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

Abstract

The product of the Nijmegen breakage syndrome gene (NBS1) plays crucial roles in DNA damage response through its association with many proteins, including MRE11 and RAD50. However, it remains to be determined exactly how NBS1 accumulates at or near DNA double-strand breaks. Here we report that MDC1 directly binds to NBS1 and targets NBS1 to the sites of DNA damage. The MDC1-NBS1 interaction occurs through a specific region (residues 200 -420) of MDC1, which contains multiple consensus casein kinase 2 (CK2) phosphorylation sites. In addition, this interaction requires both the forkhead-associated (FHA) and tandem BRCA1 C-terminal (BRCT) domains of NBS1. Disruption of the MDC1-NBS1 interaction results in failure of NBS1 accumulation at DNA doublestrand breaks and impairment of intra-S checkpoint activation. These studies provide important mechanistic insights as to how MDC1 regulates NBS1 and the intra-S-phase checkpoint in response to DNA damage.complex plays important roles in cell cycle checkpoint signaling and DNA repair. Hypomorphic mutations in NBS1 and MRE11 cause Nijmegen breakage syndrome (NBS) and ataxiatelangiectasia-like disorder (ATLD) respectively (1-3), characterized by developmental defects, immunodeficiency, and a high incidence of cancer. Cells derived from NBS and ATLD patients are hypersensitive to radiation and display impaired intra-Sphase checkpoint activation (1-3), supporting critical roles of this complex in DNA damage response.In response to DNA damage, the MRE11/RAD50/NBS1 complex regulates the activation of ATM, the protein kinase essential for the DNA damage signaling. The key component involved in ATM activation is believed to be NBS1, because NBS1 directly binds to ATM through a very C-terminal motif and modulates ATM autophosphorylation (4-7). Biochemical evidence also suggests that the MRN complex recruits ATM to the vicinity of DNA double-strand breaks (DSBs) and stimulates ATM activation (8). In addition to playing a role in ATM activation, NBS1 also functions downstream of ATM in regulating the intra-S-phase checkpoint (9-11). Besides regulating ATM-dependent phosphorylation of SMC proteins (12, 13), the mechanism by which NBS1 participates in this intra-S-phase checkpoint remains elusive.The human NBS1 gene encodes a 754-aa nuclear protein with an N-terminal forkhead-associated (FHA) domain and breast cancer BRCA1 C-terminal (BRCT) domain. Recently, a second BRCT domain (termed BRCT2) has been identified adjacent to the first BRCT domain (termed BRCT1) (14). Both FHA and BRCT domains mediate protein-protein interaction by recognizing phosphoserine (pSer) and phosphothreonine (pThr)-containing motifs, and many proteins involved in the DNA damage responses contain functional FHA and BRCT domains. Previous reports indicate that retention of NBS1 at DSBs, after DNA damage, is mediated by its interaction with MDC1 (15-19). Moreover, the FHA and BRCT domains of NBS1 are required for its recruitment to DSBs (nuclear foci) after DNA damage (20)(21)(22). Despite these results, it is st...

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?…”

“…Results show that even a single repeat can bind NBS1, as a doubly phosphorylated SDT peptide (pSDpT) could directly bind the purified MRN complex 64 or a purified FHA-tBRCT fragment of NBS1. 61 However, progressively mutating more serine and threonine residues of the SDT repeats led to gradually lower affinity of MDC1 to NBS1 and to impaired redistribution of NBS1 to foci, suggesting that the existence of multiple SDT repeats in MDC1 allows cooperative and/or redundant binding to NBS1. 62,63 Another interesting issue is the unique mode of binding between the FHA and tBRCT domains of NBS1 and the phospho-SDT motif of MDC1.…”

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).…”