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Palindromic sequences are a potent source of chromosomal instability in many organisms and are implicated in the pathogenesis of human diseases. In this study, we investigate which nucleases are responsible for cleavage of the hairpin and cruciform structures and generation of double-strand breaks at inverted repeats in Saccharomyces cerevisiae. We demonstrate that the involvement of structure-specific nucleases in palindrome fragility depends on the distance between inverted repeats and their transcriptional status. The attack by the Mre11 complex is constrained to hairpins with loops

Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11–Rad50–Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre11 2 –Rad50 2 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid.

The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 molecules interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we determined the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.

Summary: The Mre11 complex (Mre11, Rad50, and Nbs1) is integral to both DNA repair and ataxia telangiectasia mutated (ATM)-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal levels. To delineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice), which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles that abolished interaction were incompatible with viability. Conversely, a 108-amino-acid Nbs1 fragment comprising the Mre11 interface was sufficient to rescue viability and ATM activation in cultured cells and support differentiation of hematopoietic cells in vivo. These data indicate that the essential role of Nbs1 is via its interaction with Mre11 and that most of the Nbs1 protein is dispensable for Mre11 complex functions and suggest that Mre11 and Rad50 directly activate ATM. : Kim et al. find that Nbs1 promotes the proper assembly and localization of a complex containing Mre11 and Rad50. Nbs1-mediated assembly is required for the function of the complex, and a 108-amino-acid Nbs1 fragment containing the Mre11 interaction domain is sufficient for this essential role. Keywords: Nbs1mid mutants, Mre11-Nbs1 interface, ATM activation, Mre11 complex assembly

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells., Author summary A complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

We derived a mouse model in which a mutant form of Nbs1 (Nbs1mid8) exhibits severely impaired binding to the Mre11-Rad50 core of the Mre11 complex. TheNbs1mid8allele was expressed exclusively in hematopoietic lineages (inNbs1-/mid8vavmice). UnlikeNbs1flox/floxvavmice, which are Nbs1 deficient in the bone marrow,Nbs1-/mid8vavmice were viable.Nbs1-/mid8vavhematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage specific blockage of B cell development. Within six months,Nbs1-/mid8mice developed highly penetrant T cell leukemias.Nbs1-/mid8vavleukemias recapitulated mutational features of human T-ALL, containing mutations inNotch1, Trp53, Bcl6, Bcor, andIkzf1, suggesting thatNbs1mid8mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis ofNbs1-/mid8vavmalignancies showed focal amplification of 9qA2, causing overexpression ofMRE11andCHK1. We propose that overexpression compensates for the meta-stable Mre11-Nbs1mid8interaction, and that selection pressure for overexpression reflects the essential role of Nbs1 in promoting assembly and activity of the Mre11 complex.

A mechanistic understanding of the yeast telomere requires an integrated understanding of telomere chromatin structure (telosomes), telomeric origins of replications, telomere length homeostasis, and telosome epigenetics. Recent molecular and genetic studies of the yeast telosomal components Rap1, Rif1, and Rif2, the Mre11 complex, and Tel1ATM promise to increase our insight into the coordination between these processes. Here, an intricate relationship is proposed between these multiple components that has resulted in increased appreciation of the multiple levels of telomere length control and their differentiation from double-strand repair. The mre11A470 motif (A470–A482) alleles have also opened new avenues to the exploration of telosome structure and function.

International audience; DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATMflox) was crossed to hypomorphic NBS1 mutants (Nbs1ΔB/ΔB mice). Nbs1ΔB/ΔB Atm−/− hematopoietic cells derived by crossing to vavcre were viable in vivo. Nbs1ΔB/ΔB Atm−/− VAV mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. Implications: A novel mouse model was developed, by combining a vavcre-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis. Mol Cancer Res; 14(2); 185–95. ©2015 AACR.

DNA double-strand breaks (DSBs) are hazardous lesions that threaten genome integrity and cell survival. The DNA damage response (DDR) safeguards the genome by sensing DSBs, halting cell cycle progression and promoting repair through either non-homologous end joining (NHEJ) or homologous recombination (HR). The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex is central to the DDR through its structural, enzymatic, and signaling roles. The complex tethers DNA ends, activates the Tel1/ATM kinase, resolves protein-bound or hairpin-capped DNA ends, and maintains telomere homeostasis. In addition to its role at DSBs, MRX/N associates with unperturbed replication forks, as well as stalled replication forks, to ensure complete DNA synthesis and to prevent chromosome rearrangements. Here, we summarize the significant progress made in characterizing the MRX/N complex and its various activities in chromosome metabolism.

