Sedelnikova, O. A., Rogakou, E. P., Panyutin, I. G. and Bonner, W. M. Quantitative Detection of 125IdU-Induced DNA Double-Strand Breaks with γ-H2AX Antibody. Radiat. Res. 158, 486–492 (2002).
When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (γ-H2AX) demonstrates that γ-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of 125I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of 125IdU and processed immunocytochemically to determine the number of γ-H2AX foci. The numbers of 125IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of 125I decays and the number of foci per cell, consistent with the assumptions that each 125I decay yields a DNA DSB and each DNA DSB yields a visible γ-H2AX focus. Based on these findings, we conclude that γ-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.
