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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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Related in: MedlinePlus

DSB repair pathways. (A) Homologous recombination repair (HRR). After the initial sensing of the DSB by MRN and the activation of ATM, H2AX is phosphorylated, which in turn elicits a sequence of signaling events thought to ultimately cause the activation of nucleases such as Mre11 and CtIP to process the DNA ends and generate ssDNA with 3′ overhangs. ssDNA is bound by RPA, which is subsequently exchanged by Rad51 and Rad51 paralogs. This exchange is facilitated by Rad52, Rad54 and BRCA2. The Rad51-decorated DNA fiber initiates strand invasion into an intact homologous DNA molecule that leads to the formation of a Holiday junction. The DNA sequence around the DSB is copied by DNA synthesis associated with branch migration, and the process is completed by resolution of the Holiday junction. HRR is a templated repair process and is therefore error free (15–17). (B) DNA-PK-dependent nonhomologous end joining (D-NHEJ). DNA ends are recognized by Ku, which recruits, after processing by Tdp1 or PNKP, DNA-PKcs. Upon end-binding, DNA-PKcs is activated and phosphorylates itself and possibly also other proteins (like H2AX on an adjacent nucleosome). Phosphorylated DNA-PKcs is thought to be released from the DNA end, which allows the DNA ligase IV/XRCC4/XLF complex to mediate end-ligation possibly with the help of a DNA polymerase that catalyzes gap filling (19,149,150). (C) Back up pathway of nonhomologous end joining (B-NHEJ). There is evidence that cells of higher eukaryotes with defects in D-NHEJ rejoin the majority of DSBs using an alternative repair pathway that is not utilizing any of the HRR-associated activities (19). This pathway is therefore termed backup NHEJ (B-NHEJ). Although details of this pathway remain to be elucidated, there is evidence that it utilizes the PARP-1/DNA Ligase III/XRCC1 repair module known to be involved in the repair of SSB and base damages (151–155), and that its function is facilitated by the linker histone H1 (156). (D) Single strand annealing (SSA). This repair pathway shares features of HRR and NHEJ, and is best described in yeast (17). After the initial sensing of the DSBs and processing of the ends by an exonuclease, possibly the MRN complex, the generated ssDNA tails are loaded with RPA. Ends are resected until homologous regions are exposed on the two DNA strands and pairing of these regions is facilitated by Rad52. After appropriate gap filling and removal of the overhangs by the ERCC1/XPF nuclease, a ligation step restores DNA integrity. The repair pathways described in B, C and D are associated with loss (and sometimes gain) of DNA material and are by nature error prone.
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Figure 2: DSB repair pathways. (A) Homologous recombination repair (HRR). After the initial sensing of the DSB by MRN and the activation of ATM, H2AX is phosphorylated, which in turn elicits a sequence of signaling events thought to ultimately cause the activation of nucleases such as Mre11 and CtIP to process the DNA ends and generate ssDNA with 3′ overhangs. ssDNA is bound by RPA, which is subsequently exchanged by Rad51 and Rad51 paralogs. This exchange is facilitated by Rad52, Rad54 and BRCA2. The Rad51-decorated DNA fiber initiates strand invasion into an intact homologous DNA molecule that leads to the formation of a Holiday junction. The DNA sequence around the DSB is copied by DNA synthesis associated with branch migration, and the process is completed by resolution of the Holiday junction. HRR is a templated repair process and is therefore error free (15–17). (B) DNA-PK-dependent nonhomologous end joining (D-NHEJ). DNA ends are recognized by Ku, which recruits, after processing by Tdp1 or PNKP, DNA-PKcs. Upon end-binding, DNA-PKcs is activated and phosphorylates itself and possibly also other proteins (like H2AX on an adjacent nucleosome). Phosphorylated DNA-PKcs is thought to be released from the DNA end, which allows the DNA ligase IV/XRCC4/XLF complex to mediate end-ligation possibly with the help of a DNA polymerase that catalyzes gap filling (19,149,150). (C) Back up pathway of nonhomologous end joining (B-NHEJ). There is evidence that cells of higher eukaryotes with defects in D-NHEJ rejoin the majority of DSBs using an alternative repair pathway that is not utilizing any of the HRR-associated activities (19). This pathway is therefore termed backup NHEJ (B-NHEJ). Although details of this pathway remain to be elucidated, there is evidence that it utilizes the PARP-1/DNA Ligase III/XRCC1 repair module known to be involved in the repair of SSB and base damages (151–155), and that its function is facilitated by the linker histone H1 (156). (D) Single strand annealing (SSA). This repair pathway shares features of HRR and NHEJ, and is best described in yeast (17). After the initial sensing of the DSBs and processing of the ends by an exonuclease, possibly the MRN complex, the generated ssDNA tails are loaded with RPA. Ends are resected until homologous regions are exposed on the two DNA strands and pairing of these regions is facilitated by Rad52. After appropriate gap filling and removal of the overhangs by the ERCC1/XPF nuclease, a ligation step restores DNA integrity. The repair pathways described in B, C and D are associated with loss (and sometimes gain) of DNA material and are by nature error prone.

