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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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Related in: MedlinePlus

Laser-directed DNA double-strand breaks in MCF7 cells. UVA light was delivered by a 390-nm laser as described in Materials and Methods. The white lines trace the path of the laser as guided with a joystick. The percentages refer to the relative laser energy used in each transit. (A) Cells grown with BrdU. (B) Cells grown without BrdU. In both cases, cells were incubated with Hoechst dye 33258 as described in Materials and Methods.
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Figure 4: Laser-directed DNA double-strand breaks in MCF7 cells. UVA light was delivered by a 390-nm laser as described in Materials and Methods. The white lines trace the path of the laser as guided with a joystick. The percentages refer to the relative laser energy used in each transit. (A) Cells grown with BrdU. (B) Cells grown without BrdU. In both cases, cells were incubated with Hoechst dye 33258 as described in Materials and Methods.

Mentions: If each γ-H2AX focus identifies a DNA double-strand break, then the two should coincide. To determine this, advantage was taken of the finding that γ-H2AX was formed when DNA double-strand breaks were introduced into cells by the BrdU dye–UVA light procedure of Limoli and Ward 1994 (Rogakou et al. 1998). A UVA pulsed laser (390 nm) commonly used in LaserScissors™ devices can be substituted for the UVA fluorescent light source (365 nm), and has the advantage of permitting the illumination of specific partial nuclear volumes. When MCF7 cells with BrdU-containing DNA were exposed to the laser in the presence of the dye, those nuclear regions traversed by the laser at 1, 10, and 30% relative power contained γ-H2AX foci (Fig. 4 A). γ-H2AX formation was dependent on the presence of BrdU; when BrdU was absent but dye still present, γ-H2AX foci were consistently found only in the cells traversed with the laser at 30% relative power (Fig. 4 B). This experiment demonstrates that γ-H2AX foci form 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)

Laser-directed DNA double-strand breaks in MCF7 cells. UVA light was delivered by a 390-nm laser as described in Materials and Methods. The white lines trace the path of the laser as guided with a joystick. The percentages refer to the relative laser energy used in each transit. (A) Cells grown with BrdU. (B) Cells grown without BrdU. In both cases, cells were incubated with Hoechst dye 33258 as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Laser-directed DNA double-strand breaks in MCF7 cells. UVA light was delivered by a 390-nm laser as described in Materials and Methods. The white lines trace the path of the laser as guided with a joystick. The percentages refer to the relative laser energy used in each transit. (A) Cells grown with BrdU. (B) Cells grown without BrdU. In both cases, cells were incubated with Hoechst dye 33258 as described in Materials and Methods.
Mentions: If each γ-H2AX focus identifies a DNA double-strand break, then the two should coincide. To determine this, advantage was taken of the finding that γ-H2AX was formed when DNA double-strand breaks were introduced into cells by the BrdU dye–UVA light procedure of Limoli and Ward 1994 (Rogakou et al. 1998). A UVA pulsed laser (390 nm) commonly used in LaserScissors™ devices can be substituted for the UVA fluorescent light source (365 nm), and has the advantage of permitting the illumination of specific partial nuclear volumes. When MCF7 cells with BrdU-containing DNA were exposed to the laser in the presence of the dye, those nuclear regions traversed by the laser at 1, 10, and 30% relative power contained γ-H2AX foci (Fig. 4 A). γ-H2AX formation was dependent on the presence of BrdU; when BrdU was absent but dye still present, γ-H2AX foci were consistently found only in the cells traversed with the laser at 30% relative power (Fig. 4 B). This experiment demonstrates that γ-H2AX foci form 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