Choi Jun-Hyuk, Kim So-Young, Kim Sook-Kyung, Kemp Michael G, Sancar Aziz
From the Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon 305-340, South Korea, the Department of Bio-Analytical Science, University of Science & Technology, Daejeon 305-350, South Korea, and
From the Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon 305-340, South Korea.
J Biol Chem. 2015 Nov 27;290(48):28812-21. doi: 10.1074/jbc.M115.690354. Epub 2015 Oct 5.
DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.
紫外线及紫外线模拟剂造成的DNA损伤会在哺乳动物细胞中引发一系列相互关联的反应，包括DNA修复、DNA损伤检查点和细胞凋亡。传统上，这些反应是使用不同方法分别进行分析的。在此，我们描述了一种统一的方法，该方法能够使用来自同一细胞群体的裂解物并行量化所有这三种反应。我们表明，一种高度灵敏的体内切除修复测定法能够在损伤诱导后数分钟内检测多种DNA损伤（紫外线损伤、化学致癌物和化疗药物）的核苷酸切除修复。因此，该方法能够实时测量核苷酸切除修复活性，并且可以与DNA损伤反应的其他成分（包括DNA损伤检查点和凋亡信号传导）一起进行监测。因此，这种方法提供了一个方便可靠的平台，用于在单一细胞群体中同时检查DNA损伤反应的多个方面，可应用于多种致癌剂和化疗药物。
