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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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Numbers of foci in IMR90 cells. Maximum projections of fields of IMR90 cells, similar to those shown in Fig. 2A–F, were analyzed by eye for numbers of foci per nucleus. Nuclei were scored as containing 0–5, 6–10, 11–15, 16–20, or 20–25 foci. All whole nuclei in a field were included, none of which contained >25 foci.
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Figure 3: Numbers of foci in IMR90 cells. Maximum projections of fields of IMR90 cells, similar to those shown in Fig. 2A–F, were analyzed by eye for numbers of foci per nucleus. Nuclei were scored as containing 0–5, 6–10, 11–15, 16–20, or 20–25 foci. All whole nuclei in a field were included, none of which contained >25 foci.

Mentions: With IMR90 cells, foci were apparent 3 min after irradiation with 0.6 Gy (Fig. 2 B), persisted at 15–60 min (Fig. 2, C–E), then decreased in number at 180 min (Fig. 2 F). With MCF7 cells, the time course of foci appearance and disappearance was similar (Fig. 2, I–N). A more detailed analysis of IMR90 cells is presented in Fig. 3, in which individual nuclei in fields of cells were scored for the number of foci. Compared with the unirradiated control cells, which contained an average of 1.5 ± 2.4 (14 nuclei) foci per nucleus, all the IMR90 cells 3 min after exposure to 0.6 Gy contained numerous small foci, with an average of 16.3 ± 3.6 (11 nuclei) foci per nucleus. The foci became fewer in number but better defined after 15 min, 10.1 ± 3.9 (17 nuclei) foci per nucleus; 30 min, 11.6 ± 5.3 (18 nuclei) foci per nucleus; and 60 min, 11.4 ± 6.1 (15 nuclei) foci per nucleus. After 180 min recovery, the number of foci again decreased to 4.8 ± 3.3 (17 nuclei) foci per nucleus, and at 270 min there were 4.5 ± 5.3 (26 nuclei) foci per nucleus. In addition, after 270 min recovery, 2 of the 26 scored nuclei appeared to be free of foci, possibly suggesting that in these two cells all of the introduced DNA double-strand breaks had been rejoined. This time course obtained by counting foci is very similar to that obtained by Rogakou et al. 1998, in which mass formation of γ-H2AX was measured after CHO cells had been irradiated with 200 Gy. This similarity suggests that the processes of γ-H2AX appearance and disappearance are the same at these two very different amounts of ionizing radiation.


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

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

Numbers of foci in IMR90 cells. Maximum projections of fields of IMR90 cells, similar to those shown in Fig. 2A–F, were analyzed by eye for numbers of foci per nucleus. Nuclei were scored as containing 0–5, 6–10, 11–15, 16–20, or 20–25 foci. All whole nuclei in a field were included, none of which contained >25 foci.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Numbers of foci in IMR90 cells. Maximum projections of fields of IMR90 cells, similar to those shown in Fig. 2A–F, were analyzed by eye for numbers of foci per nucleus. Nuclei were scored as containing 0–5, 6–10, 11–15, 16–20, or 20–25 foci. All whole nuclei in a field were included, none of which contained >25 foci.
Mentions: With IMR90 cells, foci were apparent 3 min after irradiation with 0.6 Gy (Fig. 2 B), persisted at 15–60 min (Fig. 2, C–E), then decreased in number at 180 min (Fig. 2 F). With MCF7 cells, the time course of foci appearance and disappearance was similar (Fig. 2, I–N). A more detailed analysis of IMR90 cells is presented in Fig. 3, in which individual nuclei in fields of cells were scored for the number of foci. Compared with the unirradiated control cells, which contained an average of 1.5 ± 2.4 (14 nuclei) foci per nucleus, all the IMR90 cells 3 min after exposure to 0.6 Gy contained numerous small foci, with an average of 16.3 ± 3.6 (11 nuclei) foci per nucleus. The foci became fewer in number but better defined after 15 min, 10.1 ± 3.9 (17 nuclei) foci per nucleus; 30 min, 11.6 ± 5.3 (18 nuclei) foci per nucleus; and 60 min, 11.4 ± 6.1 (15 nuclei) foci per nucleus. After 180 min recovery, the number of foci again decreased to 4.8 ± 3.3 (17 nuclei) foci per nucleus, and at 270 min there were 4.5 ± 5.3 (26 nuclei) foci per nucleus. In addition, after 270 min recovery, 2 of the 26 scored nuclei appeared to be free of foci, possibly suggesting that in these two cells all of the introduced DNA double-strand breaks had been rejoined. This time course obtained by counting foci is very similar to that obtained by Rogakou et al. 1998, in which mass formation of γ-H2AX was measured after CHO cells had been irradiated with 200 Gy. This similarity suggests that the processes of γ-H2AX appearance and disappearance are the same at these two very different amounts of ionizing radiation.

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