Detection of DNA Double-Strand Breaks by γ-H2AX Immunodetection

DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of genetic instability as they can lead to mutations, genome rearrangements, or loss of genetic material when not properly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBs with the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated in serine 139 in mammals (S129 in yeast), is named γ-H2AX. Detection of DSBs is of high relevance in research on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association of DSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels of γ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter some techniques to detect γ-H2AX are described.