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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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H2AX in the context of chromatin. (A) Organization of DNA in chromatin. One hundred and forty-seven base pairs of DNA (red) are wrapped around a nucleosome (yellow) consisting of eight histone proteins (two H2A/H2B dimers and two H3/H4 dimers), thus forming the 11 nm nucleosome. The histones dimerize via the histone fold motif and four histone dimers form the nucleosome core. Nucleosomes are separated by linker DNA sections of 20–80 bp in length. The DNA wraps in 1.7 turns around the nucleosome forming 142 hydrogen bonds at the DNA histone interface. The histone tails protrude from the nucleosome core and can be modified, for instance by acetylation, phosphorylation or ubiquitinylation. Further condensation of chromatin, as in the 30 nm fiber, allows a 100-fold compaction of DNA (schematic representation; the actual organization of the nucleosomes in the 30 nm fiber is still under investigation). (B) All histone proteins share the highly conserved histone fold motif (displayed in color) containing the three alpha helices involved in nucleosome core organization. Alpha helical domains outside the histone fold domain are shown in gray. The structure on the right illustrates how two histone fold domains interact for dimer formation. (C) A model of the nucleosome core particle showing DNA interactions with core histones (redrawn in a modified form from Ref. (148)).The DNA entry and exit points are localized at the H2A/H2B dimer. The H2AX C-terminus, which is 14 amino acids longer than that of H2A, is drawn here (there are no structural data available and the schematic drawing is only for demonstration purposes) in black with a red arrow marking the phosphorylation site within the SQEY motif.
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Figure 1: H2AX in the context of chromatin. (A) Organization of DNA in chromatin. One hundred and forty-seven base pairs of DNA (red) are wrapped around a nucleosome (yellow) consisting of eight histone proteins (two H2A/H2B dimers and two H3/H4 dimers), thus forming the 11 nm nucleosome. The histones dimerize via the histone fold motif and four histone dimers form the nucleosome core. Nucleosomes are separated by linker DNA sections of 20–80 bp in length. The DNA wraps in 1.7 turns around the nucleosome forming 142 hydrogen bonds at the DNA histone interface. The histone tails protrude from the nucleosome core and can be modified, for instance by acetylation, phosphorylation or ubiquitinylation. Further condensation of chromatin, as in the 30 nm fiber, allows a 100-fold compaction of DNA (schematic representation; the actual organization of the nucleosomes in the 30 nm fiber is still under investigation). (B) All histone proteins share the highly conserved histone fold motif (displayed in color) containing the three alpha helices involved in nucleosome core organization. Alpha helical domains outside the histone fold domain are shown in gray. The structure on the right illustrates how two histone fold domains interact for dimer formation. (C) A model of the nucleosome core particle showing DNA interactions with core histones (redrawn in a modified form from Ref. (148)).The DNA entry and exit points are localized at the H2A/H2B dimer. The H2AX C-terminus, which is 14 amino acids longer than that of H2A, is drawn here (there are no structural data available and the schematic drawing is only for demonstration purposes) in black with a red arrow marking the phosphorylation site within the SQEY motif.

Mentions: The accommodation of ∼2 m of DNA in the ∼10 µm nucleus of a human cell is made possible through its organization into chromatin. The basic unit of chromatin, the nucleosome, consists of 147 base pairs of DNA wrapped in nearly two (1.7) left-handed superhelical turns around a ∼100 kDa octamer of histone proteins, each separated from the next one by a linker section of variable length (20–80 bp) (Figure 1A). As a result, the 6.4 × 109 bp of a human diploid cell are organized into over 30 million nucleosomes. Four small (100–135 aa), highly conserved histone proteins, H2A, H2B, H3 and H4, each sharing the histone-fold motif and present in two copies (Figure 1B), form the histone octamer and compact the DNA through the mediated wrapping by approximately 3-fold (1). For nucleosome assembly, DNA is first wrapped around the H3–H4 tetramer before the addition of two H2A–H2B dimers completes the core (2).Figure 1.


