Soong Chen-Pang, Breuer Gregory A, Hannon Ryan A, Kim Savina D, Salem Ahmed F, Wang Guilin, Yu Ruoxi, Carriero Nicholas J, Bjornson Robert, Sundaram Ranjini K, Bindra Ranjit S
Department of Internal Medicine, University of Connecticut, Farmington, CT 06030, United States.
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States.
DNA Repair (Amst). 2015 Feb;26:44-53. doi: 10.1016/j.dnarep.2014.12.002. Epub 2014 Dec 19.
Efficient DNA double-strand break (DSB) repair is a critical determinant of cell survival in response to DNA damaging agents, and it plays a key role in the maintenance of genomic integrity. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. We now understand that HR and NHEJ repair are composed of multiple sub-pathways, some of which still remain poorly understood. As such, there is great interest in the development of novel assays to interrogate these key pathways, which could lead to the development of novel therapeutics, and a better understanding of how DSBs are repaired. Furthermore, assays which can measure repair specifically at endogenous chromosomal loci are of particular interest, because of an emerging understanding that chromatin interactions heavily influence DSB repair pathway choice. Here, we present the design and validation of a novel, next-generation sequencing-based approach to study DSB repair at chromosomal loci in cells. We demonstrate that NHEJ repair "fingerprints" can be identified using our assay, which are dependent on the status of key DSB repair proteins. In addition, we have validated that our system can be used to detect dynamic shifts in DSB repair activity in response to specific perturbations. This approach represents a unique alternative to many currently available DSB repair assays, which typical rely on the expression of reporter genes as an indirect read-out for repair. As such, we believe this tool will be useful for DNA repair researchers to study NHEJ repair in a high-throughput and sensitive manner, with the capacity to detect subtle changes in DSB repair patterns that was not possible previously.
高效的DNA双链断裂（DSB）修复是细胞在应对DNA损伤剂时存活的关键决定因素，并且在维持基因组完整性方面发挥着关键作用。同源重组（HR）和非同源末端连接（NHEJ）是哺乳动物细胞中修复DSB的两条主要途径。我们现在了解到，HR和NHEJ修复由多个子途径组成，其中一些仍然了解甚少。因此，人们对开发新的检测方法来探究这些关键途径非常感兴趣，这可能会导致新型疗法的开发，并更好地理解DSB是如何修复的。此外，能够特异性测量内源性染色体位点修复的检测方法特别受关注，因为人们逐渐认识到染色质相互作用会严重影响DSB修复途径的选择。在这里，我们展示了一种基于新一代测序的新颖方法的设计和验证，用于研究细胞中染色体位点的DSB修复。我们证明可以使用我们的检测方法识别NHEJ修复“指纹”，这些指纹取决于关键DSB修复蛋白的状态。此外，我们已经验证我们的系统可用于检测DSB修复活性响应特定扰动的动态变化。这种方法是许多目前可用的DSB修复检测方法的独特替代方法，这些方法通常依赖于报告基因的表达作为修复的间接读出。因此，我们相信这个工具将有助于DNA修复研究人员以高通量和灵敏的方式研究NHEJ修复，能够检测以前不可能检测到的DSB修复模式的细微变化。
