Mdb1, a Fission Yeast Homolog of Human MDC1, Modulates DNA Damage Response and Mitotic Spindle Function

During eukaryotic DNA damage response (DDR), one of the earliest events is the phosphorylation of the C-terminal SQ motif of histone H2AX (H2A in yeasts). In human cells, phosphorylated H2AX (γH2AX) is recognized by MDC1, which serves as a binding platform for the accumulation of a myriad of DDR factors on chromatin regions surrounding DNA lesions. Despite its important role in DDR, no homolog of MDC1 outside of metazoans has been described. Here, we report the characterization of Mdb1, a protein from the fission yeast Schizosaccharomyces pombe, which shares significant sequence homology with human MDC1 in their C-terminal tandem BRCT (tBRCT) domains. We show that in vitro, recombinant Mdb1 protein binds a phosphorylated H2A (γH2A) peptide, and the phospho-specific binding requires two conserved phospho-binding residues in the tBRCT domain of Mdb1. In vivo, Mdb1 forms nuclear foci at DNA double strand breaks (DSBs) induced by the HO endonuclease and ionizing radiation (IR). IR-induced Mdb1 focus formation depends on γH2A and the phospho-binding residues of Mdb1. Deleting the mdb1 gene does not overtly affect DNA damage sensitivity in a wild type background, but alters the DNA damage sensitivity of cells lacking another γH2A binder Crb2. Overexpression of Mdb1 causes severe DNA damage sensitivity in a manner that requires the interaction between Mdb1 and γH2A. During mitosis, Mdb1 localizes to spindles and concentrates at spindle midzones at late mitosis. The spindle midzone localization of Mdb1 requires its phospho-binding residues, but is independent of γH2A. Loss of Mdb1 or mutating its phospho-binding residues makes cells more resistant to the microtubule depolymerizing drug thiabendazole. We propose that Mdb1 performs dual roles in DDR and mitotic spindle regulation.     

Dimerization, but not phosphothreonine binding, is conserved between the forkhead-associated domains of Drosophila MU2 and human MDC1

Mutator 2 (MU2) in Drosophila melanogaster has been proposed to be the ortholog of human MDC1, a key mediator in DNA damage response. The forkhead-associated (FHA) domain of MDC1 is a dimerization module regulated by trans binding to phosphothreonine 4 from another molecule. Here we present the crystal structure of the MU2 FHA domain at 1.9 Å resolution, revealing its evolutionarily conserved role in dimerization. As compared to the MDC1 FHA domain, the MU2 FHA domain dimerizes using a different and more stable interface and contains a degenerate phosphothreonine-binding pocket. Our results suggest that the MU2 dimerization is constitutive and lacks phosphorylation-mediated regulation.Structured summary of protein interactionsMU2 and MU2 bind by cosedimentation in solution (View interaction)MU2 and MU2 bind by X-ray crystallography (View interaction)MU2 and MU2 bind by molecular sieving (View interaction)     

A divalent FHA/BRCT‐binding mechanism couples the MRE11–RAD50–NBS1 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.     

Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain

MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.     

The CC1-FHA Tandem as a Central Hub for Controlling the Dimerization and Activation of Kinesin-3 KIF1A

Kinesin-3 KIF1A plays prominent roles in axonal transport and synaptogenesis. KIF1A adopts?a monomeric form in?vitro but acts as a processive dimer in?vivo. The mechanism underlying the motor dimerization is poorly understood. Here, we find that the CC1-FHA tandem of KIF1A exists as a stable dimer. The structure of CC1-FHA reveals that the linker between CC1 and FHA unexpectedly forms a β-finger hairpin, which integrates CC1 with FHA assembling a CC1-FHA homodimer. More importantly, dissociation of the CC1-FHA dimer unleashes CC1 and the β-finger, which are both essential for the motor inhibition. Thus, dimerization of the CC1-FHA tandem not only promotes the KIF1A dimer formation but also may trigger the motor activity via sequestering the CC1/β-finger region. The CC1-FHA tandem likely functions as a hub for controlling the dimerization and activation of KIF1A, which may represent?a new paradigm for the kinesin regulation shared by other kinesin-3 motors.     

The DNA damage response mediator MDC1 directly interacts with the anaphase-promoting complex/cyclosome

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (gamma-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of gamma-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.     

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.     

Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase

Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1).     

Structural delineation of MDC1-FHA domain binding with CHK2-pThr68

Mammalian MDC1 interacts with CHK2 in the regulation of DNA damage-induced S-phase checkpoint and apoptosis, which is directed by the association of MDC1-FHA and CHK2-pThr68. However, different ligand specificities of MDC1-FHA have been reported, and no structure is available. Here we report the crystal structures of MDC1-FHA and its complex with a CHK2 peptide containing pThr68. Unlike other FHA domains, MDC1-FHA exists as an intrinsic dimer in solution and in crystals. Structural and binding analyses support pThr+3 ligand specificity and provide structural insight into MDC1-CHK2 interaction.     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

Telomere capture, a rare event that stabilizes chromosome breaks, is associated with certain genetic abnormalities in humans. Studies pertaining to the generation, maintenance, and biological effects of telomere formation are limited in metazoans. A mutation, mu2^a, in Drosophila melanogaster decreases the rate of repair of double strand DNA breaks in oocytes, thus leading to chromosomes that have lost a natural telomere and gained a new telomere. Amino acid sequence, domain architecture, and protein interactions suggest that MU2 is an ortholog of human MDC1. The MU2 protein is a component of meiotic recombination foci and localizes to repair foci in S2 cells after irradiation in a manner similar to that of phosphorylated histone variant H2Av. Domain searches indicated that the protein contains an N-terminal FHA domain and a C-terminal tandem BRCT domain. Peptide pull-down studies showed that the BRCT domain interacts with phosphorylated H2Av, while the FHA domain interacts with the complex of MRE11, RAD50, and NBS. A frameshift mutation that eliminates the MU2 BRCT domain decreases the number and size of meiotic phospho-H2Av foci. MU2 is also required for the intra-S checkpoint in eye-antennal imaginal discs. MU2 participates at an early stage in the recognition of DNA damage at a step that is prerequisite for both DNA repair and cell cycle checkpoint control. We propose a model suggesting that neotelomeres may arise when radiation-induced chromosome breaks fail to be repaired, fail to arrest progression through meiosis, and are deposited in the zygote, where cell cycle control is absent and rapid rounds of replication and telomere formation ensue.     