TRF2 binds to telomeric repeats and is critical for telomere integrity. Evidence suggests that it also localizes to non-telomeric DNA damage sites. However, this recruitment appears to be precarious and functionally controversial. We find that TRF2 recruitment to damage sites occurs by a two-step mechanism: the initial rapid recruitment (phase I) and stable and prolonged association with damage sites (phase II). Phase I is poly(ADP-ribose) polymerase (PARP)-dependent and requires the N-terminal basic domain. The phase II recruitment requires the C-terminal MYB/SANT domain and the iDDR region in the hinge domain, which is mediated by the MRE11 complex and is stimulated by hTERT. PARP-dependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates two phases. TRF2 binds to the I-PpoI-induced DNA double-strand break sites, which is enhanced by the presence of complex damage and is dependent on PARP and the MRE11 complex. TRF2 depletion affects non-sister chromatid homologous recombination (HR) repair, but not HR between sister chromatids or non-homologous endjoining pathways. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.Summary StatementTRF2 is recruited to DNA double-strand break damage sites by a two-step mechanism and functions in non-sister chromatid homologous recombination repair

DNA double-strand breaks (DSBs) in cells can undergo nucleolytic degradation to generate long 3' single-stranded DNA tails. This process is termed DNA end resection, and its occurrence effectively commits to break repair via homologous recombination, which entails the acquisition of genetic information from an intact, homologous donor DNA sequence. Recent advances, prompted by the identification of the nucleases that catalyze resection, have revealed intricate layers of functional redundancy, interconnectedness, and regulation. Here, we review the current state of the field with an emphasis on the major questions that remain to be answered. Topics addressed will include how resection initiates via the introduction of an endonucleolytic incision close to the break end, the molecular mechanism of the conserved MRE11 complex in conjunction with Sae2/CtIP within such a model, the role of BRCA1 and 53BP1 in regulating resection initiation in mammalian cells, the influence of chromatin in the resection process, and potential roles of novel factors.

The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50., This study reveals novel roles for the checkpoint kinase Tel1/ATM and Rif2 in regulating the function of the MRX complex during repair of DNA double-strand breaks by nonhomologous end joining and homologous recombination., Author Summary Many tumors contain mutations that confer defects in repairing DNA double-strand breaks (DSBs). In both yeast and mammals, the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals) plays critical functions in repairing a DSB by either nonhomologous end joining (NHEJ) or homologous recombination (HR). Furthermore, it recruits the checkpoint kinase Tel1/ATM. Although ATM is considered to be a tumor suppressor, up-regulation of ATM signaling promotes chemoresistance, radioresistance and metastasis. For this reason, cancer therapies targeting ATM have been developed to increase the effectiveness of standard genotoxic treatments and/or to set up synthetic lethal approaches in cancers with DNA repair defects. We aimed to identify the precise role of ATM/Tel1 in these processes. By performing a synthetic phenotype screen, we identified a mutation (rad50-V1269M) altering the Rad50 subunit of the MRX complex, which sensitizes cells lacking Tel1 to genotoxic agents. Genetic and biochemical characterization of MRV1269MX protein complex revealed that Tel1 promotes MRX association at DSBs to allow proper MRX-DNA binding that is needed for DSB repair. The role of Tel1 in promoting MRX retention on DSBs is counteracted by Rif2, which can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes in Rad50. Our finding that MRX dysfunctions can be synthetically lethal with Tel1 loss in the presence of genotoxic agents suggests that ATM inhibitors could be beneficial in patients whose tumors have defective MRN functions.