Mentions: Modification of chromatin structure will be important for all pathways utilized by the cell to repair DSBs. Particularly, homologous recombination repair (HRR), the only error-free pathway, will require extensive chromatin modification to facilitate its essential steps: initial processing of DNA ends, search for homology, invasion into the intact homologous double helix, formation of a Holiday junction, DNA synthesis with the associated branch migration and final resolution of the Holiday junction (Figure 2A) (15–17). Also, error-prone pathways utilized in the repair of DSBs have obvious requirements for chromatin modification. Thus, the search of homology required during single strand annealing (SSA) (Figure 2D) (17), as well as the end processing step, will be facilitated by chromatin modifications. Finally, the homology-independent removal of DSBs by nonhomologous end joining (NHEJ) (18) may require chromatin modification for efficient recognition and ligation (Figure 2B), although this modification will be more limited than that required for the other repair pathways and may depend upon the actual structure of chromatin in the vicinity of the break.Figure 2.


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)

DSB repair pathways. (A) Homologous recombination repair (HRR). After the initial sensing of the DSB by MRN and the activation of ATM, H2AX is phosphorylated, which in turn elicits a sequence of signaling events thought to ultimately cause the activation of nucleases such as Mre11 and CtIP to process the DNA ends and generate ssDNA with 3′ overhangs. ssDNA is bound by RPA, which is subsequently exchanged by Rad51 and Rad51 paralogs. This exchange is facilitated by Rad52, Rad54 and BRCA2. The Rad51-decorated DNA fiber initiates strand invasion into an intact homologous DNA molecule that leads to the formation of a Holiday junction. The DNA sequence around the DSB is copied by DNA synthesis associated with branch migration, and the process is completed by resolution of the Holiday junction. HRR is a templated repair process and is therefore error free (15–17). (B) DNA-PK-dependent nonhomologous end joining (D-NHEJ). DNA ends are recognized by Ku, which recruits, after processing by Tdp1 or PNKP, DNA-PKcs. Upon end-binding, DNA-PKcs is activated and phosphorylates itself and possibly also other proteins (like H2AX on an adjacent nucleosome). Phosphorylated DNA-PKcs is thought to be released from the DNA end, which allows the DNA ligase IV/XRCC4/XLF complex to mediate end-ligation possibly with the help of a DNA polymerase that catalyzes gap filling (19,149,150). (C) Back up pathway of nonhomologous end joining (B-NHEJ). There is evidence that cells of higher eukaryotes with defects in D-NHEJ rejoin the majority of DSBs using an alternative repair pathway that is not utilizing any of the HRR-associated activities (19). This pathway is therefore termed backup NHEJ (B-NHEJ). Although details of this pathway remain to be elucidated, there is evidence that it utilizes the PARP-1/DNA Ligase III/XRCC1 repair module known to be involved in the repair of SSB and base damages (151–155), and that its function is facilitated by the linker histone H1 (156). (D) Single strand annealing (SSA). This repair pathway shares features of HRR and NHEJ, and is best described in yeast (17). After the initial sensing of the DSBs and processing of the ends by an exonuclease, possibly the MRN complex, the generated ssDNA tails are loaded with RPA. Ends are resected until homologous regions are exposed on the two DNA strands and pairing of these regions is facilitated by Rad52. After appropriate gap filling and removal of the overhangs by the ERCC1/XPF nuclease, a ligation step restores DNA integrity. The repair pathways described in B, C and D are associated with loss (and sometimes gain) of DNA material and are by nature error prone.