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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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Comparison of DSB repair kinetics as measured by pulsed-field gel electrophoresis (PFGE) with the development of γ-H2AX foci. (A) Plateau-phase A549 cells were exposed to 20 Gy or 1 Gy X-rays and analyzed by PFGE (157) or γ-H2AX immunofluorescence (158), respectively, at the indicated time points. PFGE results (squares) have been normalized to the signal measured at 0 h, while the γ-H2AX results (circles) have been normalized to the maximum number of foci scored. The number of foci per cell was quantified using the Leica Q-Win software with the help of a special routine developed for foci counting. Foci were counted on 3D picture stacks generated on a Leica SP5 confocal microscope. γ-H2AX results were normalized to the maximum number of foci scored per cell—typically reached between 30 min and 1 h after IR. (B) Typical gel used to generate the PFGE results shown in A. DNA is stained with ethidium bromide. (C) Examples of γ-H2AX immunofluorescence at different times after exposure to IR (1 Gy). Cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with a phosphospecific primary anti-γ-H2AX antibody and an Alexa 568-labeled secondary antibody. Red shows γ-H2AX foci, blue nuclei stained with DAPI.
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Figure 5: Comparison of DSB repair kinetics as measured by pulsed-field gel electrophoresis (PFGE) with the development of γ-H2AX foci. (A) Plateau-phase A549 cells were exposed to 20 Gy or 1 Gy X-rays and analyzed by PFGE (157) or γ-H2AX immunofluorescence (158), respectively, at the indicated time points. PFGE results (squares) have been normalized to the signal measured at 0 h, while the γ-H2AX results (circles) have been normalized to the maximum number of foci scored. The number of foci per cell was quantified using the Leica Q-Win software with the help of a special routine developed for foci counting. Foci were counted on 3D picture stacks generated on a Leica SP5 confocal microscope. γ-H2AX results were normalized to the maximum number of foci scored per cell—typically reached between 30 min and 1 h after IR. (B) Typical gel used to generate the PFGE results shown in A. DNA is stained with ethidium bromide. (C) Examples of γ-H2AX immunofluorescence at different times after exposure to IR (1 Gy). Cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with a phosphospecific primary anti-γ-H2AX antibody and an Alexa 568-labeled secondary antibody. Red shows γ-H2AX foci, blue nuclei stained with DAPI.

Mentions: Although several DNA damage-associated histone modifications have been described, here we focus on the most conspicuous one that has been at the center of research activities during the last several years: the modification of the H2A variant, H2AX. H2AX is one of the most conserved H2A-variants (Table 1 and Figure 3), and is present in chromatin at levels that vary between 2 and 25% of the H2A pool, depending on the cell line and tissue examined. H2AX moved to the center of cellular responses to DNA damage after the discovery that it becomes locally phosphorylated, to generate γ-H2AX, in the vicinity of DSBs (24–26). The combination of phosphospecific antibodies that recognize the phosphorylated S-139 residue of γ-H2AX with immunofluorescence microscopy documented the local phosphorylation through the formation of distinct foci in the vicinity of DSBs and allowed monitoring of their induction and repair (Figure 5C). Thus, a wide spectrum of applications could be developed ranging from mechanistic studies in biology to useful applications in oncology and radiation protection (see below).Figure 3.


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)

Comparison of DSB repair kinetics as measured by pulsed-field gel electrophoresis (PFGE) with the development of γ-H2AX foci. (A) Plateau-phase A549 cells were exposed to 20 Gy or 1 Gy X-rays and analyzed by PFGE (157) or γ-H2AX immunofluorescence (158), respectively, at the indicated time points. PFGE results (squares) have been normalized to the signal measured at 0 h, while the γ-H2AX results (circles) have been normalized to the maximum number of foci scored. The number of foci per cell was quantified using the Leica Q-Win software with the help of a special routine developed for foci counting. Foci were counted on 3D picture stacks generated on a Leica SP5 confocal microscope. γ-H2AX results were normalized to the maximum number of foci scored per cell—typically reached between 30 min and 1 h after IR. (B) Typical gel used to generate the PFGE results shown in A. DNA is stained with ethidium bromide. (C) Examples of γ-H2AX immunofluorescence at different times after exposure to IR (1 Gy). Cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with a phosphospecific primary anti-γ-H2AX antibody and an Alexa 568-labeled secondary antibody. Red shows γ-H2AX foci, blue nuclei stained with DAPI.
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Figure 5: Comparison of DSB repair kinetics as measured by pulsed-field gel electrophoresis (PFGE) with the development of γ-H2AX foci. (A) Plateau-phase A549 cells were exposed to 20 Gy or 1 Gy X-rays and analyzed by PFGE (157) or γ-H2AX immunofluorescence (158), respectively, at the indicated time points. PFGE results (squares) have been normalized to the signal measured at 0 h, while the γ-H2AX results (circles) have been normalized to the maximum number of foci scored. The number of foci per cell was quantified using the Leica Q-Win software with the help of a special routine developed for foci counting. Foci were counted on 3D picture stacks generated on a Leica SP5 confocal microscope. γ-H2AX results were normalized to the maximum number of foci scored per cell—typically reached between 30 min and 1 h after IR. (B) Typical gel used to generate the PFGE results shown in A. DNA is stained with ethidium bromide. (C) Examples of γ-H2AX immunofluorescence at different times after exposure to IR (1 Gy). Cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with a phosphospecific primary anti-γ-H2AX antibody and an Alexa 568-labeled secondary antibody. Red shows γ-H2AX foci, blue nuclei stained with DAPI.
Mentions: Although several DNA damage-associated histone modifications have been described, here we focus on the most conspicuous one that has been at the center of research activities during the last several years: the modification of the H2A variant, H2AX. H2AX is one of the most conserved H2A-variants (Table 1 and Figure 3), and is present in chromatin at levels that vary between 2 and 25% of the H2A pool, depending on the cell line and tissue examined. H2AX moved to the center of cellular responses to DNA damage after the discovery that it becomes locally phosphorylated, to generate γ-H2AX, in the vicinity of DSBs (24–26). The combination of phosphospecific antibodies that recognize the phosphorylated S-139 residue of γ-H2AX with immunofluorescence microscopy documented the local phosphorylation through the formation of distinct foci in the vicinity of DSBs and allowed monitoring of their induction and repair (Figure 5C). Thus, a wide spectrum of applications could be developed ranging from mechanistic studies in biology to useful applications in oncology and radiation protection (see below).Figure 3.

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