Recent work has highlighted the importance of alternative, error-prone mechanisms for joining DNA double-strand breaks (DSB) in mammalian cells. These noncanonical, non-homologous end joining (NHEJ) pathways threaten genomic stability but remain poorly characterized. The RAG post-cleavage complex normally prevents V(D)J recombination-associated DSBs from accessing alternative NHEJ. Because the MRE11/RAD50/NBS1 complex localizes to RAG-mediated DSBs and possesses DNA end tethering, processing and joining activities, we asked whether it plays a role in the mechanism of alternative NHEJ, or participates in regulating access of DSBs to alternative repair pathways. We find that NBS1 is required for alternative NHEJ of hairpin coding ends, suppresses alternative NHEJ of signal ends, and promotes proper resolution of inversional recombination intermediates. These data demonstrate that the MRE11 complex functions at two distinct levels, regulating repair pathway choice (likely through enhancing the stability of DNA-end complexes) and participating in alternative NHEJ of coding ends.

Central to the DNA damage response (DDR) is the highly conserved Mre11 complex consisting of Mre11, Rad50 and Nbs1. The Mre11 complex acts as a sensor of DNA double-strand breaks (DSBs) and regulates the signal transduction cascades that are triggered following damage detection.(1) Rare human genetic instability syndromes such as Ataxia-telangiectasia (A-T) and Nijmegen Breakage Syndrome (NBS) have underscored the importance of the DSB response in the suppression of tumorigenesis, as well as other severe pathologies affecting the development of both the immune system and the central nervous system. Using murine models of the human diseases, we have investigated the role of the Mre11 complex, and other modulators of the DSB response, in tumor suppression.(2,3) We found that the checkpoint kinase Chk2 is crucial for the suppression of a diverse array of tumor types in Mre11 complex mutants and uncovered multiple roles for the Mre11 complex in apoptotic signaling in parallel to Chk2.(4,5).

The Mre11 complex functions in double-strand break (DSB) repair, meiotic recombination, and DNA damage checkpoint pathways. Sae2 deficiency has opposing effects on the Mre11 complex. On one hand, it appears to impair Mre11 nuclease function in DNA repair and meiotic DSB processing, and on the other, Sae2 deficiency activates Mre11-complex-dependent DNA-damage-signaling via the Tel1–Mre11 complex (TM) pathway. We demonstrate that SAE2 overexpression blocks the TM pathway, suggesting that Sae2 antagonizes Mre11-complex checkpoint functions. To understand how Sae2 regulates the Mre11 complex, we screened for sae2 alleles that behaved as the null with respect to Mre11-complex checkpoint functions, but left nuclease function intact. Phenotypic characterization of these sae2 alleles suggests that Sae2 functions as a multimer and influences the substrate specificity of the Mre11 nuclease. We show that Sae2 oligomerizes independently of DNA damage and that oligomerization is required for its regulatory influence on the Mre11 nuclease and checkpoint functions.

The recognition and repair of DNA double-strand breaks (DSBs) is a complex process that draws upon a multitude of proteins. This is not surprising since this is a lethal lesion if left unrepaired and also contributes to genome instability and the consequential risk of cancer and other pathologies. Some of the key proteins that recognize these breaks in DNA are mutated in distinct genetic disorders that predispose to agent sensitivity, genome instability, cancer predisposition and/or neurodegeneration. These include members of the Mre11 complex (Mre11/Rad50/Nbs1) and ataxia-telangiectasia (A-T) mutated (ATM), mutated in the human genetic disorder A-T. The mre11 (MRN) complex appears to be the major sensor of the breaks and subsequently recruits ATM where it is activated to phosphorylate in turn members of that complex and a variety of other proteins involved in cell-cycle control and DNA repair. The MRN complex is also upstream of ATM and ATR (A-T-mutated and rad3-related) protein in responding to agents that block DNA replication. To date, more than 30 ATM-dependent substrates have been identified in multiple pathways that maintain genome stability and reduce the risk of disease. We focus here on the relationship between ATM and the MRN complex in recognizing and responding to DNA DSBs.

