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.

MDC1 regulates intra-S-phase checkpoint by targeting NBS1 to DNA double-strand breaks

“…While this work was under review, three related papers were published (35)(36)(37). These studies agree with our conclusion and support a role of MDC1/NBS1 interaction in DNA damage checkpoint control.…”

The cellular response to DNA damage: A focus on MDC1 and its interacting proteins

“…The mechanism by which MDC1 regulates NBS1 retention at DSBs was poorly understood until recent independent reports by four groups which demonstrated that MDC1 directly binds NBS1. [61][62][63][64] Since NBS1 also binds ATM, 29 these results suggest that MDC1 can recruit multiple ATM molecules to DSBs, both directly through its FHA domain as well as indirectly through NBS1, thus allowing a more efficient and quick positive feedback loop (Fig. 1D).…”

“…Similar results were obtained when mutations or deletions were introduced in either the SDT repeats of MDC1 or the FHA-tBRCT domains of NBS1. [61][62][63][64] Interesting questions arise from the data discussed above. Why does MDC1 contain multiple SDT repeats?…”

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

“…The FHA-BRCT fusion domain is essential for the relocation of NBS1 to DNA damage sites (Kobayashi et al 2004;Stracker and Petrini 2011). The BRCT fold of NBS1 recognizes PAR, whereas the FHA fold of NBS1 could recognize the pThr motifs in MDC1 (Chapman and Jackson 2008;Spycher et al 2008;Wu et al 2008;Lloyd et al 2009), a functional partner of gH2AX (Stucki et al 2005;Lou et al 2006). Again, in MDC1 À/À cells, the stable retention of NBS1 to DNA damage sites is impaired, and olaparib treatment abolished the relocation of NBS1 to DNA damage sites in MDC1 À/À cells (Fig.…”

“…It has been shown that the FHA domains recognize phospho-Thr (pThr) motifs (Sun et al 1998;Durocher et al 1999Durocher et al , 2000Li et al 2002;Mahajan et al 2008). For example, the FHA domain of Rad53 recognizes the pThr motif of Rad9 in budding yeast (Sun et al 1998), the FHA domain of fission yeast NBS1 recognizes the pThr of Ctp1 (Williams et al 2009), and the FHA domain of human NBS1 and RNF8 recognizes the pThr motifs of MDC1 (Chapman and Jackson 2008;Spycher et al 2008;Wu et al 2008;Lloyd et al 2009). While the BRCT domains have been shown to recognize phospho-Ser (pSer) motifs (Manke et al 2003;Yu et al 2003), the BRCA1 BRCT domain binds pSer motifs in several downstream partners Yu and Chen 2004;Kim et al 2007b;Liu et al 2007;Wang et al 2007).…”