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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.

项目总结