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)

H2AX in the context of chromatin. (A) Organization of DNA in chromatin. One hundred and forty-seven base pairs of DNA (red) are wrapped around a nucleosome (yellow) consisting of eight histone proteins (two H2A/H2B dimers and two H3/H4 dimers), thus forming the 11 nm nucleosome. The histones dimerize via the histone fold motif and four histone dimers form the nucleosome core. Nucleosomes are separated by linker DNA sections of 20–80 bp in length. The DNA wraps in 1.7 turns around the nucleosome forming 142 hydrogen bonds at the DNA histone interface. The histone tails protrude from the nucleosome core and can be modified, for instance by acetylation, phosphorylation or ubiquitinylation. Further condensation of chromatin, as in the 30 nm fiber, allows a 100-fold compaction of DNA (schematic representation; the actual organization of the nucleosomes in the 30 nm fiber is still under investigation). (B) All histone proteins share the highly conserved histone fold motif (displayed in color) containing the three alpha helices involved in nucleosome core organization. Alpha helical domains outside the histone fold domain are shown in gray. The structure on the right illustrates how two histone fold domains interact for dimer formation. (C) A model of the nucleosome core particle showing DNA interactions with core histones (redrawn in a modified form from Ref. (148)).The DNA entry and exit points are localized at the H2A/H2B dimer. The H2AX C-terminus, which is 14 amino acids longer than that of H2A, is drawn here (there are no structural data available and the schematic drawing is only for demonstration purposes) in black with a red arrow marking the phosphorylation site within the SQEY motif.
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Related In: Results  -  Collection

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Figure 1: H2AX in the context of chromatin. (A) Organization of DNA in chromatin. One hundred and forty-seven base pairs of DNA (red) are wrapped around a nucleosome (yellow) consisting of eight histone proteins (two H2A/H2B dimers and two H3/H4 dimers), thus forming the 11 nm nucleosome. The histones dimerize via the histone fold motif and four histone dimers form the nucleosome core. Nucleosomes are separated by linker DNA sections of 20–80 bp in length. The DNA wraps in 1.7 turns around the nucleosome forming 142 hydrogen bonds at the DNA histone interface. The histone tails protrude from the nucleosome core and can be modified, for instance by acetylation, phosphorylation or ubiquitinylation. Further condensation of chromatin, as in the 30 nm fiber, allows a 100-fold compaction of DNA (schematic representation; the actual organization of the nucleosomes in the 30 nm fiber is still under investigation). (B) All histone proteins share the highly conserved histone fold motif (displayed in color) containing the three alpha helices involved in nucleosome core organization. Alpha helical domains outside the histone fold domain are shown in gray. The structure on the right illustrates how two histone fold domains interact for dimer formation. (C) A model of the nucleosome core particle showing DNA interactions with core histones (redrawn in a modified form from Ref. (148)).The DNA entry and exit points are localized at the H2A/H2B dimer. The H2AX C-terminus, which is 14 amino acids longer than that of H2A, is drawn here (there are no structural data available and the schematic drawing is only for demonstration purposes) in black with a red arrow marking the phosphorylation site within the SQEY motif.
Mentions: The accommodation of ∼2 m of DNA in the ∼10 µm nucleus of a human cell is made possible through its organization into chromatin. The basic unit of chromatin, the nucleosome, consists of 147 base pairs of DNA wrapped in nearly two (1.7) left-handed superhelical turns around a ∼100 kDa octamer of histone proteins, each separated from the next one by a linker section of variable length (20–80 bp) (Figure 1A). As a result, the 6.4 × 109 bp of a human diploid cell are organized into over 30 million nucleosomes. Four small (100–135 aa), highly conserved histone proteins, H2A, H2B, H3 and H4, each sharing the histone-fold motif and present in two copies (Figure 1B), form the histone octamer and compact the DNA through the mediated wrapping by approximately 3-fold (1). For nucleosome assembly, DNA is first wrapped around the H3–H4 tetramer before the addition of two H2A–H2B dimers completes the core (2).Figure 1.

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