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.

The MRE11-RAD50-NBS1 (MRN) complex accumulates at sites of DNA double-strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1-dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation.

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail’ dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.

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