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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 formation in human cells after irradiation (maximum projections showing all recorded foci). IMR90 normal human fibroblasts (A–H) and MCF7 human breast cancer cells (I–P) were exposed to various amounts of ionizing radiation and permitted to recover for various lengths of time. (A and I) Unirradiated control, (B and J) 3 min after 0.6 Gy, (C and K) 15 min after 0.6 Gy, (D and L) 30 min after 0.6 Gy, (E and M) 60 min after 0.6 Gy, (F and N) 180 min after 0.6 Gy, (G and O) 15 min after 2 Gy, (H and P) 15 min after 22 Gy. m indicates mitotic MCF7 cells in J and M. Cells were processed for laser scanning confocal microscopy as described in Materials and Methods.
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Figure 2: γ-H2AX foci formation in human cells after irradiation (maximum projections showing all recorded foci). IMR90 normal human fibroblasts (A–H) and MCF7 human breast cancer cells (I–P) were exposed to various amounts of ionizing radiation and permitted to recover for various lengths of time. (A and I) Unirradiated control, (B and J) 3 min after 0.6 Gy, (C and K) 15 min after 0.6 Gy, (D and L) 30 min after 0.6 Gy, (E and M) 60 min after 0.6 Gy, (F and N) 180 min after 0.6 Gy, (G and O) 15 min after 2 Gy, (H and P) 15 min after 22 Gy. m indicates mitotic MCF7 cells in J and M. Cells were processed for laser scanning confocal microscopy as described in Materials and Methods.

Mentions: With the demonstration that anti-γ is specific for γ-H2AX, the distribution of γ-H2AX in irradiated cells was examined. Cells of the normal human fibroblast line IMR90 and the human breast cancer line MCF7 both responded to ionizing radiation with the formation of discrete foci containing γ-H2AX throughout the nuclei (Fig. 2). Some cells of both lines contained foci in the absence of irradiation; most of the unirradiated MCF7 cells contained one to two foci (Fig. 2 I), whereas fewer of the IMR90 cells did (Fig. 2 A). The amount of γ-H2AX present in these foci in unirradiated cells is evidently below the level of detection of the immunoblots shown in Fig. 1 A, but may account for the signal seen on highly exposed immunoblots. No foci were apparent in unirradiated or irradiated cells when 1 μM immunizing peptide was included in the first antibody solution (data not presented). The relationship between the presence of γ-H2AX in unirradiated and irradiated cells is being examined.


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 formation in human cells after irradiation (maximum projections showing all recorded foci). IMR90 normal human fibroblasts (A–H) and MCF7 human breast cancer cells (I–P) were exposed to various amounts of ionizing radiation and permitted to recover for various lengths of time. (A and I) Unirradiated control, (B and J) 3 min after 0.6 Gy, (C and K) 15 min after 0.6 Gy, (D and L) 30 min after 0.6 Gy, (E and M) 60 min after 0.6 Gy, (F and N) 180 min after 0.6 Gy, (G and O) 15 min after 2 Gy, (H and P) 15 min after 22 Gy. m indicates mitotic MCF7 cells in J and M. Cells were processed for laser scanning confocal microscopy as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: γ-H2AX foci formation in human cells after irradiation (maximum projections showing all recorded foci). IMR90 normal human fibroblasts (A–H) and MCF7 human breast cancer cells (I–P) were exposed to various amounts of ionizing radiation and permitted to recover for various lengths of time. (A and I) Unirradiated control, (B and J) 3 min after 0.6 Gy, (C and K) 15 min after 0.6 Gy, (D and L) 30 min after 0.6 Gy, (E and M) 60 min after 0.6 Gy, (F and N) 180 min after 0.6 Gy, (G and O) 15 min after 2 Gy, (H and P) 15 min after 22 Gy. m indicates mitotic MCF7 cells in J and M. Cells were processed for laser scanning confocal microscopy as described in Materials and Methods.
Mentions: With the demonstration that anti-γ is specific for γ-H2AX, the distribution of γ-H2AX in irradiated cells was examined. Cells of the normal human fibroblast line IMR90 and the human breast cancer line MCF7 both responded to ionizing radiation with the formation of discrete foci containing γ-H2AX throughout the nuclei (Fig. 2). Some cells of both lines contained foci in the absence of irradiation; most of the unirradiated MCF7 cells contained one to two foci (Fig. 2 I), whereas fewer of the IMR90 cells did (Fig. 2 A). The amount of γ-H2AX present in these foci in unirradiated cells is evidently below the level of detection of the immunoblots shown in Fig. 1 A, but may account for the signal seen on highly exposed immunoblots. No foci were apparent in unirradiated or irradiated cells when 1 μM immunizing peptide was included in the first antibody solution (data not presented). The relationship between the presence of γ-H2AX in unirradiated and irradiated cells is being examined.

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