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: DSB repair pathways. (A) Homologous recombination repair (HRR). After the initial sensing of the DSB by MRN and the activation of ATM, H2AX is phosphorylated, which in turn elicits a sequence of signaling events thought to ultimately cause the activation of nucleases such as Mre11 and CtIP to process the DNA ends and generate ssDNA with 3′ overhangs. ssDNA is bound by RPA, which is subsequently exchanged by Rad51 and Rad51 paralogs. This exchange is facilitated by Rad52, Rad54 and BRCA2. The Rad51-decorated DNA fiber initiates strand invasion into an intact homologous DNA molecule that leads to the formation of a Holiday junction. The DNA sequence around the DSB is copied by DNA synthesis associated with branch migration, and the process is completed by resolution of the Holiday junction. HRR is a templated repair process and is therefore error free (15–17). (B) DNA-PK-dependent nonhomologous end joining (D-NHEJ). DNA ends are recognized by Ku, which recruits, after processing by Tdp1 or PNKP, DNA-PKcs. Upon end-binding, DNA-PKcs is activated and phosphorylates itself and possibly also other proteins (like H2AX on an adjacent nucleosome). Phosphorylated DNA-PKcs is thought to be released from the DNA end, which allows the DNA ligase IV/XRCC4/XLF complex to mediate end-ligation possibly with the help of a DNA polymerase that catalyzes gap filling (19,149,150). (C) Back up pathway of nonhomologous end joining (B-NHEJ). There is evidence that cells of higher eukaryotes with defects in D-NHEJ rejoin the majority of DSBs using an alternative repair pathway that is not utilizing any of the HRR-associated activities (19). This pathway is therefore termed backup NHEJ (B-NHEJ). Although details of this pathway remain to be elucidated, there is evidence that it utilizes the PARP-1/DNA Ligase III/XRCC1 repair module known to be involved in the repair of SSB and base damages (151–155), and that its function is facilitated by the linker histone H1 (156). (D) Single strand annealing (SSA). This repair pathway shares features of HRR and NHEJ, and is best described in yeast (17). After the initial sensing of the DSBs and processing of the ends by an exonuclease, possibly the MRN complex, the generated ssDNA tails are loaded with RPA. Ends are resected until homologous regions are exposed on the two DNA strands and pairing of these regions is facilitated by Rad52. After appropriate gap filling and removal of the overhangs by the ERCC1/XPF nuclease, a ligation step restores DNA integrity. The repair pathways described in B, C and D are associated with loss (and sometimes gain) of DNA material and are by nature error prone.
Mentions: Modification of chromatin structure will be important for all pathways utilized by the cell to repair DSBs. Particularly, homologous recombination repair (HRR), the only error-free pathway, will require extensive chromatin modification to facilitate its essential steps: initial processing of DNA ends, search for homology, invasion into the intact homologous double helix, formation of a Holiday junction, DNA synthesis with the associated branch migration and final resolution of the Holiday junction (Figure 2A) (15–17). Also, error-prone pathways utilized in the repair of DSBs have obvious requirements for chromatin modification. Thus, the search of homology required during single strand annealing (SSA) (Figure 2D) (17), as well as the end processing step, will be facilitated by chromatin modifications. Finally, the homology-independent removal of DSBs by nonhomologous end joining (NHEJ) (18) may require chromatin modification for efficient recognition and ligation (Figure 2B), although this modification will be more limited than that required for the other repair pathways and may depend upon the actual structure of chromatin in the vicinity of the break.Figure 2.

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