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.

项目概要 在每个细胞周期中，DNA 复制机器都会遇到复制叉障碍，包括 DNA 损伤、 形成二级结构的重复序列和转录机制。也有致癌转化 扰乱正常复制并导致复制叉功能障碍，通常称为复制应激。 对复制应激的反应是细胞 DNA 损伤反应的一个重要方面， 不适当的反应会导致基因组不稳定和癌症。 我们最近发现了一种保护停滞状态所需的新监管途径 复制分叉和从复制压力中恢复。我们发现哺乳动物的复制体含有 以前未识别且完全未研究的蛋白质 RTF2（复制终止因子 2），它必须 被删除以正确应对复制压力。我们展示了 RTF2 从停滞的分叉中移除 该过程依赖于蛋白酶体穿梭蛋白 DDI1 和 DDI2，它们与 RTF2 和 蛋白酶体。 RTF2 在停滞的分叉上持续存在会导致复制叉重启缺陷、复制叉过度激活 DNA 损伤信号、单链 DNA 的积累、对复制药物的敏感性，包括 羟基脲和阿菲迪霉素，以及染色体不稳定。我们的结果表明 RTF2 的去除是 细胞管理复制压力和维持基因组完整性所必需的。 拟议研究的首要目标是充分了解 RTF2 在 DNA 复制过程中如何发挥作用。 为此，我们将使用条件敲除小鼠和细胞来充分表征没有 RTF2 的复制 模型，并确定 RTF2 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受到调节以及为什么需要将其去除 来自复制体。 RTF2 泛素化对于与 DDI1/2 相互作用是必要的，因此我们将识别 这种泛素化的调控网络以及随后从复制叉中去除 RTF2 的过程。最终目标 在这个项目中，是利用 DNA 损伤反应过程中蛋白质的去除同样重要的想法 作为将 DNA 修复蛋白招募到 DNA 损伤位点的重要手段。大多数已发表的研究都 专注于移动或招募到 DNA 损伤位点的蛋白质。然而，我们在 DDI 和 RTF2 方面的工作 表明 DNA 损伤反应网络的一个重要组成部分缺失，即必须去除的蛋白质 从损伤部位进行修复，以实现适当的 DNA 损伤反应和修复。为了鉴定其他蛋白质 在复制压力期间去除，我们将使用类似于我们在 DDI 研究中使用的方法，并且 使用最近开发的技术检测在应激复制叉处不当富集的蛋白质， 新生 DNA 上蛋白质的分离 (iPOND)。我们预计我们的研究将确定未知的监管 DNA 损伤反应过程中至关重要的网络，可防止致癌基因组的发展 不稳定。