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

DNA中的双链断裂是一种致命的基因组损伤形式，也可能导致大的- 规模染色体重排和缺失时，错误修复。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 修复在真核生物中，这些途径之间的决定可以有持久的 对细胞命运的影响。目前的DNA修复模型表明， 同源重组因子相互竞争末端结合、加工和 在细胞的S期和G2期以有利于同源重组的方式修复 周期，但这种竞争仍然是不明确的，尽管多年的研究。在这里，我们建立在我们 最近的结果表明，Mre 11-Rad 50-Nbs 1（MRN）复合体在 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）。生物化学，单分子实验， 对人类细胞中DNA修复中间体的定量研究表明，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， 而是从非同源途径到同源途径的顺序和有序过程。到 验证这些结果，并了解双链断裂的识别和处理， 机制水平，我们建议进一步研究DNA末端加工的特点 在人类细胞中的分布。我们将通过分析DNA末端的调控来验证我们的假设 通过Mre 11核酸酶活性和CtIP修饰进行处理，并将检查 DNA末端加工的特征，因为它发生在非循环细胞。最后，我们将 研究MRN的合作，多亚基末端修复复合物的结构生物学， 与DNA末端的DNA-PK形成，具有重组蛋白以及天然复合物 人类细胞。这些实验将进一步建立新的方法， DNA修复中间体，并巩固了DNA双链断裂修复的新范式， 机械地将非同源末端连接与同源末端连接的起始相联系， 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.