The Mre11/Nbs1/Rad50 complex (MRN) plays multiple roles in the maintenance of genome stability, including repair of double-stranded breaks (DSBs) and activation of the S-phase checkpoint. Here we demonstrate that MRN is required for the prevention of DNA rereplication in mammalian cells. DNA replication is strictly regulated by licensing control so that the genome is replicated once and only once per cell cycle. Inactivation of Nbs1 or Mre11 leads to a substantial increase of DNA rereplication induced by overexpression of the licensing factor Cdt1. Our studies reveal that multiple mechanisms are likely involved in the MRN-mediated suppression of rereplication. First, both Mre11 and Nbs1 are required for facilitating ATR activation when Cdt1 is overexpressed, which in turn suppresses rereplication. Second, Cdt1 overexpression induces ATR-mediated phosphorylation of Nbs1 at Ser343 and this phosphorylation depends on the FHA and BRCT domains of Nbs1. Mutations at Ser343 or in the FHA and BRCT domains lead to more severe rereplication when Cdt1 is overexpressed. Third, the interaction of the Mre11 complex with RPA is important for the suppression of rereplication. This suggests that modulating RPA activity via a direct interaction of MRN is likely one of the effector mechanisms to suppress rereplication. Moreover, we demonstrate that MRN is also required for preventing the accumulation of DSBs when rereplication is induced. Therefore, our studies suggest new roles of MRN in the maintenance of genome stability through preventing rereplication and rereplication-associated DSBs when licensing control is compromised.

During the first meiotic prophase, numerous DNA double-strand breaks (DSB) are formed in the genome in order to initiate recombination between homologous chromosomes. The conserved Mre11 complex, formed of Mre11, Rad50 and Nbs1 (Xrs2 in Saccharomyces cerevisiae) proteins, plays a crucial role in mitotic cells for sensing and repairing DSB. In meiosis the Mre11 complex is also required for meiotic recombination. Depending on the organisms, the Mre11 complex is required for the formation of the DSB catalysed by the transesterase Spo11 protein. It then plays a unique function in removing covalently attached Spo11 from the 5' extremity of the breaks through its nuclease activity, to allow further break resection. Finally, the Mre11 complex also plays a role during meiosis in bridging DNA molecules together and in sensing Spo11 DSB and activating the DNA damage checkpoint. In this article the different biochemical functions of the Mre11 complex required during meiosis are reviewed, as well as the consequences of Mre11 complex inactivation for meiosis in several organisms. Finally, I describe the meiotic phenotypes of several animal models that have been developed to model hypomorphic mutations of the Mre11 complex, involved in humans in some genetic instability disorders.

The Mre11 complex (Mre11-Rad50-Nbs1) is involved in a diverse array of DNA metabolic processes including the response to DNA double-strand breaks (DSBs). The structure of Rad50 plays a key role in the DNA-binding and end-bridging activity of the complex. An interesting feature within the central portion of the Rad50 protein is the Rad50 hook region that is defined by the highly conserved CXXC motif. The structure of the Pyrococcus furiosus Rad50 hook region revealed an intermolecular dimerization of Rad50 through the coordination of a zinc ion by the four cysteines. Biochemical and genetic analysis in Saccharomyces cerevisiae have shown that mutations in the conserved cysteines impact all functions of the Mre11 complex including interaction with Mre11, increased sensitivity to DSB inducing agents, telomere maintenance and intrachromosomal association. Mutations in the yeast hook domain can lead to increased chromosome fragmentation, suggesting that the hook domain of Rad50 is essential for the tethering of chromosome ends. In this study, we have examined the effects of mutating the key cysteine residues in the hook domain of human Rad50 (hRad50), focusing on the interactions Rad50 has with itself, Mre11 and DNA. Our results reveal that mutation of the conserved cysteine residues abrogates dimerization at the hook domain in hRad50; however, disrupting dimerization at this domain does not appear to impair the interaction of full-length hRad50 with itself and hMre11 or affect DNA-binding activity of the hMre11-Rad50 complex.

Summary The Mre11 complex (consisting of MRE11, RAD50, and NBS1/Xrs2) is required for double-strand break (DSB) formation, processing, and checkpoint signaling during meiotic cell division in S. cerevisiae [1–8]. Whereas studies of Mre11 complex mutants in S. pombe and A. thaliana indicate that the complex has other essential meiotic roles [9–11], relatively little is known regarding the functions of the complex downstream of meiotic break formation and processing or its role in meiosis in higher eukaryotes. We analyzed meiotic events in mice harboring hypomorphic Mre11 and Nbs1 mutations which, unlike null mutants, support viability [12–16]. Our studies revealed defects in the temporal progression of meiotic prophase, incomplete and aberrant synapsis of homologous chromosomes, persistence of strand exchange proteins, and alterations in both the frequency and placement of MLH1 foci, a marker of crossovers. A unique sex-dependent effect on MLH1 foci and chiasmata numbers was observed: males exhibited an increase and females a decrease in recombination levels. Thus, our findings implicate the Mre11 complex in meiotic DNA repair and synapsis in mammals and indicate that the complex may contribute to the establishment of normal sex-specific differences in meiosis.

