γH2AX foci analysis for monitoring DNA double-strand break repair Strengths, limitations and optimization

DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating gamma H2AX foci that can be visualized by immunofluorescence. There is a close correlation between gamma H2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, gamma H2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionizing radiation, gamma H2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimize the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.