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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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γ-H2AX is a specific and efficient coordinator of DDR signaling. Following the initial phosphorylation of H2AX by ATM, or DNA-PK, a nucleation reaction is initiated starting with the recruitment of MDC1 and continuing with that of the MRN complex to further activate ATM. This generates a feedback loop that leads to further phosphorylation of H2AX and the chromatin modifications required for the recruitment of 53BP1. The activation cascade culminates with the recruitment of RNF8 to phosphorylated MDC1 and the polyubiquitinylation of H2AX to recruit BRCA1/BARD1 (see text for details).
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Figure 4: γ-H2AX is a specific and efficient coordinator of DDR signaling. Following the initial phosphorylation of H2AX by ATM, or DNA-PK, a nucleation reaction is initiated starting with the recruitment of MDC1 and continuing with that of the MRN complex to further activate ATM. This generates a feedback loop that leads to further phosphorylation of H2AX and the chromatin modifications required for the recruitment of 53BP1. The activation cascade culminates with the recruitment of RNF8 to phosphorylated MDC1 and the polyubiquitinylation of H2AX to recruit BRCA1/BARD1 (see text for details).

Mentions: With the interaction between MDC1 and γ-H2AX, the site of the DSB is prepared for signaling and repair (Figure 4). This is because there is evidence that MDC1 also directly interacts in a highly dynamic manner (71) with NBS1 (74), which in the form of MRN complex is required for the activation of ATM (44,75). This interaction is mediated through phosphorylation of MDC1 by casein kinase 2 (CK2) that promotes phosphorylation-dependent interactions with NBS1 through its closely apposed FHA and twin BRCA domains (76). In this way, a positive feed-back loop is generated that extends H2AX phosphorylation to the Mbp regions described above, but how is the initial H2AX phosphorylation event induced? Based on the extremely high affinity of Ku for DNA ends, a likely scenario is phosphorylation through DNA-PK (Figure 4). However, if ATM can also be directly activated by DSBs, it could also function as the initial H2AX kinase—if mechanisms are in place to facilitate accessibility of the kinase to the DSB. In agreement with the model of a positive feedback loop, loss of MDC1 expression, or reduction of its cellular levels by siRNA treatment, reduces H2AX phosphorylation in response to IR probably as a result of a defect in recruiting ATM (65,72,77).Figure 4.


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)

γ-H2AX is a specific and efficient coordinator of DDR signaling. Following the initial phosphorylation of H2AX by ATM, or DNA-PK, a nucleation reaction is initiated starting with the recruitment of MDC1 and continuing with that of the MRN complex to further activate ATM. This generates a feedback loop that leads to further phosphorylation of H2AX and the chromatin modifications required for the recruitment of 53BP1. The activation cascade culminates with the recruitment of RNF8 to phosphorylated MDC1 and the polyubiquitinylation of H2AX to recruit BRCA1/BARD1 (see text for details).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553572&req=5

Figure 4: γ-H2AX is a specific and efficient coordinator of DDR signaling. Following the initial phosphorylation of H2AX by ATM, or DNA-PK, a nucleation reaction is initiated starting with the recruitment of MDC1 and continuing with that of the MRN complex to further activate ATM. This generates a feedback loop that leads to further phosphorylation of H2AX and the chromatin modifications required for the recruitment of 53BP1. The activation cascade culminates with the recruitment of RNF8 to phosphorylated MDC1 and the polyubiquitinylation of H2AX to recruit BRCA1/BARD1 (see text for details).
Mentions: With the interaction between MDC1 and γ-H2AX, the site of the DSB is prepared for signaling and repair (Figure 4). This is because there is evidence that MDC1 also directly interacts in a highly dynamic manner (71) with NBS1 (74), which in the form of MRN complex is required for the activation of ATM (44,75). This interaction is mediated through phosphorylation of MDC1 by casein kinase 2 (CK2) that promotes phosphorylation-dependent interactions with NBS1 through its closely apposed FHA and twin BRCA domains (76). In this way, a positive feed-back loop is generated that extends H2AX phosphorylation to the Mbp regions described above, but how is the initial H2AX phosphorylation event induced? Based on the extremely high affinity of Ku for DNA ends, a likely scenario is phosphorylation through DNA-PK (Figure 4). However, if ATM can also be directly activated by DSBs, it could also function as the initial H2AX kinase—if mechanisms are in place to facilitate accessibility of the kinase to the DSB. In agreement with the model of a positive feedback loop, loss of MDC1 expression, or reduction of its cellular levels by siRNA treatment, reduces H2AX phosphorylation in response to IR probably as a result of a defect in recruiting ATM (65,72,77).Figure 4.

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