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50S/S mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50S/S mice and found that Rad50S/S cellular attrition was strongly ATM and Chk2 dependent. The hypomorphic Mre11ATLD1 and Nbs1ΔB alleles conferred similar rescue of Rad50S/S-dependent hematopoietic failure. These data indicate that the Mre11 complex activates an ATM–Chk2-dependent apoptotic pathway. We find that apoptosis and cell cycle checkpoint activation are parallel outcomes of the Mre11 complex–ATM pathway. Conversely, the Rad50S mutation mitigated several phenotypic features of ATM deficiency. We propose that the Rad50S allele is hypermorphic for DNA damage signaling, and that the resulting constitutive low-level activation of the DNA damage response accounts for the partial suppression of ATM deficiency in Rad50S/S Atm-/- mice.

THE RAD50, XRS2, and MRE11 genes were first identified in screens for ionizing radiation (IR)-sensitive or meiotic recombination-defective mutants in Saccharomyces cerevisiae (Symington 2002). Subsequent studies revealed similar defects in meiosis, repair of IR-induced DNA damage, telomere length, nonhomologous end joining (NHEJ), and the intra-S phase checkpoint in mre11, rad50, and xrs2 null mutants (Haber 1998; D'Amours and Jackson 2002). The corresponding proteins form a high-affinity complex with an ∼2:2:1 stoichiometry of Mre11, Rad50, and Xrs2 (Anderson et al. 2001; Chen et al. 2001). Both Mre11 and Rad50 are conserved and are homologous to the Escherichia coli SbcD and SbcC proteins, respectively (Sharples and Leach 1995). Functional analogs of the Xrs2 subunit are found only in eukaryotes, e.g., the human Nbs1 protein, and the sequences of these display very limited interspecies homology (Carney et al. 1998; Varon et al. 1998; Chahwan et al. 2003; Ueno et al. 2003). Mre11 contains five sequence motifs found in a superfamily of phosphoesterases that, apart from E. coli SbcD, also includes di-metal Ser/Thr protein phosphatases. Conserved residues within these motifs are required for Mre11 nuclease activity in vitro (Furuse et al. 1998; Usui et al. 1998; Moreau et al. 1999). Mre11 displays 3′–5′ ssDNA, 3′–5′ dsDNA exonuclease, and ssDNA endonuclease activities with the endonuclease activity acting preferentially at ssDNA/dsDNA transitions (Furuse et al. 1998; Paull and Gellert 1998; Usui et al. 1998; Moreau et al. 1999; Trujillo and Sung 2001). Biochemical analyses of the Mre11-Rad50 complex suggest that one function of the complex is to tether DNA ends together (Chen et al. 2001; de Jager et al. 2001; Hopfner et al. 2002), and it has been suggested that this tethering activity could hold sister chromatids in close proximity for efficient repair (Wiltzius et al. 2005). The rapid recruitment of the Mre11-Rad50-Xrs2 (MRX) complex to double-strand breaks (DSBs) in vivo is critical for signaling DNA damage to downstream effectors, such as Tel1 and Mec1 in yeast or ATM in mammalian cells (Nelms et al. 1998; Grenon et al. 2001; Usui et al. 2001; Nakada et al. 2003; Uziel et al. 2003; Costanzo et al. 2004; Lisby et al. 2004). The rapid recruitment of the Mre11 complex to DSBs is also suggestive of an early role in the repair of DSBs. Studies with the mre11 null (mre11Δ) mutant have shown a defect in the 5′–3′ resection of DSBs suggesting a direct role of Mre11 in the nucleolytic resection process (Lee et al. 1998; Tsubouchi and Ogawa 1998). Although the exonuclease activity of Mre11 is of the opposite polarity to that observed for DSB resection (White and Haber 1990), the endonuclease activity of Mre11 could potentially function in concert with a helicase unwinding the DNA duplex and the Mre11 endonuclease resecting the 5′ ends. Analysis of mre11 nuclease-defective (mre11-nd) mutants has led to conflicting views on the role of the Mre11 nuclease in end resection because of the different phenotypes conferred by these alleles. All of the mre11-nd mutants share the property of sporulation deficiency due to an inability