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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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Immunoblots. After exposure to the indicated amount of ionizing radiation and a 30-min recovery, cells were harvested. Extracts were prepared and analyzed by gel electrophoresis and immunoblotting on polyvinylidene difluoride (PVDF) membranes as described in Materials and Methods. (A) Human MCF7 breast cancer cells. Blots of fractionated total SDS extracts were probed as indicated with anti-γ preimmune serum (pre) or with anti-γ containing 1 μM immunizing peptide (P-pep). The left-most lane shows the protein staining pattern on SDS gels. (B and C) Human SF268 astrocytoma cells. Cultures were irradiated with 100 Gy and analyzed on high-resolution two-dimension acetic acid gels. (D–G) Other eucaryotes. The position of migration of the respective H2AX homologues, H2AX in M. muntjak (D) and X. laevis (E), H2AvD in D. melanogaster (F), and H2A in S. cerevisiae (G) are indicated by γ. Cultures of S. cerevisiae strain BY4733 were irradiated and allowed to recover for 30 min at 30°C. Nuclei were prepared from spheroplasts (Celis 1998), and histones were extracted as described (Ueda and Tanaka 1995). For MCF7, M. muntjak, X. laevis, and S. cerevisiae extracts were prepared with SDS and fractionated on 12% NuPage SDS gels (Novex Novel Technology). For D. melanogaster, extracts were prepared with 0.5 N HCl and fractionated on 12% acetic acid–urea–Triton X-100 gels.
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Figure 1: Immunoblots. After exposure to the indicated amount of ionizing radiation and a 30-min recovery, cells were harvested. Extracts were prepared and analyzed by gel electrophoresis and immunoblotting on polyvinylidene difluoride (PVDF) membranes as described in Materials and Methods. (A) Human MCF7 breast cancer cells. Blots of fractionated total SDS extracts were probed as indicated with anti-γ preimmune serum (pre) or with anti-γ containing 1 μM immunizing peptide (P-pep). The left-most lane shows the protein staining pattern on SDS gels. (B and C) Human SF268 astrocytoma cells. Cultures were irradiated with 100 Gy and analyzed on high-resolution two-dimension acetic acid gels. (D–G) Other eucaryotes. The position of migration of the respective H2AX homologues, H2AX in M. muntjak (D) and X. laevis (E), H2AvD in D. melanogaster (F), and H2A in S. cerevisiae (G) are indicated by γ. Cultures of S. cerevisiae strain BY4733 were irradiated and allowed to recover for 30 min at 30°C. Nuclei were prepared from spheroplasts (Celis 1998), and histones were extracted as described (Ueda and Tanaka 1995). For MCF7, M. muntjak, X. laevis, and S. cerevisiae extracts were prepared with SDS and fractionated on 12% NuPage SDS gels (Novex Novel Technology). For D. melanogaster, extracts were prepared with 0.5 N HCl and fractionated on 12% acetic acid–urea–Triton X-100 gels.

Mentions: To examine the spatial distribution of γ-H2AX in the chromatin of irradiated cells, a polyclonal antibody (anti-γ) was raised in rabbits against a synthetic phosphorylated peptide containing the mammalian γ-H2AX COOH-terminal sequence. On immunoblots of total protein extracts from irradiated MCF7 cells, anti-γ detected one band at the position expected for γ-H2AX (Fig. 1 A). No binding was detected in irradiated samples when the immunizing peptide was present as competitor (Fig. 1 A, P-pep) or with preimmune serum (Fig. 1 A, pre). Although anti-γ binding was not apparent in unirradiated MCF7 samples at film exposures optimal for γ-H2AX detection (Fig. 1 A, 0 Gy), small amounts of binding were detectable on highly exposed immunoblots. Results from our laboratory show that γ-H2AX is present in apoptotic cells with fragmented DNA (Rogakou, E.P., W. Nieves-Neira, C. Boon, Y. Pommier, and W.M. Bonner, manuscript submitted for publication), indicating a possible source of γ-H2AX in unirradiated cultures. Another possibility is that anti-γ cross-reacts slightly with unmodified H2AX. However, two-dimension gels which separate these two protein species (Fig. 1 B) showed that anti-γ bound only γ-H2AX (Fig. 1 C, solid line) with no detectable cross-reaction to unmodified H2AX. This result indicates that the binding of anti-γ in extracts of unirradiated cells is due to the presence of γ-H2AX in some of the cells in those cultures.