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.     

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

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.     

The BRCA1 RING and BRCT domains cooperate in targeting BRCA1 to ionizing radiation-induced nuclear foci

BRCA1 accumulates in nuclear foci during S-phase and reassembles into DNA repair-associated foci after DNA damage, reflecting its role in genome maintenance. BRCA1 comprises a RING domain at the N terminus and a BRCT domain at the C terminus, through which it associates with DNA repair proteins. The key sequences that target BRCA1 to DNA damage-induced foci have not been identified. Here, we mapped the BRCA1 foci-targeting domains of yellow fluorescence protein (YFP)-tagged BRCA1 in MCF-7 breast cancer cells exposed to ionizing radiation (IR). Cancer mutations specific to the BRCT domain, but not the RING domain, abolished BRCA1 recruitment to IR-induced foci. The YFP-BRCT domain itself, however, localized poorly at IR-induced foci, and the RING domain and other sequences were negative. We discovered that only when the RING and BRCT domains were combined was foci targeting restored to levels observed for wild-type BRCA1. The RING-BRCT fusion co-localized at foci with the MDC1 DNA damage response factor and inhibited entry of endogenous BRCA1 into nuclear foci. Our results explain why exon 11-deficient BRCA1 splice variants are targeted to IR-induced foci even though they are incapable of repairing DNA damage. We propose that both RING and BRCT domains together target BRCA1 to large focal assemblies at DNA double-stranded breaks.     

MDC1/NFBD1: a key regulator of the DNA damage response in higher eukaryotes

The protein MDC1/NFBD1 contains a forkhead-associated (FHA) domain and two BRCA1 carboxyl-terminal (BRCT) domains. It interacts with several proteins involved in DNA damage repair and checkpoint signalling, and is phosphorylated in response to DNA damage and during mitosis. Upon treatment of cultured human cells with DNA damaging agents, MDC1/NFBD1 translocates to sites of DNA lesions, where it collaborates with other proteins and with phosphorylated histone H2AX to mediate the accumulation of checkpoint and repair factors into nuclear foci. Down-regulation of MDC1/NFBD1 expression levels by small interfering RNA (siRNA) renders cells hyper-sensitive to DNA damaging agents and leads to defects in cell cycle checkpoint activation and apoptosis. Thus, MDC1/NFBD1 appears to be a key regulator of the DNA damage response in mammalian cells.     

Homo-oligomerization is the essential function of the tandem BRCT domains in the checkpoint protein Crb2

BRCT (BRCA1 C terminus) domains are frequently found as a tandem repeat in proteins involved in DNA damage responses, such as Saccharomyces cerevisiae Rad9, human 53BP1 and BRCA1. Tandem BRCT domains mediate protein-protein and protein-DNA interactions. However, the functional significance of these interactions is largely unknown. Here we report the oligomerization of Schizosaccharomyces pombe checkpoint protein Crb2 through its tandem BRCT domains. Truncated Crb2 without BRCT domains is defective in DNA damage checkpoint signaling. However, addition of either of two heterologous dimerization motifs largely restores the functions of truncated Crb2 without BRCT domains. Replacement of Crb2 BRCT domains with a dimerization motif also renders cells resistant to the dominant negative effect of overexpressing Crb2 BRCT domains. These results demonstrate that the crucial function of the tandem BRCT domains is to oligomerize Crb2.     

NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain

DNA double-strand breaks represent the most potentially serious damage to a genome; hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Here, we show that NBS1, the gene product defective in Nijmegen breakage syndrome (NBS), physically interacts with histone, rather than damaged DNA, by direct binding to gamma-H2AX. We also demonstrate that NBS1 binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells and was also delayed in AT cells, which lack the kinase activity for phosphorylation of H2AX. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to gamma-H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant gamma-H2AX. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for relocalization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage.     

The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50[middle dot]hMRE11[middle dot]NBS1 complex DNA repair activity

NBS1 (p95), the protein responsible for Nijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain. The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination. Here, we show in vivo direct evidence that NBS1 recruits the hMRE11 nuclease complex into the cell nucleus and leads to the formation of foci by utilizing different functions from several domains. The amino acid sequence at 665-693 on the C terminus of NBS1, where a novel identical sequence with yeast Xrs2 protein was found, is essential for hMRE11 binding. The hMRE11-binding region is necessary for both nuclear localization of the complex and for cellular radiation resistance. On the other hand, the FHA domain regulates nuclear foci formation of the multiprotein complex in response to DNA damage but is not essential for nuclear transportation of the complex and radiation resistance. Because the FHA/BRCA1 C terminus domain is widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, and DNA repair, the foci formation could be associated with many phenotypes of Nijmegen breakage syndrome other than radiation sensitivity.     

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.     

Structural basis for phosphorylation-dependent signaling in the DNA-damage response.

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.