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

• 批准号: 10613908
• 负责人: Kristina Schmidt
• 金额: $ 29.63万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613908
• 关键词: Binding; Biological Assay; Biological Models; Bromodeoxyuridine; Bypass; Cell Cycle; Cells; Chromatin; Co-Immunoprecipitations; Complex; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Synthesis Inhibition; DNA biosynthesis; DNA replication fork; DNA-Binding Proteins; Data; Disease; Eukaryotic Cell; Experimental Designs; Family; Genetic Recombination; Genome; Genomic Instability; Human; Label; Learning; Lesion; Licensing; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Mutation; N-terminal; Orthologous Gene; Pathway interactions; 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; biological adaptation to stress; cell type; design; gel electrophoresis; genome integrity; genomic locus; helicase; homologous recombination; insight; mutant; novel; origin recognition complex; preservation; prevent; recruit; repaired; replication stress; response; synthetic lethal interaction; tumorigenesis; Binding; Biological Assay; Biological Models; Bromodeoxyuridine; Bypass; Cell Cycle; Cells; Chromatin; Co-Immunoprecipitations; Complex; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Synthesis Inhibition; DNA biosynthesis; DNA replication fork; DNA-Binding Proteins; Data; Disease; Eukaryotic Cell; Experimental Designs; Family; Genetic Recombination; Genome; Genomic Instability; Human; Label; Learning; Lesion; Licensing; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Mutation; N-terminal; Orthologous Gene; Pathway interactions; 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; biological adaptation to stress; cell type; design; gel electrophoresis; genome integrity; genomic locus; helicase; homologous recombination; insight; mutant; novel; origin recognition complex; preservation; prevent; recruit; repaired; replication stress; response; synthetic lethal interaction; tumorigenesis

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.

项目摘要 细胞在复制应力中限制DNA复制的能力对于保留基因组完整性至关重要。 我们最近发现，缺乏RRM3解旋酶的酵母细胞不会阻止DNA合成 复制应力。我们发现（1）这种新的RRM3功能与其解旋酶活性无关，而是 （2）地图到结合原点识别ORC5的N端尾部的区域 复杂的，（3）N末端尾部对于RRM3与起源的关联至关重要 复制应力，但不在未扰动的细胞中。我们假设ORC通过其N末端招募RRM3到 恢复复合物，并且在复制过程中抑制DNA合成需要这种关联 压力。 RRM3被认为使用其解旋酶活性来“扫除”复制体之前的DNA以辅助 复制叉进展。因此，我们认为缺乏RRM3的酵母是一个出色的模型 揭示对复制叉暂停的细胞响应的系统。确实，使用定量蛋白质组学我们 确定同源重组因子RDH54和RAD5介导的无误差途径 病变旁路在RRM3缺陷型细胞的染色质部分中进行了更新，并且缺乏两者，RRM3 和rad5，累积的DNA双链断裂（DSB）。此外，叉子保护综合体和 在缺乏RAD5的细胞中，聚合酶因染色质而丢失。根据这些发现，我们假设 RAD5定义了一种主要的DSB预防机制，该机制需要克服和可能崩溃 RRM3Δ突变体中暂停的叉子。我们进一步假设Rad5通过介导PCNA实现了这一目标 多泛素化以调节叉子块的无错误旁路，例如积聚在DNA结合的蛋白 DNA在没有RRM3甜p活性的情况下，（ii）通过稳定复制体组件 协调重新启动所需。旨在检验这些假设的实验将（1）确定 RRM3在复制应力期间限制DNA合成的机制，（2）确定机制 RRM3-ORC5结合在复制过程中调节原点关联，起源活性和DNA合成 压力和（3）定义了对复制叉暂停增加的细胞反应。我们希望完成 该提案的目的将为维持DNA完整性的基本机制提供新的启示 真核细胞中的复制计划和伸长率。我们希望我们的发现将RRM3建立为 不仅是复制体的组成部分，而且是起源前的发射络合物的组成部分。我们对 RRM3在防止复制叉块以及RAD5和RDH54修复这些作用中的作用 通过无错误机制阻塞的叉子也将有助于阐明人类细胞如何处理复制叉 块并更好地定义了Rad5直系同源物HLTF在抑制肿瘤发生中的作用。