Limits...
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.

Show MeSH

Related in: MedlinePlus

γ-H2AX foci developing on M. muntjak mitotic chromosomes (maximum projections). M. muntjak cell cultures were exposed to 0.6 Gy on ice, covered with growth media at 37°C, and allowed to recover for 0.3 (A), 1 (B), 3 (C), 9 (D), 30 (E), and 90 min (F) before fixation. Fields were scanned by eye using phase optics for mitotic cells. Selected cells were optically sectioned at 0.5-μm intervals. The sections were recombined into a maximum projection. The green channel is amplified to the same extent in all samples to visualize the nascent foci at 1 and 3 min; however, this results in some overexposed foci in the 9-, 30-, and 90-min samples.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169482&req=5

Figure 6: γ-H2AX foci developing on M. muntjak mitotic chromosomes (maximum projections). M. muntjak cell cultures were exposed to 0.6 Gy on ice, covered with growth media at 37°C, and allowed to recover for 0.3 (A), 1 (B), 3 (C), 9 (D), 30 (E), and 90 min (F) before fixation. Fields were scanned by eye using phase optics for mitotic cells. Selected cells were optically sectioned at 0.5-μm intervals. The sections were recombined into a maximum projection. The green channel is amplified to the same extent in all samples to visualize the nascent foci at 1 and 3 min; however, this results in some overexposed foci in the 9-, 30-, and 90-min samples.

Mentions: A study was performed with muntjac cell cultures exposed to 0.6 Gy and permitted to recover for various times (Fig. 6). The fields were searched for mitotic cells, which were imaged. A mitotic figure with discretely visible arms taken from each recovery time period is presented in Fig. 6. γ-H2AX foci, although not detectable after 0.3 min (Fig. 6 A), were detectable as small punctate foci after 1 min and continued to grow in number and size until 30 min (Fig. 6, B–E). At 90 min, the γ-H2AX foci were fewer in number but similar in size to those seen at 30 min. Since muntjac cells contain ∼90% of the DNA per cell as human cells (Levy et al. 1993), similar numbers of γ-H2AX foci per Gy would be expected in the two. If each half mitotic figure contains the G1 complement of DNA, one would expect ∼6 initial and 6 persistent DNA double-strand breaks in each. The number of γ-H2AX foci visible in each half of the 9- and 30-min mitotic figures (Fig. 6D and Fig. E) is higher than that found in the IMR90 cells (Fig. 2C and Fig. D, and Fig. 3), and nearer the expected value for initial rather than persistent DNA double-strand breaks. This difference might reflect a greater sensitivity of detection of small γ-H2AX foci in mitotic cells due to the greater compaction of the chromatin; mitotic cells often display more distinct foci than do interphase cells (compare the mitotic and interphase cells in Fig. 2J and Fig. M). The difference could also be due to differences in DNA double-strand break detection and rejoining between interphase and mitotic cells, or to differences between human and muntjac cell metabolism. The IMR90 cells contained more foci at 3 min after 0.6 Gy than later (Fig. 2B and Fig. C), whereas the opposite was the case for the muntjac mitotic cells (Fig. 6C and Fig. D). These findings indicate that these types of differences do exist, but whether they are due to differences in detection sensitivity, DNA compaction, species metabolism, or other factors requires further study.


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 developing on M. muntjak mitotic chromosomes (maximum projections). M. muntjak cell cultures were exposed to 0.6 Gy on ice, covered with growth media at 37°C, and allowed to recover for 0.3 (A), 1 (B), 3 (C), 9 (D), 30 (E), and 90 min (F) before fixation. Fields were scanned by eye using phase optics for mitotic cells. Selected cells were optically sectioned at 0.5-μm intervals. The sections were recombined into a maximum projection. The green channel is amplified to the same extent in all samples to visualize the nascent foci at 1 and 3 min; however, this results in some overexposed foci in the 9-, 30-, and 90-min samples.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: γ-H2AX foci developing on M. muntjak mitotic chromosomes (maximum projections). M. muntjak cell cultures were exposed to 0.6 Gy on ice, covered with growth media at 37°C, and allowed to recover for 0.3 (A), 1 (B), 3 (C), 9 (D), 30 (E), and 90 min (F) before fixation. Fields were scanned by eye using phase optics for mitotic cells. Selected cells were optically sectioned at 0.5-μm intervals. The sections were recombined into a maximum projection. The green channel is amplified to the same extent in all samples to visualize the nascent foci at 1 and 3 min; however, this results in some overexposed foci in the 9-, 30-, and 90-min samples.
Mentions: A study was performed with muntjac cell cultures exposed to 0.6 Gy and permitted to recover for various times (Fig. 6). The fields were searched for mitotic cells, which were imaged. A mitotic figure with discretely visible arms taken from each recovery time period is presented in Fig. 6. γ-H2AX foci, although not detectable after 0.3 min (Fig. 6 A), were detectable as small punctate foci after 1 min and continued to grow in number and size until 30 min (Fig. 6, B–E). At 90 min, the γ-H2AX foci were fewer in number but similar in size to those seen at 30 min. Since muntjac cells contain ∼90% of the DNA per cell as human cells (Levy et al. 1993), similar numbers of γ-H2AX foci per Gy would be expected in the two. If each half mitotic figure contains the G1 complement of DNA, one would expect ∼6 initial and 6 persistent DNA double-strand breaks in each. The number of γ-H2AX foci visible in each half of the 9- and 30-min mitotic figures (Fig. 6D and Fig. E) is higher than that found in the IMR90 cells (Fig. 2C and Fig. D, and Fig. 3), and nearer the expected value for initial rather than persistent DNA double-strand breaks. This difference might reflect a greater sensitivity of detection of small γ-H2AX foci in mitotic cells due to the greater compaction of the chromatin; mitotic cells often display more distinct foci than do interphase cells (compare the mitotic and interphase cells in Fig. 2J and Fig. M). The difference could also be due to differences in DNA double-strand break detection and rejoining between interphase and mitotic cells, or to differences between human and muntjac cell metabolism. The IMR90 cells contained more foci at 3 min after 0.6 Gy than later (Fig. 2B and Fig. C), whereas the opposite was the case for the muntjac mitotic cells (Fig. 6C and Fig. D). These findings indicate that these types of differences do exist, but whether they are due to differences in detection sensitivity, DNA compaction, species metabolism, or other factors requires further study.

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