Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage

Abstract

Mammalian cells respond to DNA double-strand breaks (DSBs) by recruiting DNA repair and cell-cycle checkpoint proteins to such sites. Central to these DNA damage response (DDR) events is the DNA damage mediator protein MDC1. MDC1 interacts with several DDR proteins, including the MRE11-RAD50-NBS1 (MRN) complex. Here, we show that MDC1 is phosphorylated on a cluster of conserved repeat motifs by casein kinase 2 (CK2). Moreover, we establish that this phosphorylation of MDC1 promotes direct, phosphorylation-dependent interactions with NBS1 in a manner that requires the closely apposed FHA and twin BRCT domains in the amino terminus of NBS1. Finally, we show that these CK2-targeted motifs in MDC1 are required to mediate NBS1 association with chromatin-flanking sites of unrepaired DSBs. These findings provide a molecular explanation for the MDC1-MRN interaction and yield insights into how MDC1 coordinates the focal assembly and activation of several DDR factors in response to DNA damage.

Figure 1

Phosphorylated MDC1 SDTD motifs bind to MRN. (A) Domain architecture of MDC1 and alignment of the SDTD region from MDC1 proteins in human (hMDC1), chimpanzee (ptMDC1), pig (ssNFBD1), dog (cfMDC1) and mouse (mmMDC1). RBM indicates RNF8-binding motifs. Identical and similar residues are boxed in black and grey, respectively. CK2 consensus sites are underlined, and residues identified as phosphorylated (Beausoleil et al, 2004; Olsen et al, 2006) are numbered (′phosphorylated residues identified in both studies). (B) Silver-stained SDS–polyacrylamide gel of an SDTD peptide pull-down. PP, S329 T331 doubly phosphorylated peptide and −, its non-phosphorylated equivalent; B, bead-interacting proteins removed from extracts in a pre-clearing step; M, molecular weight markers. (C) Immunoblot of SDTD and histone H2AX phosphopeptide-interacting proteins. INP, input (5% of total); γ, phosphorylated H2AX peptide. CK2, casein kinase 2; MRN, MRE11–RAD50–NBS1; SDTD, Ser–Asp–Thr–Asp motif.

Figure 2

Casein kinase 2-dependent phosphorylation of MDC1 SDTD motifs induces interaction with MRN. (A) Left panels: SDTD peptide interacting proteins, isolated from HeLa nuclear extracts (as in Fig 1C), were subjected to phosphatase or mock treatment, and then analysed with the indicated antibodies. Right panels: whole-cell extracts (WCE) from osteosarcoma (U2OS) cells 1 h after 5 Gy irradiation or control cells were immunoblotted with the indicated antibodies. (B) At 72 h after control (CNTL) or MDC1-targeting siRNA, control cells or cells incubated for 2 h following 5 Gy of X-rays were processed for immunofluorescence with MDC1 and MDC1 pS329pT331 antibodies. (C) Immunoblot analysis of purified GST-SDTD₂ and GST-SDAD₂ after in vitro phosphorylation by recombinant CK2, or mock treatment. (D) A 250 ng portion of purified GST-SDTD₂ or GST-SDAD₂ was mock treated or phosphorylated by CK2 in vitro and then incubated with HeLa nuclear extracts. GST fusions and interacting proteins were captured on glutathione-Sepharose beads and immunoblotted. (E) Expression plasmids encoding HA-SDTD₆ were transfected into U2OS cells. After 48 h, cells were treated with 4,5,6,7-tetrabromo-benzimidazole (TBB; 75 μM; Sigma-Aldrich (Poole, Dorset, UK), T0826) or dimethyl sulphoxide (DMSO) for 6 h. Extracts were immunoprecipitated (IP) with monoclonal antibodies against HA or GFP (CNTL) and immunoblotted. CK2, casein kinase 2; DAPI, 4′,6–diamidino–2–phenylindole; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, haemagglutinin; INP, input; MRN, MRE11–RAD50–NBS1; SDAD, Ser–Asp–Ala–Ser motif; SDTD, Ser–Asp–Thr–Asp motif; siRNA, small interfering RNA.

Figure 3

Binding of MRN to MDC1 phospho-SDTD motifs requires the NBS FHA and BRCT₂ domains. (A) MRN interacts directly with phosphorylated SDTD peptides. SDTD and H2AX peptide-coupled beads were incubated with 250 ng of recombinant human MRN, and interacting proteins were analysed by western blotting. INP, input (20%); PP, MDC1 S329/T331 double phosphorylated peptide; γ, phosphorylated H2AX peptide; −, non-phosphorylated equivalent peptides. (B) A 200 ng portion of CK2-phosphorylated or mock-phosphorylated GST-SDTD₆ was incubated in pull-down reactions with 25 μl of in vitro-translated (IVT) HA fusions corresponding to amino-acid residues 1–348 of NBS1 (HA-fNBS1), or analogous proteins bearing point mutations predicted to abolish either FHA (R28A/H45A) or BRCT₂ (K160M) phosphorylation-dependent interactions. INP, input (20%). (C) A 2.0 mg portion of cell extracts prepared from MRC5, NBS (ILB1) or NBS (ILB1) fibroblasts stably expressing NBS1 or NBS1^R28A was incubated with GST-SDAD₂ treated as in (B). GST fusions were retrieved (as in Fig 2C) and interacting proteins were immunoblotted. INP, input (2.5%). CK2, casein kinase 2; GST, glutathione S-transferase; HA, haemagglutinin; MRN, MRE11–RAD50–NBS1; SDTD, Ser–Asp–Thr–Asp motif.

Figure 4

The MDC1 SDTD–MRN interaction recruits NBS1 to damaged chromatin. (A) SDTD deletion relative to the MDC1 domain architecture. (B) Indicated expression constructs were transfected into human embryonic kidney 293 cells. After 48 h, extracts were prepared, immunoprecipitated (IP) with GFP antibodies and immunoblotted. Immunoprecipitations and washes were performed at 150 mM salt; INP, input (5%). (C,D) Indicated osteosarcoma (U2OS) cell lines were treated with two rounds of control (CNTL) or MDC1-targeting siRNA for 72 h. Cells were then treated with 5 Gy of X-rays and processed for immunofluorescence 4 h later with MDC1, (C) NBS1 or (D) 53BP1 antibodies (non-irradiated cells are shown in supplementary Fig S4B,C online). (E) U2OS cells stably expressing siRNA-resistant MDC1^wt (a) or MDC1^SDTDΔ (b) were treated with siRNA against MDC1 as above, and then the nuclei were subjected to laser micro-irradiation. After 30 min, cells were pre-extracted with detergent, fixed and immunostained with antibodies against MDC1 and NBS1. Asterisks indicate resistance to MDC1 siRNA. GFP, green fluorescent protein; MRN, MRE11–RAD50–NBS1; SDTD, Ser–Asp–Thr–Asp motif; siRNA, small interfering RNA.