括完整的 终止所需的蛋白质完成，包括至关重要但尚不重要的蛋白质 发现。这种方法导致了终止的生化模型，该模型涉及 复制叉与以前想到的停滞相反。我也产生了一个大的 终止潜在调节剂的蛋白质组学数据集。我的实验室将利用这些专业知识来解决三个 关于复制终止的广泛问题： 1。拓扑压力如何影响终止？ 2。终止如何调节后续细胞周期事件？ 3。复制终止所需的全套蛋白质是什么？ 总体而言，这项工作将确定与终止有关的新机制和蛋白质，并扩大我们对 终止以包括在完成DNA合成之前和之后发生的新阶段。这项工作提供了 我实验室的一些长期目标的基础，这些目标是： 1。了解终止和细胞携带的溶液所带来的生化问题 2。确定终止如何塑造细胞周期并影响基因组稳定性 3。重建脊椎动物中DNA复制的终止