Analyzing heterochromatic DNA double strand break (DSB) repair in response to ionizing radiation

Abstract

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.