Limits...
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.

Show MeSH

Related in: MedlinePlus

Direct interaction between the phosphorylated MDC1 N terminus and the MRN complex is mediated by the NBS1 FHA domain. (A) MRN proteins were purified as described in Materials and methods. Proteins were separated on SDS-polyacrylamide gels and stained with silver. MR, MRE11–RAD50 subcomplex; N, partially purified NBS1. (B and C) Purified GST-MDC1 fragment M-3 comprising part of the SDT region was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex (B), purified MR subcomplex (C, top), and partially purified NBS1 (C, bottom) followed by GST pull-down analysis. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11. (D, top) Schematic representation of NBS1 with its functional domains. (bottom) Purified GST-MDC1 fragment M-3 was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex where the NBS1 subunit was either wild type or contained a point mutation in the FHA domain (R28A). Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with a polyclonal antibody against NBS1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2315671&req=5

fig3: Direct interaction between the phosphorylated MDC1 N terminus and the MRN complex is mediated by the NBS1 FHA domain. (A) MRN proteins were purified as described in Materials and methods. Proteins were separated on SDS-polyacrylamide gels and stained with silver. MR, MRE11–RAD50 subcomplex; N, partially purified NBS1. (B and C) Purified GST-MDC1 fragment M-3 comprising part of the SDT region was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex (B), purified MR subcomplex (C, top), and partially purified NBS1 (C, bottom) followed by GST pull-down analysis. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11. (D, top) Schematic representation of NBS1 with its functional domains. (bottom) Purified GST-MDC1 fragment M-3 was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex where the NBS1 subunit was either wild type or contained a point mutation in the FHA domain (R28A). Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with a polyclonal antibody against NBS1.

Mentions: To determine whether the MRN complex interacts directly with the phosphorylated MDC1 N terminus, we coexpressed all three MRN subunits and a subcomplex consisting of MRE11 and RAD50 (MR) in Sf9 cells by means of recombinant baculovirus infection followed by purification of the recombinant proteins to near homogeneity (Fig. 3 A, MRN and MR). We also isolated partially purified NBS1 alone (Fig. 3 A, N). Interaction studies with purified recombinant MRN, MR, and NBS1 were complicated by the fact that these proteins exhibited a significant unspecific binding activity toward the glutathione–Sepharose beads used in this analysis (Fig. 3 B, beads alone). Nevertheless, we consistently observed a significant enrichment of purified MRN and partially purified NBS1 when we used CK2-phosphorylated fragment M-3 in the pull-down assay (Fig. 3, B and C; bottom). Untreated M-3 or GST alone did not result in such enrichment. Similarly, neither phosphorylated nor unphosphorylated M-3 was capable of efficiently binding to MRE11/RAD50 in the absence of NBS1 (Fig. 3 C, top). In summary, these results indicate that the NBS1 subunit of the MRN complex directly associates with the phosphorylated M-3 fragment of MDC1 and mediates the interaction between MDC1 and the MRN complex.


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)

Direct interaction between the phosphorylated MDC1 N terminus and the MRN complex is mediated by the NBS1 FHA domain. (A) MRN proteins were purified as described in Materials and methods. Proteins were separated on SDS-polyacrylamide gels and stained with silver. MR, MRE11–RAD50 subcomplex; N, partially purified NBS1. (B and C) Purified GST-MDC1 fragment M-3 comprising part of the SDT region was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex (B), purified MR subcomplex (C, top), and partially purified NBS1 (C, bottom) followed by GST pull-down analysis. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11. (D, top) Schematic representation of NBS1 with its functional domains. (bottom) Purified GST-MDC1 fragment M-3 was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex where the NBS1 subunit was either wild type or contained a point mutation in the FHA domain (R28A). Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with a polyclonal antibody against NBS1.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Direct interaction between the phosphorylated MDC1 N terminus and the MRN complex is mediated by the NBS1 FHA domain. (A) MRN proteins were purified as described in Materials and methods. Proteins were separated on SDS-polyacrylamide gels and stained with silver. MR, MRE11–RAD50 subcomplex; N, partially purified NBS1. (B and C) Purified GST-MDC1 fragment M-3 comprising part of the SDT region was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex (B), purified MR subcomplex (C, top), and partially purified NBS1 (C, bottom) followed by GST pull-down analysis. Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with antibodies against RAD50, NBS1, and MRE11. (D, top) Schematic representation of NBS1 with its functional domains. (bottom) Purified GST-MDC1 fragment M-3 was preincubated with CK2 and ATP. The fragment was incubated with purified MRN complex where the NBS1 subunit was either wild type or contained a point mutation in the FHA domain (R28A). Bound proteins were separated on SDS-polyacrylamide gels followed by immunoblotting. The blots were probed with a polyclonal antibody against NBS1.
Mentions: To determine whether the MRN complex interacts directly with the phosphorylated MDC1 N terminus, we coexpressed all three MRN subunits and a subcomplex consisting of MRE11 and RAD50 (MR) in Sf9 cells by means of recombinant baculovirus infection followed by purification of the recombinant proteins to near homogeneity (Fig. 3 A, MRN and MR). We also isolated partially purified NBS1 alone (Fig. 3 A, N). Interaction studies with purified recombinant MRN, MR, and NBS1 were complicated by the fact that these proteins exhibited a significant unspecific binding activity toward the glutathione–Sepharose beads used in this analysis (Fig. 3 B, beads alone). Nevertheless, we consistently observed a significant enrichment of purified MRN and partially purified NBS1 when we used CK2-phosphorylated fragment M-3 in the pull-down assay (Fig. 3, B and C; bottom). Untreated M-3 or GST alone did not result in such enrichment. Similarly, neither phosphorylated nor unphosphorylated M-3 was capable of efficiently binding to MRE11/RAD50 in the absence of NBS1 (Fig. 3 C, top). In summary, these results indicate that the NBS1 subunit of the MRN complex directly associates with the phosphorylated M-3 fragment of MDC1 and mediates the interaction between MDC1 and the MRN complex.

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