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¹ Institute of Veterinary Biochemistry and Molecular Biology and ² Institute of Molecular Cancer Research, University of Zürich, 8057 Zürich, Switzerland
³ Cancer Research UK Institute for Cancer Studies, Birmingham University, Birmingham B15 2TT, England, UK
⁴ Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK

Correspondence to Manuel Stucki: m.stucki{at}vetbio.uzh.ch

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.

Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; BRCT, BRCA1 C terminal; CK, caseine kinase; DDR, DNA damage response; DSB, double-strand break; FHA, forkhead associated; IR, ionizing radiation; MEF, mouse embryonic fibroblast; MR, MRE11 and RAD50; MRN, MRE11–RAD50–NBS1; NBS, Nijmegen breakage syndrome; PIKK, phosphoinositide-3-kinase–related protein kinase; PNK, polynucleotide kinase; pSDpT, phosphorylated SDT peptide; PST, Pro-Ser-Thr; SDT, Ser-Asp-Thr; TBB, tetrabromo-2-azabenzimidazole.

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