title: Constitutive phosphorylation of MDC1 physically links the MRE11-RAD50-NBS1 complex to damaged chromatin.
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.
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, 8057 Zürich, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Spycher
authors.forename: Edward S
authors.initials: ES
authors.lastname: Miller
authors.forename: Kelly
authors.initials: K
authors.lastname: Townsend
authors.forename: Lucijana
authors.initials: L
authors.lastname: Pavic
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Morrice
authors.forename: Pavel
authors.initials: P
authors.lastname: Janscak
authors.forename: Grant S
authors.initials: GS
authors.lastname: Stewart
authors.forename: Manuel
authors.initials: M
authors.lastname: Stucki
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D017766:Acid Anhydride Hydrolases; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D047390:Casein Kinase II / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics*; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000378:metabolism*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D000076228:MRE11 Homologue Protein; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
chemical_list: D018528:ATP-Binding Cassette Transporters; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C508937:MDC1 protein, mouse; C512689:Mre11a protein, mouse; C112156:NBN protein, human; C466108:Nijmegen breakage syndrome 1 protein, mouse; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D047390:Casein Kinase II; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C495198:Rad50 protein, mouse; D045643:DNA Repair Enzymes