The role of DNA damage tolerance pathways in human cells

DNA损伤耐受途径在人类细胞中的作用

基本信息

批准号：
10170386
负责人：
Anja-Katrin Bielinsky
金额：
$ 44.43万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-14 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10170386
关键词：
Address Alleles Bypass CRISPR/Cas technology Cancer Etiology Cancer-Predisposing Gene Cell division Cells Chromosomal Rearrangement Chromosomes Comb animal structure Complex DNA DNA Adducts DNA Damage DNA Sequence Rearrangement DNA Structure DNA biosynthesis DNA lesion DNA replication fork DNA-Directed DNA Polymerase Defect Event Exposure to Fanconi Anemia pathway Fanconi Anemia-BRCA Pathway Fiber Generations Genes Genetic Recombination Genome Stability Genomic Instability Genomics HCT116 Cells Human Laboratories Lead Length Lysine Malignant Neoplasms Mediating Modification Molecular Monitor Monoubiquitination Mutation Nucleotides Oncogenes Optics Pathway interactions Phenotype Point Mutation Polymerase Process Proteins Radial Regulation Role Sister Chromatid Sister Chromatid Exchange Source Structure Sumoylation Pathway Technology Telomerase Telomere Maintenance Telomere Shortening Testing Ubiquitination Variant Work base blocking factor brca gene cancer prevention cancer therapy carcinogenesis cell transformation detection method genetic information genetic variant genome editing genome integrity imaging approach innovation malignant breast neoplasm mutant novel preservation recruit replication stress single molecule structural genomics telomere translocase ubiquitin-protein ligase

项目摘要

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.

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表明 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。