1] Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan. [2] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki University, Nagasaki, Japan. [3] Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.
1] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki University, Nagasaki, Japan. [2] Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.
Nat Protoc. 2015 Jan;10(1):12-24. doi: 10.1038/nprot.2014.194. Epub 2014 Dec 4.
DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete.
DNA 修复系统可保护细胞免受基因组不稳定性和致癌作用的影响。因此，用于测量 DNA 修复活性的测定法不仅对于 DNA 修复缺陷疾病的临床诊断具有重要价值，而且对于基础研究和抗癌药物开发也具有重要价值。两种常用的测定法是 UDS（非计划 DNA 合成，需要精确测量极少量的修复 DNA 合成）和 RRS（DNA 损伤后 RNA 合成的恢复）。UDS 和 RRS 都是评估核苷酸切除修复（NER）活性的主要终点，NER 是最通用的 DNA 修复过程。传统的 UDS 和 RRS 测定法既繁琐又耗时，因为它们测量与 NER 相关的放射性核苷的掺入。在这里，我们描述了一种通过研究炔基缀合核苷类似物的掺入，然后进行荧光叠氮化物偶联点击化学反应来监测非放射性 UDS 和 RRS 的综合方案。该系统也适用于快速测量细胞对 DNA 损伤试剂的敏感性，以及基于慢病毒的互补测定法，可用于系统地确定与 DNA 修复缺陷疾病相关的致病基因。使用原代成纤维细胞进行典型的 UDS 或 RRS 测定（包括病毒互补测试），整个过程需要 1 周时间才能完成。
