Detection of DNA Double-Strand Breaks byγ-H2AX Immunodetection

DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of geneticinstability as they can lead to mutations, genome rearrangements, or loss of genetic material when notproperly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBswith the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated inserine 139 in mammals (S129 in yeast), is namedγ-H2AX. Detection of DSBs is of high relevance inresearch on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association ofDSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels ofγ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter sometechniques to detectγ-H2AX are described.