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:10491038
- 负责人:
- 金额:$ 33.9万
- 依托单位:
- 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:
- 关键词:AphidicolinCell CycleCell DeathCell modelCellsChIP-seqChromosomal InstabilityChromosome Fragile SitesDNADNA DamageDNA Repair GeneDNA biosynthesisDNA lesionDNA replication forkDataDefectDevelopmentDiagnosisElementsEnzymesEssential GenesEventExcisionFunctional disorderFutureGenesGenetic MaterialsGenetic TranscriptionGenomeGenome StabilityGenomic InstabilityGenomicsGoalsHematopoietic SystemHumanKnowledgeMaintenanceMalignant NeoplasmsMammalian CellMass Spectrum AnalysisMediatingMediator of activation proteinModelingMusMutagenesisMutateOkazaki fragmentsOncogenicOutcomeParticipantPersonsPharmaceutical PreparationsPositioning AttributeProcessProteinsPublishingRecoveryRegulationRegulatory PathwayRepetitive SequenceReplication InitiationResearchRibonucleotidesRoleSignal TransductionSingle-Stranded DNASiteStressStructureSystemTechniquesTestingTravelUbiquitinationUnited StatesWorkbiological adaptation to stresscancer therapychromatin immunoprecipitationconditional knockoutgenetic approachgenome integrityhydroxyureainhibitorinsightliquid chromatography mass spectrometrymembermouse modelmulticatalytic endopeptidase complexnovelnucleasepreventprotein degradationrecruitrepairedreplication stressresponsestress managementtermination factortumorubiquitin-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复制机制遇到复制叉障碍,包括DNA损伤,
二级结构形成重复序列和转录机制。致癌性转化还
干扰正常复制并导致通常称为复制应激的复制叉功能障碍。
对复制应激的反应是细胞中DNA损伤反应的一个重要方面,
不适当反应的后果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,这是保护停滞的
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含有一个
以前未鉴定和完全未研究的蛋白质,RTF 2(复制终止因子2),它必须
以适当应对复制压力。我们展示了RTF 2在一个
这一过程依赖于蛋白酶体穿梭蛋白DDI1和DDI2,它们与RTF 2相互作用,
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,
DNA损伤信号传导、单链DNA的积累、对复制药物的敏感性,包括
羟基脲和蚜霉素,以及染色体不稳定性。我们的研究结果表明,去除RTF 2是
细胞管理复制压力和维持基因组完整性所必需的。
这项研究的第一个目标是全面了解RTF 2在DNA复制过程中的功能。
为此,我们将使用条件性敲除小鼠和细胞,
模型,并确定如何RTF2调节DNA复制在未受干扰的条件下的机制。的
第二个目标是确定RTF2在复制应激下如何调节自身,以及为什么需要将其去除
来自复制体。RTF2泛素化是与DDI 1/2相互作用所必需的,因此我们将鉴定
这种泛素化的调控网络和随后从复制叉中去除RTF 2。最终目标
在这个项目中,是利用这样一个想法,即在DNA损伤反应过程中蛋白质的去除同样是
重要的是将DNA修复蛋白募集到DNA损伤位点。大多数已发表的研究报告
集中在蛋白质的运输或招募到DNA损伤的位点。然而,我们在DDI和RTF 2上的工作
这表明DNA损伤反应网络的一个重要组成部分缺失,即必须被去除的蛋白质。
以允许适当的DNA损伤反应和修复。为了识别其他蛋白质
在复制压力期间移除,我们将使用与DDI研究相似的方法,
使用最近开发的技术检测在应激复制叉处不适当富集的蛋白质,
新生DNA上的蛋白质分离(iPOND)。我们设想我们的研究将确定未知的监管
在DNA损伤反应过程中至关重要的网络,防止致癌基因组的发展
不稳定
项目成果
期刊论文数量(0)
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Agata Smogorzewska的其他文献
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{{ truncateString('Agata Smogorzewska', 18)}}的其他基金
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:
10689130 - 财政年份:2021
- 资助金额:
$ 33.9万 - 项目类别:
Functions of human RAD51 and its paralogs during DNA interstrand crosslink repair
人类 RAD51 及其旁系同源物在 DNA 链间交联修复过程中的功能
- 批准号:
9399639 - 财政年份:2017
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The role of nucleases in interstrand crosslink repair
核酸酶在链间交联修复中的作用
- 批准号:
8612988 - 财政年份:2014
- 资助金额:
$ 33.9万 - 项目类别:
The role of nucleases in interstrand crosslink repair
核酸酶在链间交联修复中的作用
- 批准号:
8792867 - 财政年份:2014
- 资助金额:
$ 33.9万 - 项目类别:
The role of nucleases in interstrand crosslink repair
核酸酶在链间交联修复中的作用
- 批准号:
9208149 - 财政年份:2014
- 资助金额:
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