Role of Pif1 family DNA helicase Rrm3 in regulating DNA synthesis during replication stress

Pif1家族DNA解旋酶Rrm3在复制应激期间调节DNA合成中的作用

基本信息

批准号：
10397011
负责人：
Kristina Schmidt
金额：
$ 29.65万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10397011
关键词：
Binding Binding Proteins Biological Assay Biological Models Bromodeoxyuridine Bypass Cell Cycle Cells Chromatin Co-Immunoprecipitations Complex Coupled DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair DNA Synthesis Inhibition DNA biosynthesis DNA replication fork Data Disease Electron Transport Complex III Eukaryotic Cell Family Genetic Recombination Genome Genomic Instability Human Label Lead Learning Lesion Light Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Mutation N-terminal Orthologous Gene Pathway interactions Play Polymerase Polyubiquitination Pre-Replication Complex Prevention Process Proteome Proteomics RAD52 gene Replication Initiation Replication Origin Role S phase SMARCA3 gene Saccharomycetales Site Sumoylation Pathway Tail Testing Ubiquitination Up-Regulation Yeasts base biological adaptation to stress design experimental study gel electrophoresis genome integrity genomic locus helicase homologous recombination insight mutant novel origin recognition complex preservation prevent recruit repaired replication stress response tumorigenesis

项目摘要

PROJECT SUMMARY The ability of cells to restrict DNA replication during replication stress is critical to preserving genome integrity. We recently discovered that yeast cells lacking the Rrm3 helicase do not arrest DNA synthesis during replication stress. We found (1) that this new Rrm3 function is independent of its helicase activity and instead (2) maps to a region of the poorly characterized N-terminal tail that binds Orc5 of the origin recognition complex, and (3) that the N-terminal tail is essential for Rrm3 association with origins in the presence of replication stress, but not in unperturbed cells. We hypothesize that ORC recruits Rrm3 via its N-terminal tail to pre-replication complexes and that this association is required for inhibition of DNA synthesis during replication stress. Rrm3 is thought to use its helicase activity to ‘sweep’ the DNA ahead of the replisome clear to aid replication fork progression. We reasoned therefore that yeast that lacks Rrm3 makes an excellent model system for revealing the cellular response to replication fork pausing. Indeed, using quantitative proteomics we determined that the homologous recombination factor Rdh54 and the Rad5-mediated pathway for error-free lesion bypass are upregulated in the chromatin fraction of rrm3-deficient cells and that cells lacking both, Rrm3 and Rad5, accumulate DNA double strand breaks (DSBs). Moreover, the fork protection complex and polymerase are lost from the chromatin in cells lacking Rad5. Based on these findings we hypothesize that Rad5 defines a major DSB prevention mechanism that is required to overcome stalling and possibly collapse of paused forks in the rrm3∆ mutant. We further hypothesize that Rad5 accomplishes this by mediating PCNA polyubiquitination to regulate error-free bypass of fork blocks, such as DNA-bound proteins that accumulate on DNA in the absence of the Rrm3 sweepase activity, and (ii) by stabilizing replisome components that are required for coordinated restart. The experiments designed to test these hypotheses will (1) identify the mechanism by which Rrm3 restricts DNA synthesis during replication stress, (2) determine the mechanism by which Rrm3-Orc5 binding regulates origin association, origin activity, and DNA synthesis during replication stress and (3) define the cellular response to increased replication fork pausing. We expect that accomplishing the aims of this proposal will shed new light on fundamental mechanisms that maintain the integrity of DNA replication initiation and elongation in eukaryotic cells. We expect our findings to establish Rrm3 as a component not only of the replisome, but also of the pre-initiation complex at origins. What we learn about the role of Rrm3 in preventing replication fork blocks and about the role of Rad5 and Rdh54 in repairing these blocked forks by an error-free mechanism will also help to clarify how human cells deal with replication fork blocks and better define the role of the Rad5 ortholog HLTF in suppressing tumorigenesis.

项目概要细胞在复制应激期间限制 DNA 复制的能力对于保持基因组完整性至关重要。我们最近发现，缺乏 Rrm3 解旋酶的酵母细胞在解旋过程中不会阻止 DNA 合成。我们发现 (1) 这种新的 Rrm3 功能与其解旋酶活性无关，而是独立于其解旋酶活性。 (2) 映射到结合起源识别的 Orc5 的 N 末端尾部区域 (3) N 末端尾部对于 Rrm3 与起源的关联是必需的我们捕获到 ORC 通过其 N 末端尾部招募 Rrm3 来进行复制应激。复制前复合体，并且这种关联是复制过程中抑制 DNA 合成所必需的人们认为 Rrm3 利用其解旋酶活性“清除”复制体之前的 DNA，以提供帮助。因此，我们推断缺乏 Rrm3 的酵母是一个很好的模型。事实上，我们使用定量蛋白质组学来揭示细胞对复制叉暂停的反应。确定同源重组因子 Rdh54 和 Rad5 介导的途径无错误 rrm3 缺陷细胞和同时缺乏 Rrm3 的细胞的染色质部分中病变旁路上调 Rad5，积累DNA双链断裂（DSB）此外，叉保护复合物和。在缺乏 Rad5 的细胞中，聚合酶从染色质中丢失。根据这些发现，我们发现这一点。 Rad5 定义了一个主要的 DSB 预防机制，需要克服失速和可能的崩溃我们进一步发现 Rad5 通过介导 PCNA 来实现这一点。多泛素化来调节叉块的无差错旁路，例如积累在 DNA 在缺乏 Rrm3 清除酶活性的情况下，以及 (ii) 通过稳定复制体组分协调重启所需的实验旨在测试这些假设将（1）确定 Rrm3 在复制应激期间限制 DNA 合成的机制，(2) 通过以下方式确定机制 Rrm3-Orc5 结合调节复制过程中的起源关联、起源活性和 DNA 合成压力和（3）定义细胞对复制叉暂停增加的反应，我们期望实现这一点。该提案的目的将为维护 DNA 完整性的基本机制提供新的启示我们期望我们的研究结果能够将 Rrm3 确定为真核细胞中的复制起始和延伸。不仅是复制体的组成部分，而且也是起始前复合物的组成部分。 Rrm3 在防止复制叉阻断中的作用以及 Rad5 和 Rdh54 在修复这些复制叉阻断中的作用通过无差错机制阻止复制叉也将有助于阐明人类细胞如何处理复制叉阻断并更好地定义 Rad5 直系同源物 HLTF 在抑制肿瘤发生中的作用。