The role of DNA damage tolerance pathways in human cells
The role of DNA damage tolerance pathways in human cells
批准号:
10436922
负责人:
Anja-Katrin Bielinsky
金额:
$29.43万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2019
资助国家:
美国
项目状态:
已结题
起止时间:
2019-08-14 至 2022-11-14
关键词:
AddressAllelesBypassCRISPR/Cas technologyCancer EtiologyCancer-Predisposing GeneCell divisionCellsChromosomal RearrangementChromosomesComb animal structureComplexDNADNA AdductsDNA DamageDNA Sequence RearrangementDNA StructureDNA biosynthesisDNA lesionDNA replication forkDNA-Directed DNA PolymeraseDefectEventExposure toFanconi Anemia pathwayFanconi Anemia-BRCA PathwayFiberGenerationsGenesGenetic RecombinationGenome StabilityGenomic InstabilityGenomicsHCT116 CellsHumanLaboratoriesLeadLengthLysineMalignant NeoplasmsMediatingModificationMolecularMonitorMonoubiquitinationMutationNucleotidesOncogenesOpticsPathway interactionsPhenotypePoint MutationPolymeraseProcessProteinsRadialRegulationRoleSister ChromatidSister Chromatid ExchangeSourceSumoylation PathwayTechnologyTelomeraseTelomere MaintenanceTelomere ShorteningTestingUbiquitinationVariantWorkbaseblocking factorbrca genecancer preventioncancer therapycarcinogenesiscell transformationdetection methodgenetic informationgenetic variantgenome editinggenome integrityimaging approachinnovationmalignant breast neoplasmmutantnovelpreservationrecruitreplication stresssingle moleculetelomeretranslocaseubiquitin-protein ligase
中文摘要
项目概要
准确的 DNA 复制对于保持基因组稳定性和保护基因组至关重要。
抗癌作用。 DNA 复制的障碍,例如 DNA 加合物、次级
DNA 结构或核苷酸耗尽会阻止 DNA 聚合酶的进展,
从而阻止复制叉,从而导致复制叉崩溃和 DNA 断裂。由于这是一个
点突变和结构变异的来源,了解复制压力是
对于癌症的预防和治疗都很重要。停滞的复制叉可能是
相反,这是一个稳定停滞叉的过程,但也使它们暴露于核解作用
降解,除非通过 FA-BRCA 途径加载 RAD51 进行保护。
PCNA 是一种重要的复制叉成分,一旦暴露于 DNA
损伤在赖氨酸 (K) 164 处泛素化。该事件控制 DNA 的两种机制
病灶旁路、跨病灶合成和模板切换。虽然传统的 PCNA
单泛素化被认为是 DNA 损伤诱导的事件,我们最近的工作
实验室和其他人建议 PCNA 泛素化在
复制。为了机械地解决这个问题,我们采用了 CRISPR/Cas9 基因组
编辑技术在内源性 PCNA 等位基因中引入 K164R 突变
HEK293T、RPE1 和 HCT116 细胞。这些细胞的初步表征揭示
意想不到的基因组不稳定特征,包括端粒侵蚀、复制叉
降解和总染色体重排。基于这些初步
结果,我们提出 PCNA 泛素化在以下方面具有以前未被认识到的作用:
调节基因组稳定性。我们将通过三个具体目标来解决这个问题:
目标 1 将研究 PCNA 修饰 K164 在端粒中的作用
维护。目标 2 将研究 K164 修饰的 PCNA 在抑制
复制叉退化。目标 3 将阐明 PCNA 的功能影响
K164 基因组重排的修饰。我们的工作即将揭开新奇的面纱
PCNA K164 修饰调节细胞机制,并将产生显着的影响
影响我们对基因组完整性的理解。
英文摘要
Project Summary
Accurate DNA replication is essential for preserving genomic stability and protecting
against carcinogenesis. Obstacles to DNA replication, such as DNA adducts, secondary
DNA structures, or nucleotide depletion block the progression of DNA polymerases and
thus arrest replication forks, which can lead to fork collapse and DNA breaks. As this is a
source for point mutations and structural variations, understanding replication stress is
important for both cancer prevention and treatment. Stalled replication forks can be
reversed, a process which stabilizes stalled forks, but also exposes them to nucleolytical
degradation, unless protected through loading of RAD51 by the FA-BRCA pathway.
PCNA is an essential replication fork component, which upon exposure to DNA
damage is ubiquitinated at lysine (K) 164. This event controls two mechanisms of DNA
lesion bypass, translesion synthesis and template switching. While traditionally PCNA
mono-ubiquitination was considered a DNA damage-induced event, recent work by our
laboratories and others suggested a more broad impact of PCNA ubiquitination during
replication. To mechanistically address this, we employed the CRISPR/Cas9 genome
editing technology to introduce the K164R mutation in the endogenous PCNA alleles in
HEK293T, RPE1, and HCT116 cells. Preliminary characterization of these cells revealed
unexpected genomic instability features, including telomere erosion, replication fork
degradation, and gross chromosomal rearrangements. Based on these preliminary
results, we propose that PCNA ubiquitination has previously unrecognized roles in
regulating genomic stability. We will address this in three specific aims:
Aim 1 will investigate the role of PCNA modification at K164 in telomere
maintenance. Aim 2 will investigate the role of K164-modified PCNA in suppressing
replication fork degradation. Aim 3 will elucidate the functional impact of PCNA
modification at K164 on genomic rearrangements. Our work is poised to uncover novel
cellular mechanisms regulated by PCNA modification at K164, and will have a significant
impact on our understanding of genome integrity.
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海外基金