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: 10436922
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 29.43万
• 依托单位: UNIVERSITY OF MINNESOTA
• 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 2022-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436922
• 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; 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; telomere; translocase; ubiquitin-protein ligase

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稳定性和保护 对抗致癌作用DNA复制的障碍，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，这可能导致叉崩溃和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 对癌症预防和治疗都很重要。停止的复制分叉可以 相反，这一过程稳定了停滞的叉子，但也使它们暴露在核溶解中。 除非通过FA-BRCA途径加载RAD 51来保护，否则这可能导致降解。 PCNA是一种重要的复制叉成分，当接触DNA时， 损伤在赖氨酸（K）164处被泛素化。这一事件控制着DNA的两种机制 病变旁路、经病变合成和模板转换。传统意义上的PCNA 单泛素化被认为是一种DNA损伤诱导的事件，我们的研究人员最近的工作表明， 实验室和其他人提出了PCNA泛素化的更广泛的影响， 复制的为了从机制上解决这个问题，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K164 R突变， HEK 293 T、RPE 1和HCT 116细胞。这些细胞的初步特征显示， 意想不到的基因组不稳定性特征，包括端粒侵蚀，复制叉 降解和严重的染色体重排基于这些初步 结果，我们提出PCNA泛素化在细胞增殖中具有以前未认识到的作用， 调节基因组稳定性。我们将在三个具体目标中解决这一问题： 目的1探讨端粒K164位点PCNA修饰在端粒形成中的作用 上维护目的2探讨K164修饰的PCNA在抑制肿瘤细胞增殖中的作用。 复制叉降级。目的3将阐明PCNA的功能影响， 在K164处的修饰对基因组重排的影响。我们的工作是准备揭开新的 K164的PCNA修饰调节细胞机制，并将具有显著的 影响了我们对基因组完整性的理解。