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

• 批准号: 10750291
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 14.59万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 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/10750291
• 关键词: role; DNA; damage; tolerance; pathways

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.

项目摘要 准确的DNA复制对于保持基因组的稳定性和保护至关重要 抗致癌作用。DNA复制障碍，如DNA加合物，继发性 DNA结构，或核苷酸枯竭，阻止DNA聚合酶和 从而阻止复制分叉，这可能导致分叉崩溃和DNA断裂。因为这是一个 点突变和结构变异的来源，理解复制应激是 对癌症预防和治疗都很重要。停滞的复制分叉可能是 相反，这是一种稳定失速叉子的过程，但也会使它们暴露在溶解核物质的环境中 降解，除非通过FA-BRCA途径装载RAD51来保护。 增殖细胞核抗原是一种重要的复制分叉成分，当接触到DNA时， 在赖氨酸(K)164，损伤是泛素化的。此事件控制DNA的两种机制 病变旁路、跨病变合成和模板转换。虽然传统上，增殖细胞核抗原 单一泛素化被认为是一种DNA损伤诱导的事件，我们最近的工作 实验室和其他机构认为，增殖细胞核抗原泛素化在 复制。为了机械地解决这个问题，我们使用了CRISPR/Cas9基因组 将K164R突变引入内源性增殖细胞核抗原等位基因的编辑技术 HEK293T、RPE1和HCT116细胞。对这些细胞的初步表征揭示了 意想不到的基因组不稳定特征，包括端粒侵蚀、复制分叉 退化和总的染色体重排。基于这些初步的 结果，我们认为增殖细胞核抗原泛素化具有以前未被认识到的作用 调节基因组稳定性。我们将从三个具体目标来解决这个问题： 目的1研究端粒K164处增殖细胞核抗原修饰的作用 维修。目的2研究K164修饰的增殖细胞核抗原在抑制 复制分叉降级。目标3将阐明增殖细胞核抗原的功能影响 K164基因对基因组重排的修饰。我们的工作即将揭开小说的面纱 K164处的增殖细胞核抗原修饰调控的细胞机制，将具有显著的 对我们理解基因组完整性的影响。