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© The Rockefeller University Press, 0021-9525/1999//905 $5.00
The Journal of Cell Biology, Volume 146, Number 5, , 1999 905-916

Original Article

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



Emmy P. Rogakoua, Chye Boona, Christophe Redona, and William M. Bonnera

a Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
NIH-NCI, Bldg. 37, Rm. 5D17, Bethesda, MD 20892.(301) 402-0752(301) 496-5942

wmbonner{at}helix.nih.gov

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.

Key Words: ionizing radiation • histone H2AX • DNA double-strand breaks • phosphorylation • indirect immunofluorescence

© 1999 The Rockefeller University Press

1.used in this paper: BrdU, bromo deoxyuridine


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