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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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Phosphorylation-dependent interaction between the MDC1 N terminus and the MRN complex. (A) Sequence alignment of the region in MDC1 that is essential for MRN foci. The conserved acidic motifs are highlighted by horizontal bars. Phosphorylated residues identified by in vivo phosphorylation site mapping are highlighted by arrowheads (Beausoleil et al., 2004; Olsen et al., 2006; Villen et al., 2007). (B, top) Representation of human MDC1 and the overlapping GST fragments. (bottom) Two fragments at the N terminus of MDC1 are phosphorylated by CK2 in vitro. Purified GST-MDC1 fragments were incubated with purified recombinant CK2 in the presence of radioactive ATP. Proteins were separated by SDS-PAGE, and dried gels were subjected to autoradiography. A Coomassie blue–stained gel of the purified GST fragments is shown on top of the autoradiograph. Note that fragment M-6 (PST repeat region) was not expressed in bacteria. (C) Purified GST-MDC1 fragments (M-1–5) were preincubated with and without recombinant CK2 in the presence of ATP. (D) Purified GST-MDC1 fragment M-3 was preincubated with CK2 either in the presence or absence of the CK2 inhibitor TBB. (C and D) The fragments were then used to pull down proteins from HeLa nuclear extract. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11.
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fig2: Phosphorylation-dependent interaction between the MDC1 N terminus and the MRN complex. (A) Sequence alignment of the region in MDC1 that is essential for MRN foci. The conserved acidic motifs are highlighted by horizontal bars. Phosphorylated residues identified by in vivo phosphorylation site mapping are highlighted by arrowheads (Beausoleil et al., 2004; Olsen et al., 2006; Villen et al., 2007). (B, top) Representation of human MDC1 and the overlapping GST fragments. (bottom) Two fragments at the N terminus of MDC1 are phosphorylated by CK2 in vitro. Purified GST-MDC1 fragments were incubated with purified recombinant CK2 in the presence of radioactive ATP. Proteins were separated by SDS-PAGE, and dried gels were subjected to autoradiography. A Coomassie blue–stained gel of the purified GST fragments is shown on top of the autoradiograph. Note that fragment M-6 (PST repeat region) was not expressed in bacteria. (C) Purified GST-MDC1 fragments (M-1–5) were preincubated with and without recombinant CK2 in the presence of ATP. (D) Purified GST-MDC1 fragment M-3 was preincubated with CK2 either in the presence or absence of the CK2 inhibitor TBB. (C and D) The fragments were then used to pull down proteins from HeLa nuclear extract. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11.

Mentions: Efficient accumulation of the MRN complex in foci at sites of DSBs is critically dependent on MDC1 (Goldberg et al., 2003; Lukas et al., 2004). To determine the region of MDC1 that mediates MRN foci formation, we transfected MDC1−/− mouse embryonic fibroblasts (MEFs; Lou et al., 2006) with a series of N-terminal deletion mutants of mouse MDC1 and assessed MRN accumulation by indirect immunofluorescence using an antibody specific for mouse NBS1 (Celeste et al., 2003). Consistent with published data (Lou et al., 2006), MDC1−/− MEFs were completely defective for NBS1 accumulation (unpublished data), but transient transfection of these cells with HA-tagged full-length mouse MDC1 readily restored NBS1 foci formation in response to 5 Gy of ionizing radiation (IR; Fig. 1 B, WT). Deletion of 153 and 295 N-terminal amino acids of MDC1 did not result in any detectable reduction of NBS1 foci (Fig. 1 B, ΔN1 and ΔN2), but deletion of 452 and 645 amino acids led to a complete loss of NBS1 accumulation (Fig. 1 B, ΔN3 and ΔN4). This indicated that the region in MDC1 essential for mediating NBS1 accumulation is located somewhere between amino acids 295 and 452. Indeed, internal deletion of this 157–amino acid region completely abolished the ability of MDC1 to mediate NBS1 foci formation (Fig. 1 B, ΔI1). Preliminary sequence analysis of this region revealed a significant abundance of acidic amino acids as compared with other regions of MDC1 (Fig. 2 A).


