Understanding Replication Stress Response in Mammalian Cells

了解哺乳动物细胞的复制应激反应

• 批准号: 10491038
• 负责人: Agata Smogorzewska
• 金额: $ 33.9万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10491038
• 关键词: Aphidicolin; Cell Cycle; Cell Death; Cell model; Cells; ChIP-seq; Chromosomal Instability; Chromosome Fragile Sites; DNA; DNA Damage; DNA Repair Gene; DNA biosynthesis; DNA lesion; DNA replication fork; Data; Defect; Development; Diagnosis; Elements; Enzymes; Essential Genes; Event; Excision; Functional disorder; Future; Genes; Genetic Materials; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Hematopoietic System; Human; Knowledge; Maintenance; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Modeling; Mus; Mutagenesis; Mutate; Okazaki fragments; Oncogenic; Outcome; Participant; Persons; Pharmaceutical Preparations; Positioning Attribute; Process; Proteins; Publishing; Recovery; Regulation; Regulatory Pathway; Repetitive Sequence; Replication Initiation; Research; Ribonucleotides; Role; Signal Transduction; Single-Stranded DNA; Site; Stress; Structure; System; Techniques; Testing; Travel; Ubiquitination; United States; Work; biological adaptation to stress; cancer therapy; chromatin immunoprecipitation; conditional knockout; genetic approach; genome integrity; hydroxyurea; inhibitor; insight; liquid chromatography mass spectrometry; member; mouse model; multicatalytic endopeptidase complex; novel; nuclease; prevent; protein degradation; recruit; repaired; replication stress; response; stress management; termination factor; tumor; ubiquitin-protein ligase

Project Summary In each cell cycle, DNA replication machinery encounters replication fork barriers including DNA lesions, secondary structure-forming repetitive sequences, and transcriptional machinery. Oncogenic transformation also perturbs normal replication and results in replication fork dysfunction commonly referred to as replication stress. Response to replication stress is an essential aspect of the DNA damage response in cells, and the consequences of inappropriate response results in genome instability and cancer. We have recently identified a novel regulatory pathway that is required for the protection of stalled replication forks and recovery from replication stress. We showed that the mammalian replisome contains a previously unidentified and completely unstudied protein, RTF2 (Replication Termination Factor 2), which must be removed for proper response to replication stress. We showed that RTF2 is removed from stalled forks in a process that is dependent on the proteasomal shuttle proteins DDI1 and DDI2, which interact with RTF2 and the proteasome. Persistence of RTF2 at stalled forks resulted in replication fork restart defects, hyperactivation of the DNA damage signaling, accumulation of single stranded DNA, sensitivity to replication drugs including hydroxyurea and aphidicolin, and chromosome instability. Our results establish that removal of RTF2 is necessary for cells to manage replication stress and maintain genome integrity. The first goal of the proposed studies is to fully understand how RTF2 functions during DNA replication. To this end, we will fully characterize replication without RTF2, using a conditional knockout mouse and cell model, and identify the mechanism of how RTF2 regulates DNA replication during unperturbed conditions. The second goal is to determine how RTF2 is itself regulated under replication stress and why it needs to be removed from the replisome. RTF2 ubiquitination is necessary for interaction with DDI1/2, thus we will identify the regulatory network of this ubiquitination and subsequent removal of RTF2 from the replication fork. The final goal in this project, is to leverage the idea that the removal of proteins during DNA damage response is as equally important as recruitment of DNA repair proteins to sites of DNA damage. Most published studies have concentrated on proteins traveling or recruited to sites of DNA damage. However, our work on DDIs and RTF2 suggests a large component of the DNA damage response network is missing, i.e. proteins that must be removed from the sites of damage to allow for proper DNA damage response and repair. In order to identify other proteins removed during replication stress, we will use an approach similar to the one we used for our DDI studies and detect proteins inappropriately enriched at stressed replication forks using a recently-developed technique, Isolation of Proteins On Nascent DNA (iPOND). We envision that our studies will identify yet unknown regulatory networks essential during the DNA damage response that prevents development of cancer-causing genome instability.

项目摘要 在每个细胞周期中，DNA复制机制遇到复制叉屏障，包括DNA病变， 二级结构形成重复序列和转录机械。也有致癌的转化 正常复制并导致复制叉功能障碍通常称为复制应力。 对复制应力的反应是细胞中DNA损伤反应的重要方面，而 不适当反应的后果导致基因组不稳定性和癌症。 我们最近确定了一种新型的监管途径，该途径是保护失速所需的 复制叉和从复制应力中恢复。我们证明了哺乳动物的重生体包含一个 以前未识别且完全未研究的蛋白质RTF2（复制终止因子2），必须 删除以适当响应复制应力。我们表明RTF2从停滞的叉子中删除 取决于蛋白酶体班车蛋白DDI1和DDI2的过程，与RTF2相互作用 蛋白酶体。 RTF2在失速叉上的持久性导致复制叉重新启动缺陷，过度激活 DNA损伤信号传导，单链DNA的积累，对复制药物的敏感性，包括 羟基脲和蚜虫蛋白，以及染色体不稳定性。我们的结果表明，删除RTF2是 细胞管理复制应力和维持基因组完整性所必需的。 拟议的研究的第一个目标是充分了解RTF2在DNA复制过程中的功能。 为此，我们将使用条件基因敲除鼠标和单元格完全表征没有RTF2的复制 模型，并确定RTF2如何调节在不受干扰的条件下如何调节DNA复制的机制。这 第二个目标是确定RTF2本身如何在复制压力下进行调节，以及为什么需要删除RTF2 来自重新质体。 RTF2泛素化对于与DDI1/2的互动是必需的，因此我们将确定 这种泛素化和随后从复制叉中删除RTF2的调节网络。最终目标 在这个项目中，是要利用以下观点：DNA损伤响应期间蛋白质的去除同样同样 重要的是将DNA修复蛋白募集到DNA损伤部位。大多数已发表的研究都有 集中于行进或招募到DNA损伤部位的蛋白质。但是，我们在DDI和RTF2上的工作 建议缺少DNA损伤响应网络的大部分，即必须去除的蛋白质 从损坏部位，可以进行适当的DNA损伤响应和修复。为了识别其他蛋白质 在复制压力期间删除，我们将使用类似于DDI研究的方法， 检测蛋白质不适当地使用最近发育的技术，在应力复制叉上富集 蛋白质在新生DNA上的分离（IPOND）。我们设想我们的研究将确定但未知的监管 在DNA损伤反应中必不可少的网络阻止了引起癌症基因组的发展 不稳定。