Klement Karolin, Goodarzi Aaron A
Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, Department of Biochemistry and Molecular Biology and Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada, T2N 4N1.
Methods Mol Biol. 2017;1599:303-315. doi: 10.1007/978-1-4939-6955-5_22.
DNA damaging agents such as ionizing irradiation induce lesions in the DNA such as double strand breaks (DSBs). Depending on cell type, 10-25% of these DSBs are induced in heterochromatin. Heterochromatic DSBs are resolved with slow kinetics (compared to DSBs in euchromatin) and require ATM activity for repair. Investigating the underlying causes of the slow component of DSB repair and the role of individual response factors in this process provides insight into DSB response pathways and will further the understanding of diseases where such pathways are dysfunctional due to mutation. Here, we describe a method to detect DSB repair foci in the heterochromatin of human cells. We provide a detailed protocol for cell culture preparation, immunofluorescence microscopy, and a computer-assisted approach to analyze overlap between DSB foci and heterochromatin.
诸如电离辐射之类的DNA损伤剂会在DNA中诱导产生损伤，例如双链断裂（DSB）。根据细胞类型的不同，这些双链断裂中有10%-25%是在异染色质中诱导产生的。异染色质双链断裂的修复动力学较慢（与常染色质中的双链断裂相比），并且需要ATM活性来进行修复。研究双链断裂修复缓慢成分的潜在原因以及个体反应因子在此过程中的作用，有助于深入了解双链断裂反应途径，并将进一步加深对因突变导致此类途径功能失调的疾病的理解。在此，我们描述了一种检测人类细胞异染色质中双链断裂修复灶的方法。我们提供了细胞培养准备、免疫荧光显微镜检查以及一种计算机辅助方法的详细方案，用于分析双链断裂灶与异染色质之间的重叠情况。
