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

DNA双链断裂是一种致命的基因组损伤形式，也可能导致大规模的 衡量染色体重排和错误修复时的缺失。非同源末端 连接和同源重组是DNA双链断裂的两条主要途径 在真核生物中进行修复，这些途径之间的决定可能会持续很长时间 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 在细胞的S和G2期，以有利于同源重组的方式进行修复 尽管经过多年的研究，但这项比赛的定义仍然不明确。在这里，我们建立在我们的 最近的结果表明，Mre11-Rad50-Nbs1(MRN)复合体在 取决于核心非同源末端连接复合体的生理条件，DNA- 依赖蛋白激酶(DNA-PK)。整体生物化学，单分子实验，以及 对人类细胞DNA修复中间产物的定量研究表明，MRN介导的末端 加工发生在DNA-PK结合端，由细胞周期调节的修复因子促进 CtIP。这些结果表明，DNA双链断裂修复的“选择”不是一种竞争， 而是一个从非同源路径到同源路径的顺序和有序过程。至 验证这些结果并了解双链断裂的识别和处理 在机制层面上，我们建议进一步研究DNA末端加工的特点 在全球范围内的人类细胞中。我们将通过分析DNA末端的调节来检验我们的假设 通过Mre11核酸酶活性和CtIP修饰进行处理，并将检查 DNA在非周期细胞中的末端加工特征。最后，我们将 研究MRN协同的多亚单位末端修复复合体的结构生物学 DNA末端带有DNA-PK的形式，既有重组蛋白，也有天然复合体 来自人类细胞。这些实验将进一步建立新的方法来表征 DNA修复中间体和巩固了DNA双链断裂修复的新范式 非同源末端的机械连接与同源末端的启动 重组。

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.