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

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

Immunoblots. After exposure to the indicated amount of ionizing radiation and a 30-min recovery, cells were harvested. Extracts were prepared and analyzed by gel electrophoresis and immunoblotting on polyvinylidene difluoride (PVDF) membranes as described in Materials and Methods. (A) Human MCF7 breast cancer cells. Blots of fractionated total SDS extracts were probed as indicated with anti-γ preimmune serum (pre) or with anti-γ containing 1 μM immunizing peptide (P-pep). The left-most lane shows the protein staining pattern on SDS gels. (B and C) Human SF268 astrocytoma cells. Cultures were irradiated with 100 Gy and analyzed on high-resolution two-dimension acetic acid gels. (D–G) Other eucaryotes. The position of migration of the respective H2AX homologues, H2AX in M. muntjak (D) and X. laevis (E), H2AvD in D. melanogaster (F), and H2A in S. cerevisiae (G) are indicated by γ. Cultures of S. cerevisiae strain BY4733 were irradiated and allowed to recover for 30 min at 30°C. Nuclei were prepared from spheroplasts (Celis 1998), and histones were extracted as described (Ueda and Tanaka 1995). For MCF7, M. muntjak, X. laevis, and S. cerevisiae extracts were prepared with SDS and fractionated on 12% NuPage SDS gels (Novex Novel Technology). For D. melanogaster, extracts were prepared with 0.5 N HCl and fractionated on 12% acetic acid–urea–Triton X-100 gels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169482&req=5

Figure 1: Immunoblots. After exposure to the indicated amount of ionizing radiation and a 30-min recovery, cells were harvested. Extracts were prepared and analyzed by gel electrophoresis and immunoblotting on polyvinylidene difluoride (PVDF) membranes as described in Materials and Methods. (A) Human MCF7 breast cancer cells. Blots of fractionated total SDS extracts were probed as indicated with anti-γ preimmune serum (pre) or with anti-γ containing 1 μM immunizing peptide (P-pep). The left-most lane shows the protein staining pattern on SDS gels. (B and C) Human SF268 astrocytoma cells. Cultures were irradiated with 100 Gy and analyzed on high-resolution two-dimension acetic acid gels. (D–G) Other eucaryotes. The position of migration of the respective H2AX homologues, H2AX in M. muntjak (D) and X. laevis (E), H2AvD in D. melanogaster (F), and H2A in S. cerevisiae (G) are indicated by γ. Cultures of S. cerevisiae strain BY4733 were irradiated and allowed to recover for 30 min at 30°C. Nuclei were prepared from spheroplasts (Celis 1998), and histones were extracted as described (Ueda and Tanaka 1995). For MCF7, M. muntjak, X. laevis, and S. cerevisiae extracts were prepared with SDS and fractionated on 12% NuPage SDS gels (Novex Novel Technology). For D. melanogaster, extracts were prepared with 0.5 N HCl and fractionated on 12% acetic acid–urea–Triton X-100 gels.
Mentions: To examine the spatial distribution of γ-H2AX in the chromatin of irradiated cells, a polyclonal antibody (anti-γ) was raised in rabbits against a synthetic phosphorylated peptide containing the mammalian γ-H2AX COOH-terminal sequence. On immunoblots of total protein extracts from irradiated MCF7 cells, anti-γ detected one band at the position expected for γ-H2AX (Fig. 1 A). No binding was detected in irradiated samples when the immunizing peptide was present as competitor (Fig. 1 A, P-pep) or with preimmune serum (Fig. 1 A, pre). Although anti-γ binding was not apparent in unirradiated MCF7 samples at film exposures optimal for γ-H2AX detection (Fig. 1 A, 0 Gy), small amounts of binding were detectable on highly exposed immunoblots. Results from our laboratory show that γ-H2AX is present in apoptotic cells with fragmented DNA (Rogakou, E.P., W. Nieves-Neira, C. Boon, Y. Pommier, and W.M. Bonner, manuscript submitted for publication), indicating a possible source of γ-H2AX in unirradiated cultures. Another possibility is that anti-γ cross-reacts slightly with unmodified H2AX. However, two-dimension gels which separate these two protein species (Fig. 1 B) showed that anti-γ bound only γ-H2AX (Fig. 1 C, solid line) with no detectable cross-reaction to unmodified H2AX. This result indicates that the binding of anti-γ in extracts of unirradiated cells is due to the presence of γ-H2AX in some of the cells in those cultures.

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