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Constitutive phosphorylation of MDC1 physically links the MRE11-RAD50-NBS1 complex to damaged chromatin.

Spycher C, Miller ES, Townsend K, Pavic L, Morrice NA, Janscak P, Stewart GS, Stucki M - J. Cell Biol. (2008)

Bottom Line: We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner.Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo.Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, 8057 Zürich, Switzerland.

ABSTRACT
The MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.

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CK2 is essential for the interaction between MDC1 and NBS1 and for the accumulation of NBS1 at sites of DSBs in vivo. (A) Down-regulation of CK2α and CK2α′ by siRNA triggers a prolonged DDR. 72 h after transfection with siRNA duplexes, cells were irradiated and harvested at the indicated time points. Extracts were prepared and resolved by SDS-PAGE followed by immunoblotting. The blots were probed with the indicated antibodies. (B) Down-regulation of CK2α and CK2α′ by siRNA abrogates NBS1 accumulation at sites of DSBs. 72 h after transfection with siRNA duplexes, cells were irradiated, fixed with methanol, and stained with antibodies against γ-H2AX and NBS1. (C) Down-regulation of CK2α by siRNA disrupts the interaction between MDC1 and NBS1 in vivo. 72 h after transfection with siRNA duplexes, cells were lysed, and immunoprecipitation was performed using the indicated antibodies. Proteins were separated by SDS-PAGE followed by immunoblotting. The blots were probed with antibodies against MDC1, NBS1, and CK2α. Black lines indicate that intervening lanes have been spliced out. (D) MDC1 is associated with CK2 in vivo. HeLa cell extracts were used to immunoprecipitate proteins with the indicated antibodies. The immunocomplexes were separated by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against CK2α and CK2α′. Bar, 10 μm.
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fig5: CK2 is essential for the interaction between MDC1 and NBS1 and for the accumulation of NBS1 at sites of DSBs in vivo. (A) Down-regulation of CK2α and CK2α′ by siRNA triggers a prolonged DDR. 72 h after transfection with siRNA duplexes, cells were irradiated and harvested at the indicated time points. Extracts were prepared and resolved by SDS-PAGE followed by immunoblotting. The blots were probed with the indicated antibodies. (B) Down-regulation of CK2α and CK2α′ by siRNA abrogates NBS1 accumulation at sites of DSBs. 72 h after transfection with siRNA duplexes, cells were irradiated, fixed with methanol, and stained with antibodies against γ-H2AX and NBS1. (C) Down-regulation of CK2α by siRNA disrupts the interaction between MDC1 and NBS1 in vivo. 72 h after transfection with siRNA duplexes, cells were lysed, and immunoprecipitation was performed using the indicated antibodies. Proteins were separated by SDS-PAGE followed by immunoblotting. The blots were probed with antibodies against MDC1, NBS1, and CK2α. Black lines indicate that intervening lanes have been spliced out. (D) MDC1 is associated with CK2 in vivo. HeLa cell extracts were used to immunoprecipitate proteins with the indicated antibodies. The immunocomplexes were separated by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against CK2α and CK2α′. Bar, 10 μm.

Mentions: To test the latter possibility, we took an siRNA approach to down-regulate the two catalytic subunits of CK2 (CK2α and CK2α′) in U2OS cells. 72 h after siRNA transfection, CK2α and CK2α′ expression reached background levels (Fig. 5 A). At the same time, we observed massive cell death and severe mitotic defects, corroborating the essential role of CK2 in the cellular metabolism and life cycle. Interestingly, we also observed a prolonged phosphorylation of ATM substrates in response to IR (Fig. 5 A), suggesting that down-regulation of CK2 by siRNA may cause a repair defect.


Constitutive phosphorylation of MDC1 physically links the MRE11-RAD50-NBS1 complex to damaged chromatin.

Spycher C, Miller ES, Townsend K, Pavic L, Morrice NA, Janscak P, Stewart GS, Stucki M - J. Cell Biol. (2008)

CK2 is essential for the interaction between MDC1 and NBS1 and for the accumulation of NBS1 at sites of DSBs in vivo. (A) Down-regulation of CK2α and CK2α′ by siRNA triggers a prolonged DDR. 72 h after transfection with siRNA duplexes, cells were irradiated and harvested at the indicated time points. Extracts were prepared and resolved by SDS-PAGE followed by immunoblotting. The blots were probed with the indicated antibodies. (B) Down-regulation of CK2α and CK2α′ by siRNA abrogates NBS1 accumulation at sites of DSBs. 72 h after transfection with siRNA duplexes, cells were irradiated, fixed with methanol, and stained with antibodies against γ-H2AX and NBS1. (C) Down-regulation of CK2α by siRNA disrupts the interaction between MDC1 and NBS1 in vivo. 72 h after transfection with siRNA duplexes, cells were lysed, and immunoprecipitation was performed using the indicated antibodies. Proteins were separated by SDS-PAGE followed by immunoblotting. The blots were probed with antibodies against MDC1, NBS1, and CK2α. Black lines indicate that intervening lanes have been spliced out. (D) MDC1 is associated with CK2 in vivo. HeLa cell extracts were used to immunoprecipitate proteins with the indicated antibodies. The immunocomplexes were separated by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against CK2α and CK2α′. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2315671&req=5

fig5: CK2 is essential for the interaction between MDC1 and NBS1 and for the accumulation of NBS1 at sites of DSBs in vivo. (A) Down-regulation of CK2α and CK2α′ by siRNA triggers a prolonged DDR. 72 h after transfection with siRNA duplexes, cells were irradiated and harvested at the indicated time points. Extracts were prepared and resolved by SDS-PAGE followed by immunoblotting. The blots were probed with the indicated antibodies. (B) Down-regulation of CK2α and CK2α′ by siRNA abrogates NBS1 accumulation at sites of DSBs. 72 h after transfection with siRNA duplexes, cells were irradiated, fixed with methanol, and stained with antibodies against γ-H2AX and NBS1. (C) Down-regulation of CK2α by siRNA disrupts the interaction between MDC1 and NBS1 in vivo. 72 h after transfection with siRNA duplexes, cells were lysed, and immunoprecipitation was performed using the indicated antibodies. Proteins were separated by SDS-PAGE followed by immunoblotting. The blots were probed with antibodies against MDC1, NBS1, and CK2α. Black lines indicate that intervening lanes have been spliced out. (D) MDC1 is associated with CK2 in vivo. HeLa cell extracts were used to immunoprecipitate proteins with the indicated antibodies. The immunocomplexes were separated by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against CK2α and CK2α′. Bar, 10 μm.
Mentions: To test the latter possibility, we took an siRNA approach to down-regulate the two catalytic subunits of CK2 (CK2α and CK2α′) in U2OS cells. 72 h after siRNA transfection, CK2α and CK2α′ expression reached background levels (Fig. 5 A). At the same time, we observed massive cell death and severe mitotic defects, corroborating the essential role of CK2 in the cellular metabolism and life cycle. Interestingly, we also observed a prolonged phosphorylation of ATM substrates in response to IR (Fig. 5 A), suggesting that down-regulation of CK2 by siRNA may cause a repair defect.

Bottom Line: We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner.Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo.Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, 8057 Zürich, Switzerland.

ABSTRACT
The MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.

Show MeSH
Related in: MedlinePlus