The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, CB2 1QN, England, UK.
J Cell Biol. 2013 Aug 5;202(3):579-95. doi: 10.1083/jcb.201303073. Epub 2013 Jul 29.
DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents.
DNA 双链断裂（DSBs）是所有基因组损伤中最具毒性的，处理其信号转导和修复的途径对于预防癌症和免疫系统的发展至关重要。尽管进行了深入的研究，但由于我们无法在细胞中的 DSB 处检测到主要的修复因子，因此我们对这些途径的了解在技术上受到限制。在本文中，我们提出了一种原始方法，该方法涉及核糖核酸酶和去污剂预处理的组合以及高分辨率显微镜。该方法允许直接可视化以前隐藏的修复复合物，包括主要的 DSB 传感器 Ku，几乎可以在任何类型的 DSB 处，包括抗癌剂诱导的 DSB 处。我们通过将其与激光微照射、超分辨率显微镜和单分子计数相结合来展示其广泛的应用，以研究修复工厂的空间组织和组成。此外，我们使用我们的方法来监测 DNA 修复并确定修复途径选择的机制，我们还展示了它在定义细胞对抗癌剂的敏感性和耐药机制方面的效用。
