DNA end processing by the Mre11/Rad50/Nbs1 complex in human cells

人类细胞中 Mre11/Rad50/Nbs1 复合物的 DNA 末端加工

• 批准号: 10415125
• 负责人: TANYA T PAULL
• 金额: $ 31.16万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10415125
• 关键词: Affect; Agreement; Aspergillus Nuclease S1; Binding; Biochemistry; Biological Assay; C-terminal; Catalytic Domain; Cell Cycle; Cell Cycle Regulation; Cells; Characteristics; Chromosomal Rearrangement; Chromosome Deletion; Collaborations; Complex; Cyclin-Dependent Kinases; Cytolysis; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA analysis; DNA biosynthesis; DNA-PKcs; DNA-dependent protein kinase; Data; Double Strand Break Repair; Electron Microscopy; Eukaryota; Event; Excision; G1 Phase; G2 Phase; G22P1 gene; Genome; Goals; Human; In Vitro; Lead; Ligation; Link; Mammalian Cell; Mass Spectrum Analysis; Mediating; Methods; Modeling; Modification; Molecular Conformation; Monitor; Nonhomologous DNA End Joining; Organism; Outcome; Pathway interactions; Pattern; Phase; Phosphorylation; Phosphorylation Site; Physiological; Play; Procedures; Process; Production; Proteins; Recombinant Proteins; Recombinants; Recovery; Regulation; Repair Complex; Role; S phase; Sequence Homology; Series; Single-Stranded DNA; Site; Structure; Testing; Work; XRCC5 gene; base; chromatin immunoprecipitation; crosslink; experimental study; homologous recombination; in vitro activity; inhibitor; next generation sequencing; novel; nuclease; protein complex; protein kinase inhibitor; recruit; repaired; restriction enzyme; single molecule; structural biology

Double-strand breaks in DNA are a lethal form of genome damage that can also lead to large- scale chromosomal rearrangements and deletions when mis-repaired. Non-homologous end joining and homologous recombination are the two major pathways of DNA double-strand break repair in eukaryotes, and the decision between these pathways can have long-lasting consequences for cell fate. Current models of DNA repair suggest that non-homologous and homologous recombination factors compete with each other for end binding, processing, and repair in a manner that favors homologous recombination during the S and G2 phases of the cell cycle, but this competition is still ill-defined despite many years of study. Here we build upon our recent results showing that the Mre11-Rad50-Nbs1 (MRN) complex performs end processing in physiological conditions that depends on the core non-homologous end joining complex, DNA- dependent protein kinase (DNA-PK). Ensemble biochemistry, single-molecule experiments, and quantitation of DNA repair intermediates in human cells shows that MRN-mediated end processing occurs at DNA-PK-bound ends, promoted by the cell cycle-regulated repair factor CtIP. These results suggest that DNA double-strand break repair "choice" is not a competition, but rather a sequential and ordered process from non-homologous to homologous pathways. To validate these results and understand double-strand break recognition and processing at a mechanistic level, we propose to further investigate the characteristics of DNA end processing globally in human cells. We will test our hypotheses by analyzing the regulation of DNA end processing by by Mre11 nuclease activity and CtIP modifications, and will examine the characteristics of DNA end processing as it occurs in non-cycling cells. Lastly, we will investigate the structural biology of the cooperative, multi-subunit end repair complex that MRN forms with DNA-PK on DNA ends, with both recombinant proteins as well as native complexes from human cells. These experiments will further establish novel methods for characterizing DNA repair intermediates and solidify a new paradigm in DNA double-strand break repair that mechanistically links non-homologous end joining with the initiation of homologous recombination.

DNA中的双链断裂是基因组损伤的致命形式，也可能导致大量 刻度染色体重排和删除时，缩放染色。非同源结局 连接和同源重组是DNA双链断裂的两个主要途径 真核生物的维修以及这些途径之间的决定可以长期持久 细胞命运的后果。当前的DNA修复模型表明，非同源和 同源重组因素相互竞争，以争取结合，加工和 以有利于在细胞的S和G2相期间有利于同源重组的方式进行修复 循环，但是尽管研究了多年，但这项竞争仍然不明显。在这里我们建立在我们的基础上 最近的结果表明，MRE11-RAD50-NBS1（MRN）复合物在 依赖核心非同源末端的生理条件连接复合物，DNA- 依赖性蛋白激酶（DNA-PK）。集合生物化学，单分子实验和 人类细胞中DNA修复中间体的定量表明MRN介导的端 处理在DNA-PK结合的末端发生，由细胞周期调节因子促进 ctip。这些结果表明，DNA双链休息修复“选择”不是竞争， 而是从非同源到同源途径的顺序和有序的过程。到 验证这些结果并了解双链断裂的识别和处理 机械水平，我们建议进一步研究DNA终端加工的特征 在人类细胞中全球。我们将通过分析DNA端的调节来检验我们的假设 通过MRE11核酸酶活性和CTIP修饰处理，将检查 DNA终端加工的特征是在非循环细胞中发生的。最后，我们会的 调查合作，多生产末端修复复合物的结构生物学 在DNA末端具有DNA-PK的形式，重组蛋白和天然复合物均具有 来自人类细胞。这些实验将进一步建立表征的新方法 DNA修复中间体并在DNA双链断裂修复中巩固新的范式 机械上的非同源末端与同源的启动联系起来 重组。