Mechanisms of replication termination in vertebrates

脊椎动物复制终止机制

• 批准号: 9751904
• 负责人: James M Dewar
• 金额: $ 49.43万
• 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/9751904
• 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; Genomics; 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; 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

项目总结 DNA复制是由被称为复制叉的大分子结构执行的，复制叉加载在 被称为“起源”的离散站点。当两个复制分叉时，DNA复制终止(‘终止 来自相邻来源的DNA迎头汇聚到同一段DNA上，大约会发生60,000个终止事件 一个典型的人类细胞中的每个细胞周期。终止包括完成DNA合成，解除连接 子代染色体，以及复制蛋白的卸载。未能终止DNA复制会导致 染色体错误分离和/或容易出错的DNA修复通路激活，从而提高突变 DNA复制率。因此，终止缺陷预计会导致基因组不稳定，这是一种 癌细胞的特征。此外，终止涉及拓扑异构酶II和p97，它们是靶向 在癌症化疗期间。总体而言，我们了解终端的工作原理是至关重要的。然而， 终止的特征很差，很大程度上是因为无法在单元中监视终止。这 出现技术限制是因为终止是异步的并且不局限于特定的基因组位点， 由于随机的原产地使用和可变的复制叉率。为了克服这一限制，我最近 开发了一种生化方法来监测非洲爪哇卵提取物中的同步、局部终止。 这种方法允许对终止事件进行广泛的机械分析，并包括完整的 终止所需的蛋白质的补充，包括非常重要但还不是的蛋白质 被发现了。这种方法导致了终止的生化模型，该模型涉及快速收敛 复制分叉，而不是之前认为会发生的停滞。我还产生了一个巨大的 潜在终末调节因子的蛋白质组数据集。我的实验室将利用这些专业知识解决三个问题 有关复制终止的广泛问题： 1.拓扑应力对终止有何影响？ 2.终止如何调节后续的细胞周期事件？ 3.终止复制所需的全套蛋白质是什么？ 总体而言，这项工作将确定参与终止的新机制和蛋白质，并扩大我们对 终止包括在DNA合成完成之前和之后发生的新阶段。这项工作提供了 为我的实验室的一些长期目标奠定基础，这些目标包括： 1.了解终止所带来的生化问题以及细胞所采用的解决方案 2.确定终止如何影响细胞周期和影响基因组稳定性 3.重建脊椎动物DNA复制的终止性