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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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Disruption of the MDC1–NBS1 interaction triggers a partial G2/M checkpoint defect. (A) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were irradiated, fixed with methanol, and stained with antibodies against MDC1 and NBS1. (B) Whole cell extracts of NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type (WT) NBS1 and R28A mutant NBS1 were resolved by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against NBS1 and tubulin. (C) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were left untreated or irradiated with 1 Gy and 3 Gy, respectively. Cells were harvested 1 h after irradiation, fixed with methanol, and stained with an antibody against phosphorylated H3 (P-H3) and propidium iodine. The percentage of phosphorylated H3–positive cells was determined by FACS analysis. Error bars represent SD. Bar, 10 μm.
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fig6: Disruption of the MDC1–NBS1 interaction triggers a partial G2/M checkpoint defect. (A) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were irradiated, fixed with methanol, and stained with antibodies against MDC1 and NBS1. (B) Whole cell extracts of NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type (WT) NBS1 and R28A mutant NBS1 were resolved by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against NBS1 and tubulin. (C) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were left untreated or irradiated with 1 Gy and 3 Gy, respectively. Cells were harvested 1 h after irradiation, fixed with methanol, and stained with an antibody against phosphorylated H3 (P-H3) and propidium iodine. The percentage of phosphorylated H3–positive cells was determined by FACS analysis. Error bars represent SD. Bar, 10 μm.

Mentions: As shown in Fig. 3 D, the R28A mutation in the NBS1 FHA domain triggers a severe defect in the association of NBS1 with the CK2-phosphorylated MDC1 SDT region in vitro (Fig. 3 D). Consistent with previous studies (Cerosaletti and Concannon, 2003; Lee et al., 2003; Horejsi et al., 2004), we also observed that this mutant is unable to accumulate in foci in stably transduced NBS fibroblasts, whereas MDC1 accumulation is not affected (Fig. 6 A). It was previously shown that the R28A mutant was not capable of rescuing the radiation sensitivity phenotype of NBS cells, whereas it did fully rescue the intra–S-phase checkpoint defect, at least at higher doses of irradiation (Lee et al., 2003). In contrast, primary B cells derived from a humanized mouse model in which another key amino acid at the phosphopeptide recognition interface of the NBS1 FHA domain had been mutated to Ala (H45A) showed partial G2/M and intra–S-phase checkpoint defects specifically at lower doses of irradiation (Difilippantonio et al., 2007). To test whether the R28A mutation causes a similar checkpoint defect, we measured alterations in the mitotic index in response to low (sublethal) doses of irradiation (1–3 Gy) in NBS fibroblasts stably transduced with full-length wild-type and R28A NBS1. Consistent with previous findings (Falck et al., 2005), NBS-iLB1 fibroblasts displayed a clear G2/M checkpoint defect in this dose range (Fig. 6 C). Stable transduction with wild-type NBS1 fully rescued the G2/M checkpoint arrest in response to 1–3 Gy of IR. However, stable transduction with R28A mutant NBS1 only partially restored the G2/M checkpoint, which is similar to the situation in the mouse B cells expressing the H45A mutant (Fig. 6 C; Difilippantonio et al., 2007). Notably, this checkpoint defect was not caused by lower expression levels of the mutant transgene as compared with the wild type (Fig. 6 B). Collectively, these results suggest that the constitutive CK2-dependent association of the MRN complex with MDC1 plays an important role in eliciting a full cell cycle checkpoint arrest.


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)

Disruption of the MDC1–NBS1 interaction triggers a partial G2/M checkpoint defect. (A) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were irradiated, fixed with methanol, and stained with antibodies against MDC1 and NBS1. (B) Whole cell extracts of NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type (WT) NBS1 and R28A mutant NBS1 were resolved by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against NBS1 and tubulin. (C) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were left untreated or irradiated with 1 Gy and 3 Gy, respectively. Cells were harvested 1 h after irradiation, fixed with methanol, and stained with an antibody against phosphorylated H3 (P-H3) and propidium iodine. The percentage of phosphorylated H3–positive cells was determined by FACS analysis. Error bars represent SD. Bar, 10 μm.
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Related In: Results  -  Collection

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fig6: Disruption of the MDC1–NBS1 interaction triggers a partial G2/M checkpoint defect. (A) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were irradiated, fixed with methanol, and stained with antibodies against MDC1 and NBS1. (B) Whole cell extracts of NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type (WT) NBS1 and R28A mutant NBS1 were resolved by SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against NBS1 and tubulin. (C) NBS-iLB1 fibroblasts and NBS-iLB1 fibroblasts stably transduced with wild-type NBS1 and R28A mutant NBS1 were left untreated or irradiated with 1 Gy and 3 Gy, respectively. Cells were harvested 1 h after irradiation, fixed with methanol, and stained with an antibody against phosphorylated H3 (P-H3) and propidium iodine. The percentage of phosphorylated H3–positive cells was determined by FACS analysis. Error bars represent SD. Bar, 10 μm.
Mentions: As shown in Fig. 3 D, the R28A mutation in the NBS1 FHA domain triggers a severe defect in the association of NBS1 with the CK2-phosphorylated MDC1 SDT region in vitro (Fig. 3 D). Consistent with previous studies (Cerosaletti and Concannon, 2003; Lee et al., 2003; Horejsi et al., 2004), we also observed that this mutant is unable to accumulate in foci in stably transduced NBS fibroblasts, whereas MDC1 accumulation is not affected (Fig. 6 A). It was previously shown that the R28A mutant was not capable of rescuing the radiation sensitivity phenotype of NBS cells, whereas it did fully rescue the intra–S-phase checkpoint defect, at least at higher doses of irradiation (Lee et al., 2003). In contrast, primary B cells derived from a humanized mouse model in which another key amino acid at the phosphopeptide recognition interface of the NBS1 FHA domain had been mutated to Ala (H45A) showed partial G2/M and intra–S-phase checkpoint defects specifically at lower doses of irradiation (Difilippantonio et al., 2007). To test whether the R28A mutation causes a similar checkpoint defect, we measured alterations in the mitotic index in response to low (sublethal) doses of irradiation (1–3 Gy) in NBS fibroblasts stably transduced with full-length wild-type and R28A NBS1. Consistent with previous findings (Falck et al., 2005), NBS-iLB1 fibroblasts displayed a clear G2/M checkpoint defect in this dose range (Fig. 6 C). Stable transduction with wild-type NBS1 fully rescued the G2/M checkpoint arrest in response to 1–3 Gy of IR. However, stable transduction with R28A mutant NBS1 only partially restored the G2/M checkpoint, which is similar to the situation in the mouse B cells expressing the H45A mutant (Fig. 6 C; Difilippantonio et al., 2007). Notably, this checkpoint defect was not caused by lower expression levels of the mutant transgene as compared with the wild type (Fig. 6 B). Collectively, these results suggest that the constitutive CK2-dependent association of the MRN complex with MDC1 plays an important role in eliciting a full cell cycle checkpoint arrest.

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