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.

项目摘要即使不复制一小段DNA也会导致染色体错误分析和误差激活俯卧的DNA修复途径。因此，必须确保完成DNA合成至关重要。但是， DNA合成的完成不受细胞周期检查点的监视。另外，虽然缺陷可以通过新的启动事件来克服早期复制阶段仍然存在未复制的DNA。因此，需要精确有效的DNA合成机制来确保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。在当前的建议中，我们专注于通常支持完成的以下机制 DNA合成以及对DNA损伤响应DNA复制的机制：（1）当两个收敛复制叉在相同的时间段相遇时，复制终止发生 DNA，是DNA合成通常完成的方式。我们最近发现拓扑压力可能导致将复制叉融合到失速的叉子，这是由拓扑异构酶II依赖性和 - 独立机制。但是，目前尚不清楚为什么其他拓扑异构酶无法弥补拓扑异构酶II以及如何有效参与这些不同的机制以确保快速分叉收敛。我们将确定不同的拓扑异构酶在DNA复制过程中扮演的角色以及它们如何各自角色有助于复制终止。我们还将研究如何促进叉子的不同蛋白质融合认识到他们的目标，以确保有效克服终止障碍。（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错误的方式绕过DNA病变。但是，目前尚不清楚NSD究竟如何贡献重新启动反向叉。我们最近开发了一种新的方法来诱导有效的叉子逆转和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在重新启动的反向叉子中扮演的角色。（3）断裂诱导的复制（BIR）从双链DNA端重新启动DNA合成。然而，目前尚不清楚BIR最终如何导致DNA合成的完成以及全套所需蛋白是。另外，有丝分裂DNA合成（MIDAS）是一个BIR样过程，在有丝分裂过程中起作用，但目前尚不清楚 MIDAS和BIR之间的选择如何确定。我们已经开发了一种诱导和监视的方法 BIR，这使我们能够独特地解决这些问题。我们将确定BIR如何完成合成和涉及的蛋白质。我们还将确定BIR和MIDAS之间的选择决定。