Mechanisms that ensure the completion of DNA synthesis

确保DNA合成完成的机制

基本信息

批准号：
10623020
负责人：
James M Dewar
金额：
$ 46.76万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2028-07-31
项目状态：
未结题

项目摘要

PROJECT SUMMARY Failure to replicate even a short stretch of DNA leads to chromosome missegregation and activation of error prone DNA repair pathways. It is therefore crucial to ensure completion of DNA synthesis. However, the completion of DNA synthesis is not under surveillance by cell cycle checkpoints. Additionally, while defects in earlier stages of replication can be overcome by new initiation events, this is essentially impossible when short stretches of unreplicated DNA remain. Thus, exquisitely effective DNA synthesis mechanisms are needed to ensure the completion of DNA synthesis. The long-term goal of my lab is to elucidate and understand these mechanisms. In the current proposal, we focus on the following mechanisms that normally support completion of DNA synthesis, as well as mechanisms that restart DNA replication in response to DNA damage: (1) Replication termination occurs when two converging replication forks meet on the same stretch of DNA and is how DNA synthesis is normally completed. We recently found that topological stress can cause converging replication forks to stall and that this is overcome by both topoisomerase II-dependent and - independent mechanisms. However, it is unclear why other topoisomerases cannot compensate for loss of topoisomerase II and how these different mechanisms are efficiently engaged to ensure rapid fork convergence. We will determine the roles that different topoisomerases play during DNA replication and how their respective roles contribute to replication termination. We will also investigate how the different proteins that promote fork convergence recognize their targets to ensure that obstacles to termination are efficiently overcome. (2) Replication fork reversal and Nascent Strand Degradation (NSD) are thought to allow replication forks to bypass DNA lesions in an essentially error-free manner. However, it is unclear how exactly NSD contributes to restart of reversed forks. We recently developed a new approach to induce efficient fork reversal and NSD and identified new steps involved in this process. We will leverage our approach and insights to determine the role that NSD plays in restart of reversed forks. (3) Break-Induced Replication (BIR) restarts DNA synthesis from a double-stranded DNA end. However, it is unclear how BIR ultimately leads to completion of DNA synthesis and what the full set of required proteins is. Additionally, Mitotic DNA Synthesis (MiDAS) is a BIR-like process that operates during mitosis but it is unclear how the choice between MiDAS and BIR is determined. We have developed an approach to induce and monitor BIR, which makes us uniquely equipped to address these questions. We will determine how BIR completes synthesis and the proteins involved. We will also determine how the choice between BIR and MiDAS is determined.

项目概要即使一小段 DNA 无法复制也会导致染色体错误分离和错误激活容易发生的 DNA 修复途径。因此，确保 DNA 合成的完成至关重要。然而， DNA 合成的完成不受细胞周期检查点的监控。此外，虽然缺陷复制的早期阶段可以通过新的起始事件来克服，当时间很短时这基本上是不可能的未复制的 DNA 片段仍然存在。因此，需要极其有效的 DNA 合成机制保证DNA合成的完成。我实验室的长期目标是阐明和理解这些机制。在当前的提案中，我们重点关注以下通常支持完成的机制 DNA 合成的过程，以及响应 DNA 损伤而重新启动 DNA 复制的机制： (1) 当两个聚合复制叉在同一段上相遇时，就会发生复制终止 DNA 是 DNA 合成通常完成的方式。我们最近发现拓扑应力会导致聚合复制叉停止，这可以通过拓扑异构酶 II 依赖性和 - 独立机制。然而，尚不清楚为什么其他拓扑异构酶不能补偿拓扑异构酶的损失拓扑异构酶 II 以及这些不同的机制如何有效地参与以确保叉子快速收敛。我们将确定不同拓扑异构酶在 DNA 复制过程中所发挥的作用以及它们各自的作用角色有助于复制终止。我们还将研究促进分叉的不同蛋白质如何一致认识到其目标，以确保有效克服终止的障碍。 (2) 复制叉逆转和新生链降解 (NSD) 被认为允许复制叉以基本无差错的方式绕过 DNA 损伤。然而，目前尚不清楚 NSD 究竟有何贡献重新启动反向货叉。我们最近开发了一种新方法来诱导有效的分叉反转和 NSD 并确定了此过程中涉及的新步骤。我们将利用我们的方法和见解来确定 NSD 在反向分叉重启中扮演的角色。 (3) 断裂诱导复制 (BIR) 从双链 DNA 末端重新开始 DNA 合成。然而，目前尚不清楚 BIR 最终如何导致 DNA 合成完成以及所需的全套蛋白质是什么是。此外，有丝分裂 DNA 合成 (MiDAS) 是一种类似 BIR 的过程，在有丝分裂期间运行，但尚不清楚如何确定 MiDAS 和 BIR 之间的选择。我们开发了一种方法来诱导和监控 BIR，这使我们有能力解决这些问题。我们将确定 BIR 如何完成合成和所涉及的蛋白质。我们还将确定如何在 BIR 和 MiDAS 之间进行选择决定。