Mechanisms of replication termination in vertebrates

脊椎动物复制终止机制

• 批准号: 9762271
• 负责人: James M Dewar
• 金额: $ 5.94万
• 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/9762271
• 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

项目概要 DNA 复制是通过称为“复制叉”的大分子结构进行的，其加载位置 当两个复制分叉时，会发生称为“起源”的离散位点。 来自相邻起源的DNA 迎面汇聚到同一条DNA 上，并且发生了约60,000 次终止事件 典型人类细胞中的每个细胞周期的终止都涉及 DNA 合成的完成、DNA 的解链。 子染色体和复制蛋白的卸载失败会导致 DNA 复制失败。 染色体错误分离和/或激活容易出错的 DNA 修复途径，从而提升突变 因此，终止缺陷预计会导致基因组不稳定，这是一个问题。 癌细胞的标志。此外，终止涉及拓扑异构酶 II 和 p97，它们是靶向的 总的来说，了解终止的工作原理至关重要。 终止的特征很差，很大程度上是因为无法在细胞中监测终止。 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， 由于随机来源使用和可变复制分叉率，我最近克服了这一限制。 开发了一种生化方法来监测爪蟾卵提取物的同步、局部终止。 这种方法允许对终止事件进行广泛的机械剖析，并包括完整的 终止所需的蛋白质补充，包括至关重要但尚未实现的蛋白质 发现这种方法导致了终止的生化模型，其中涉及快速收敛。 与之前认为会发生的停滞相比，复制分叉也产生了很大的影响。 我的实验室将利用这些专业知识来解决三个问题。 关于复制终止的广泛问题： 1. 拓扑应力如何影响终止？ 2. 终止如何调节随后的细胞周期事件？ 3. 复制终止所需的全套蛋白质是什么？ 总的来说，这项工作将确定参与终止的新机制和蛋白质，并扩大我们的视野 这项工作提供了终止包括 DNA 合成完成之前和之后发生的新阶段。 为我的实验室的一些长期目标奠定了基础，这些目标是： 1. 了解终止所带来的生化问题以及细胞采用的解决方案 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 DNA 复制的终止