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Megabase chromatin domains involved in DNA double-strand breaks in vivo.

Rogakou EP, Boon C, Redon C, Bonner WM - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

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γ-H2AX foci on defective M. muntjak mitotic figures (maximum projections). Selected mitotic figures were imaged in M. muntjak cell cultures that had been exposed to 0.6 Gy on ice and covered with growth media at 37°C for 90 min before fixation. Green arrows point to ends of isolated chromosome arms with γ-H2AX foci. In A and B, transmitted light was collected to show the outline of the cell membrane.
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Figure 7: γ-H2AX foci on defective M. muntjak mitotic figures (maximum projections). Selected mitotic figures were imaged in M. muntjak cell cultures that had been exposed to 0.6 Gy on ice and covered with growth media at 37°C for 90 min before fixation. Green arrows point to ends of isolated chromosome arms with γ-H2AX foci. In A and B, transmitted light was collected to show the outline of the cell membrane.

Mentions: These findings show that DNA double-strand breaks are rapidly detected and marked in condensed chromosomes as well as in interphase chromatin. The mitotic cells imaged in Fig. 6 appear to have been dividing normally when fixed. However, in cultures fixed at 90 min after irradiation, defective mitotic cells could be found (Fig. 7, A–C). Three mitotic cells containing six isolated chromosomal arm fragments are shown, each with a large γ-H2AX focus at one end (indicated by green arrows). In addition, no isolated chromosomal arms lacking a terminal γ-H2AX focus were found in any mitotic figure. These results provide direct visual confirmation that γ-H2AX forms en masse at the sites of DNA double-strand breaks.


Megabase chromatin domains involved in DNA double-strand breaks in vivo.

Rogakou EP, Boon C, Redon C, Bonner WM - J. Cell Biol. (1999)

γ-H2AX foci on defective M. muntjak mitotic figures (maximum projections). Selected mitotic figures were imaged in M. muntjak cell cultures that had been exposed to 0.6 Gy on ice and covered with growth media at 37°C for 90 min before fixation. Green arrows point to ends of isolated chromosome arms with γ-H2AX foci. In A and B, transmitted light was collected to show the outline of the cell membrane.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169482&req=5

Figure 7: γ-H2AX foci on defective M. muntjak mitotic figures (maximum projections). Selected mitotic figures were imaged in M. muntjak cell cultures that had been exposed to 0.6 Gy on ice and covered with growth media at 37°C for 90 min before fixation. Green arrows point to ends of isolated chromosome arms with γ-H2AX foci. In A and B, transmitted light was collected to show the outline of the cell membrane.
Mentions: These findings show that DNA double-strand breaks are rapidly detected and marked in condensed chromosomes as well as in interphase chromatin. The mitotic cells imaged in Fig. 6 appear to have been dividing normally when fixed. However, in cultures fixed at 90 min after irradiation, defective mitotic cells could be found (Fig. 7, A–C). Three mitotic cells containing six isolated chromosomal arm fragments are shown, each with a large γ-H2AX focus at one end (indicated by green arrows). In addition, no isolated chromosomal arms lacking a terminal γ-H2AX focus were found in any mitotic figure. These results provide direct visual confirmation that γ-H2AX forms en masse at the sites of DNA double-strand breaks.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

Show MeSH
Related in: MedlinePlus