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Understanding Replication Stress Response in Mammalian Cells

了解哺乳动物细胞的复制应激反应

基本信息

批准号：
10689130
负责人：
Agata Smogorzewska
金额：
$ 33.9万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
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/10689130
关键词：
Aphidicolin Cell Cycle Cell Death Cell model Cells 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 Knockout Mice Knowledge Maintenance Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Mediating Mediator Modeling Mus Mutagenesis Mutate Okazaki fragments Oncogenic Outcome Participant Persons Pharmaceutical Preparations Phosphorylation 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

项目摘要

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复制机制遇到复制分叉障碍，包括DNA损伤，二级结构-形成重复序列，以及转录机制。致癌转化也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，而不适当反应的后果会导致基因组不稳定和癌症。我们最近发现了一种新的调控途径，它是保护失速的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包含一个以前未知和完全没有研究过的蛋白质，RTF2(复制终止因子2)，它必须为了对复制压力做出正确的反应，将其移除。我们展示了RTF2是从停滞的叉子上移除的依赖于蛋白酶体穿梭蛋白DDI1和DDI2的过程，这两个蛋白与RTF2和蛋白酶体。RTF2在停滞的分叉上的持久性导致复制分叉重新启动缺陷、过度激活 DNA损伤信号、单链DNA的积累、对复制药物的敏感性羟基脲和除草剂，以及染色体不稳定性。我们的结果表明，RTF2的去除是是细胞管理复制压力和维持基因组完整性所必需的。拟议研究的第一个目标是充分了解RTF2在DNA复制过程中的作用。为此，我们将使用有条件的基因敲除小鼠和细胞，在没有RTF2的情况下，充分描述复制的特征模型，并确定RTF2如何在不受干扰的条件下调节DNA复制的机制。这个第二个目标是确定RTF2自身在复制压力下是如何调节的，以及为什么需要移除它来自复制体。RTF2泛素化是与DDI1/2相互作用所必需的，因此我们将识别这种泛素化和随后RTF2从复制叉子中移除的调控网络。最终目标在这个项目中，是利用这样一种想法，即在DNA损伤反应过程中蛋白质的去除重要的是将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在运输或招募到DNA损伤部位的蛋白质上。然而，我们在DDIS和RTF2上的工作表明DNA损伤反应网络的一大部分缺失，即必须移除的蛋白质以允许适当的DNA损伤反应和修复。为了鉴定其他蛋白质在复制压力期间删除，我们将使用类似于我们用于DDI研究的方法和使用最近开发的技术检测在应激复制叉处不适当地富含的蛋白质，新生DNA上蛋白质的分离(IPOND)。我们设想，我们的研究将确定未知的监管在阻止致癌基因组发展的DNA损伤反应中至关重要的网络不稳定。