Pathway Choice of DNA Double-Strand Break Repair

DNA双链断裂修复的途径选择

• 批准号: 9517767
• 负责人: Anthony J Davis
• 金额: $ 36.19万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-09 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9517767
• 关键词: Affinity; Alanine; Apoptosis; Attenuated; Binding; CHEK1 gene; CHEK2 gene; CRISPR/Cas technology; Cancer Etiology; Cell Cycle; Cell Cycle Checkpoint; Cell Line; Cells; Chromosomal Stability; Chromosome abnormality; Clinical; Complex; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Sequence Alteration; DNA lesion; DNA-PKcs; Data; Dissociation; Double Strand Break Repair; Drug Targeting; Etiology; Event; Excision; Frequencies; G22P1 gene; Genetic Diseases; Genome Stability; Genomic Instability; Goals; Heterozygote; Homozygote; Human; Human Cell Line; Human Genetics; Impairment; Knock-in; Knock-in Mouse; Lead; Lesion; Maintenance; Mammalian Cell; Measures; Mediating; Molecular; Molecular Conformation; Monitor; Mus; Mutation; Nonhomologous DNA End Joining; Organism; Pathway interactions; Phase; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Predisposition; Process; Progress Reports; Proteins; Regulation; Role; S Phase; Signal Transduction; Site; System; Telomere Maintenance; Testing; Translating; Tumorigenicity; V(D)J Recombination; Work; XRCC5 gene; Yeast Model System; cancer therapy; carcinogenesis; gene repair; genome integrity; homologous recombination; in vivo; insight; mouse model; novel; prevent; recombinational repair; recruit; repaired; response; senescence; tumor; tumorigenesis

PROJECT SUMMARY/ABSTRACT A large number of DNA lesions occur within a cell each day jeopardizing the integrity of the genome, of which DNA double strand breaks (DSBs) are the most toxic lesions. Unrepaired or misrepaired DSBs can result in senescence, inducted apoptosis, or chromosomal aberrations, including translocations and deletions. These chromosomal aberrations can lead to genomic instability and tumorigenesis. To counter DSBs, organisms have evolved a complex mechanism of DNA damage response that includes recognition of the broken DNA molecule, cellular signaling including modulation of the cell cycle via checkpoints, and ultimately the repair of the DNA lesion. Two prominent pathways mediate the repair of DSBs in mammalian cells: homologous recombination (HR) and non-homologous end-joining (NHEJ). Although much work has been performed to identify and characterize the factors of each DSB repair pathway, important questions regarding cross-talk amongst pathways remain unresolved. These include elucidating factors that initially bind to the DSB ends and stabilize them, whether these factors influence recruitment of downstream factors and whether these factors modulate pathway choice/switching between NHEJ and HR for the repair of DSBs. In this study, we will take a novel “Ku-centric” view of DSB repair pathway choice in mammalian systems, which challenges the paradigm established in yeast models. We hypothesize that Ku binds to DSBs in all cell cycle phases to protect DNA ends, and that phosphorylation-mediated dissociation of Ku from DSBs is one of the key mechanisms responsible for modulating pathway choice between NHEJ and HR in S phase. Furthermore, we postulate that dysregulation of this process will result in genomic instability and increased susceptibility to tumorigenesis and most importantly, will provide an insight into the etiology of spontaneous tumors. To test this hypothesis, we propose the following specific aims: 1) To determine the kinase(s) responsible for Ku70 phosphorylation and the mechanism mediating Ku dissociation from DSBs in response to DNA double-strand breaks; 2) To test the hypothesis that Ku phosphorylation is essential for homologous recombination repair (HR), DNA damage response (DDR), and maintenance of genome stability in human cells; and 3) To test the hypothesis that Ku phosphorylation-dead mutations cause genome instability and lead to tumorigenicity in mouse model. Finally, since a number of human genetic diseases and initiation of carcinogenesis are directly associated with impaired or misrepaired DNA lesions and mutations in DSB repair genes, we believe basic mechanistic insights into DSB repair mechanisms and regulation of DSB repair pathway choice could lead to the identification of possible drug targets, which would ultimately translate into clinical benefits.

项目摘要/摘要 每天在一个细胞中发生大量DNA病变，危害基因组的完整性，其中的完整性 DNA双链断裂（DSB）是最有毒的病变。未修复或缩小的DSB可能会导致 衰老，诱导的凋亡或染色体畸变，包括易位和缺失。这些 染色体畸变会导致基因组不稳定性和肿瘤发生。为了反抗DSB，有机体 已经发展出复杂的DNA损伤响应机制，其中包括识别损坏的DNA 分子，细胞信号传导，包括通过检查点对细胞周期的调节，并最终修复 DNA病变。两个突出的途径介导哺乳动物细胞中DSB的修复：同源 重组（HR）和非理论终端连接（NHEJ）。虽然已经进行了很多工作 识别和表征每个DSB修复途径的因素，有关串扰的重要问题 在途径中，仍未解决。这些包括阐明最初与DSB末端结合的因素和 稳定它们，这些因素是否影响下游因素的募集以及这些因素是否影响 调节NHEJ和HR之间选择/切换DSB的修复。在这项研究中，我们将 哺乳动物系统中DSB修复途径选择的小说“以KU为中心”的观点，这挑战了范式 在酵母模型中建立。我们假设KU在所有细胞周期阶段与DSB结合以保护DNA 末端，磷酸化介导的KU与DSB的解离是关键机制之一 负责在S相中调节NHEJ和HR之间的途径选择。此外，我们假设 该过程的失调将导致基因组不稳定性，并增加对肿瘤发生的易感性和 最重要的是，将提供对赞助肿瘤病因的见解。为了检验这一假设，我们 提案以下具体目的：1）确定导致KU70磷酸化和的激酶 响应DNA双链断裂的介导KU解离的机制； 2）测试 假设KU磷酸化对于同源重组修复（HR），DNA损伤至关重要 反应（DDR）和人类细胞中基因组稳定性的维持； 3）检验KU的假设 磷酸化死亡突变会导致基因组不稳定性，并导致小鼠模型中的肿瘤性。最后， 由于许多人类遗传疾病和癌变的主动性与 DSB修复基因中的受损或缩写的DNA病变和突变，我们认为基本机械 对DSB修复机制的见解和DSB修复途径选择的调节可能会导致 识别可能的药物靶标，最终将转化为临床益处。