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Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.

Kinner A, Wu W, Staudt C, Iliakis G - Nucleic Acids Res. (2008)

Bottom Line: In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX.This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs.We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany.

ABSTRACT
DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

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Effect of the phosphatase inhibitor Calyculin A on DSB repair kinetics and γ-H2AX foci development and decay. (A) Plateau-phase A549 cells were incubated with 0 or 50 nM Calyculin A 15 min prior to exposure to 20 or 1 Gy X-rays for PFGE or γ-H2AX immunofluorescence, respectively, and allowed to repair at 37°C for the indicated periods of time (other details of experimental design as in Figure 5). Results are shown normalized as described in Figure 5. (B, C) Typical PFGE gels used to generate the results shown in A for cells treated with 0 or 50 nM Calyculin A. DNA is stained with ethidium bromide. (D, E) γ-H2AX immunofluorescence at different times after irradiation and incubation with 0 or 50 nM Calyculin A. Other details as in Figure 5.
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Figure 6: Effect of the phosphatase inhibitor Calyculin A on DSB repair kinetics and γ-H2AX foci development and decay. (A) Plateau-phase A549 cells were incubated with 0 or 50 nM Calyculin A 15 min prior to exposure to 20 or 1 Gy X-rays for PFGE or γ-H2AX immunofluorescence, respectively, and allowed to repair at 37°C for the indicated periods of time (other details of experimental design as in Figure 5). Results are shown normalized as described in Figure 5. (B, C) Typical PFGE gels used to generate the results shown in A for cells treated with 0 or 50 nM Calyculin A. DNA is stained with ethidium bromide. (D, E) γ-H2AX immunofluorescence at different times after irradiation and incubation with 0 or 50 nM Calyculin A. Other details as in Figure 5.

Mentions: The disparity between the actual removal of DSBs, as measured by physical methods of DSB detection, and the removal of γ-H2AX foci may increase when chemical or genetic manipulations that affect the phosphorylation cascade of H2AX are employed as tools, and may further depend on the severity of the DSB (121). Thus, use of PIKK or phosphatase inhibitors, or genetic manipulation of their activity, may alter foci formation and decay in a way that further uncouples it from the physical removal of the DSBs. This is experimentally illustrated in Figure 6, where we exposed A549 cells to 50 nM Calyculin A, a nonspecific inhibitor of PP2A, a phosphatase involved in the dephosphorylation of γ-H2AX (54). Although under the experimental conditions employed treatment with Calyculin A leads to a complete stop in γ-H2AX foci decay (122), it only has a small effect on the physical removal of DSBs as measured by PFGE. Experiments with elutriated HeLa, G1 cells show similar trends, suggesting that the effect is not cell line specific, although others have arrived to different conclusions (123). It would be inaccurate to conclude on the basis of γ-H2AX foci data shown in Figure 6 that DSB rejoining is completely inhibited by Calyculin A. Notably, delayed and stage-specific phosphorylation of H2AX was also observed in irradiated mouse embryos (124).Figure 6.


Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.

Kinner A, Wu W, Staudt C, Iliakis G - Nucleic Acids Res. (2008)

Effect of the phosphatase inhibitor Calyculin A on DSB repair kinetics and γ-H2AX foci development and decay. (A) Plateau-phase A549 cells were incubated with 0 or 50 nM Calyculin A 15 min prior to exposure to 20 or 1 Gy X-rays for PFGE or γ-H2AX immunofluorescence, respectively, and allowed to repair at 37°C for the indicated periods of time (other details of experimental design as in Figure 5). Results are shown normalized as described in Figure 5. (B, C) Typical PFGE gels used to generate the results shown in A for cells treated with 0 or 50 nM Calyculin A. DNA is stained with ethidium bromide. (D, E) γ-H2AX immunofluorescence at different times after irradiation and incubation with 0 or 50 nM Calyculin A. Other details as in Figure 5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Effect of the phosphatase inhibitor Calyculin A on DSB repair kinetics and γ-H2AX foci development and decay. (A) Plateau-phase A549 cells were incubated with 0 or 50 nM Calyculin A 15 min prior to exposure to 20 or 1 Gy X-rays for PFGE or γ-H2AX immunofluorescence, respectively, and allowed to repair at 37°C for the indicated periods of time (other details of experimental design as in Figure 5). Results are shown normalized as described in Figure 5. (B, C) Typical PFGE gels used to generate the results shown in A for cells treated with 0 or 50 nM Calyculin A. DNA is stained with ethidium bromide. (D, E) γ-H2AX immunofluorescence at different times after irradiation and incubation with 0 or 50 nM Calyculin A. Other details as in Figure 5.
Mentions: The disparity between the actual removal of DSBs, as measured by physical methods of DSB detection, and the removal of γ-H2AX foci may increase when chemical or genetic manipulations that affect the phosphorylation cascade of H2AX are employed as tools, and may further depend on the severity of the DSB (121). Thus, use of PIKK or phosphatase inhibitors, or genetic manipulation of their activity, may alter foci formation and decay in a way that further uncouples it from the physical removal of the DSBs. This is experimentally illustrated in Figure 6, where we exposed A549 cells to 50 nM Calyculin A, a nonspecific inhibitor of PP2A, a phosphatase involved in the dephosphorylation of γ-H2AX (54). Although under the experimental conditions employed treatment with Calyculin A leads to a complete stop in γ-H2AX foci decay (122), it only has a small effect on the physical removal of DSBs as measured by PFGE. Experiments with elutriated HeLa, G1 cells show similar trends, suggesting that the effect is not cell line specific, although others have arrived to different conclusions (123). It would be inaccurate to conclude on the basis of γ-H2AX foci data shown in Figure 6 that DSB rejoining is completely inhibited by Calyculin A. Notably, delayed and stage-specific phosphorylation of H2AX was also observed in irradiated mouse embryos (124).Figure 6.

Bottom Line: In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX.This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs.We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany.

ABSTRACT
DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

Show MeSH
Related in: MedlinePlus