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

基本信息

批准号：
10584584
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
项目类别：
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/10584584
关键词：
Affect Agreement Aspergillus Nuclease S1 Binding Biochemistry Biological Assay C-terminal Cell Cycle Cell Cycle Regulation Cells Characteristics Chromosomal Rearrangement Chromosome Deletion Collaborations Collecting Cell 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 Enzyme Induction Eukaryota Event Excision G1 Phase G2 Phase G22P1 gene Genome Goals Human In Vitro 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 Resected Role S phase Sequence Homology Series Single-Stranded DNA Site Structure Testing Work XRCC5 gene 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

项目摘要

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一种致命形式，也可能导致大当错误修复时，染色体会发生重排和缺失。非同源末端连接和同源重组是DNA双链断裂的两个主要途径真核生物中的修复，这些途径之间的决定可以产生持久的影响对细胞命运的影响。目前的 DNA 修复模型表明，非同源和同源重组因子相互竞争末端结合、加工和以有利于细胞 S 期和 G2 期同源重组的方式进行修复周期，但尽管经过多年的研究，这种竞争仍然没有明确的定义。在这里，我们以我们的最近的结果表明，Mre11-Rad50-Nbs1 (MRN) 复合物在生理条件取决于核心非同源末端连接复合物，DNA- 依赖性蛋白激酶（DNA-PK）。整体生物化学、单分子实验和人类细胞中 DNA 修复中间体的定量表明 MRN 介导的末端加工发生在 DNA-PK 结合末端，由细胞周期调节的修复因子促进 CtIP。这些结果表明DNA双链断裂修复“选择”不是竞争，而是从非同源途径到同源途径的连续有序过程。到验证这些结果并了解双链断裂识别和处理机制层面，我们建议进一步研究DNA末端加工的特征全球范围内的人类细胞。我们将通过分析DNA末端的调控来检验我们的假设通过 Mre11 核酸酶活性和 CtIP 修饰进行处理，并将检查非循环细胞中 DNA 末端加工的特征。最后，我们将研究 MRN 合作的多亚基末端修复复合物的结构生物学在 DNA 末端与重组蛋白和天然复合物形成 DNA-PK 来自人体细胞。这些实验将进一步建立表征的新方法 DNA 修复中间体并巩固 DNA 双链断裂修复的新范例机械地连接非同源末端连接与同源末端连接的起始重组。