Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo

TLDR

Ionizing radiation–induced DNA double‑strand breaks trigger phosphorylation of histone H2AX at serine 139, producing the marker gamma‑H2AX. An antibody against gamma‑H2AX detects phosphorylated H2AX in irradiated cells of multiple species and shows that laser‑induced breaks in specific nuclear subvolumes generate localized gamma‑H2AX foci. Gamma‑H2AX foci appear within one minute of irradiation, match the number of DNA double‑strand breaks, form band‑like structures on broken chromosome arms in mitosis, and provide direct visual evidence that gamma‑H2AX accumulates at chromosomal break sites, implying higher‑order chromatin units monitor DNA integrity.

Abstract

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.