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)

Phosphorylation-dependent interaction between the MDC1 N terminus and the MRN complex. (A) Sequence alignment of the region in MDC1 that is essential for MRN foci. The conserved acidic motifs are highlighted by horizontal bars. Phosphorylated residues identified by in vivo phosphorylation site mapping are highlighted by arrowheads (Beausoleil et al., 2004; Olsen et al., 2006; Villen et al., 2007). (B, top) Representation of human MDC1 and the overlapping GST fragments. (bottom) Two fragments at the N terminus of MDC1 are phosphorylated by CK2 in vitro. Purified GST-MDC1 fragments were incubated with purified recombinant CK2 in the presence of radioactive ATP. Proteins were separated by SDS-PAGE, and dried gels were subjected to autoradiography. A Coomassie blue–stained gel of the purified GST fragments is shown on top of the autoradiograph. Note that fragment M-6 (PST repeat region) was not expressed in bacteria. (C) Purified GST-MDC1 fragments (M-1–5) were preincubated with and without recombinant CK2 in the presence of ATP. (D) Purified GST-MDC1 fragment M-3 was preincubated with CK2 either in the presence or absence of the CK2 inhibitor TBB. (C and D) The fragments were then used to pull down proteins from HeLa nuclear extract. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2315671&req=5

fig2: Phosphorylation-dependent interaction between the MDC1 N terminus and the MRN complex. (A) Sequence alignment of the region in MDC1 that is essential for MRN foci. The conserved acidic motifs are highlighted by horizontal bars. Phosphorylated residues identified by in vivo phosphorylation site mapping are highlighted by arrowheads (Beausoleil et al., 2004; Olsen et al., 2006; Villen et al., 2007). (B, top) Representation of human MDC1 and the overlapping GST fragments. (bottom) Two fragments at the N terminus of MDC1 are phosphorylated by CK2 in vitro. Purified GST-MDC1 fragments were incubated with purified recombinant CK2 in the presence of radioactive ATP. Proteins were separated by SDS-PAGE, and dried gels were subjected to autoradiography. A Coomassie blue–stained gel of the purified GST fragments is shown on top of the autoradiograph. Note that fragment M-6 (PST repeat region) was not expressed in bacteria. (C) Purified GST-MDC1 fragments (M-1–5) were preincubated with and without recombinant CK2 in the presence of ATP. (D) Purified GST-MDC1 fragment M-3 was preincubated with CK2 either in the presence or absence of the CK2 inhibitor TBB. (C and D) The fragments were then used to pull down proteins from HeLa nuclear extract. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11.
Mentions: Efficient accumulation of the MRN complex in foci at sites of DSBs is critically dependent on MDC1 (Goldberg et al., 2003; Lukas et al., 2004). To determine the region of MDC1 that mediates MRN foci formation, we transfected MDC1−/− mouse embryonic fibroblasts (MEFs; Lou et al., 2006) with a series of N-terminal deletion mutants of mouse MDC1 and assessed MRN accumulation by indirect immunofluorescence using an antibody specific for mouse NBS1 (Celeste et al., 2003). Consistent with published data (Lou et al., 2006), MDC1−/− MEFs were completely defective for NBS1 accumulation (unpublished data), but transient transfection of these cells with HA-tagged full-length mouse MDC1 readily restored NBS1 foci formation in response to 5 Gy of ionizing radiation (IR; Fig. 1 B, WT). Deletion of 153 and 295 N-terminal amino acids of MDC1 did not result in any detectable reduction of NBS1 foci (Fig. 1 B, ΔN1 and ΔN2), but deletion of 452 and 645 amino acids led to a complete loss of NBS1 accumulation (Fig. 1 B, ΔN3 and ΔN4). This indicated that the region in MDC1 essential for mediating NBS1 accumulation is located somewhere between amino acids 295 and 452. Indeed, internal deletion of this 157–amino acid region completely abolished the ability of MDC1 to mediate NBS1 foci formation (Fig. 1 B, ΔI1). Preliminary sequence analysis of this region revealed a significant abundance of acidic amino acids as compared with other regions of MDC1 (Fig. 2 A).

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