to process Spo11-induced DSBs during meiosis, as well as synthetic mitotic lethality with rad27Δ (Furuse et al. 1998; Tsubouchi and Ogawa 1998; Usui et al. 1998; Moreau et al. 1999; Debrauwere et al. 2001). The Mre11-58 protein has two point mutations in phosphoesterase motif IV (H213Y, L225I), consistently lacks nuclease activity in vitro, and is furthermore defective in complex formation with Rad50 and Xrs2 (Usui et al. 1998). The mre11-58 allele confers a phenotype similar to that of an mre11Δ allele for sensitivity to ionizing radiation, processing of HO-induced DSBs, and sensitivity to HU and telomere length, but is proficient for meiotic DSB formation (Tsubouchi and Ogawa 1998; Usui et al. 1998; D'Amours and Jackson 2001). The mre11-D16A mutant (phosphoesterase motif I) is less sensitive to IR than the mre11Δ mutant and the telomeres are of intermediate length between the mre11Δ and MRE11 strains (Furuse et al. 1998). This mutant is proficient for end-joining repair of cohesive ends, and the purified Mre11-D16A protein interacts with Rad50 and Xrs2 in vitro (Lewis et al. 2004). However, the recruitment of Rad50 to telomeres is impaired in the mre11-D16A strain and there is a defect in complex formation between the Schizosaccharomyces pombe Rad32-D25A protein (equivalent to Mre11-D16A) and Rad50 in vivo (Tomita et al. 2003; Takata et al. 2005). In contrast, the mre11-D56N and mre11-H125N strains (phosphoesterase motifs II and III, respectively) show only a three- to fourfold decrease in survival to 500 Gy IR compared to MRE11 strains, exhibit no defect in processing HO-induced DSBs, and have telomeres of the same length as MRE11 strains (Moreau et al. 1999; Llorente and Symington 2004). These results have led to the suggestion that the Mre11-D56N and Mre11-H125N proteins retain sufficient residual nuclease activity to process DSBs in vivo (Lewis et al. 2004). However, in genetic assays for endonucleolytic processing of hairpin structures in yeast, the mre11-D56N and mre11-H125N strains were as defective as the mre11Δ strain, indicating that there is little, if any, residual endonuclease activity (Rattray et al. 2001; Lobachev et al. 2002; Farah et al. 2005). Biochemical studies of the human Mre11-3 protein, which has two amino acid substitutions within phosphoesterase motif III, have shown a complete loss of nuclease activity even though in the crystal structure of the Pyrococcus furiosus Mre11-3, the overall protein fold and active site conformation is the same as those in the wild-type protein (Arthur et al. 2004). In vivo, the yeast mre11-3 mutant shows similar sensitivity to IR as the mre11-H125N strain and normal resection of an HO-induced DSB (Bressan et al. 1998; Moreau et al. 1999; Lee et al. 2002). Another possibility to account for the more severe phenotypes conferred by the mre11-D16A allele is a defect in complex assembly, as shown for the mre11-58 allele (Usui et al. 1998). Although the Mre11-D16A protein binds to Rad50 and Xrs2 in vitro, it is possible that a subtle defect in complex formation may not have been apparent at high protein concentrations (Lewis et al. 2004). To test these possibilities, we have compared several phenotypes, nuclease activity, and complex formation of six mre11 mutants with substitutions of invariant residues within phosphoesterase motifs I, II, and III. All six mutants were equally impaired for the exonuclease activity of Mre11. Consistent with previous studies (Furuse et al. 1998; Lewis et al. 2004), we find that substitutions of Asp16 within motif I confer the most severe DNA repair and telomere length defects. However, these defects appear to be related to a deficiency in complex formation as interactions between Rad50 or Xrs2 and Mre11-D16A or Mre11-D16N are severely compromised, whereas the mre11 alleles with less severe DNA repair and telomere maintenance defects exhibit stable complex formation.

The early transcriptional region 4 (E4) of adenovirus type 5 (Ad5) encodes gene products that modulate splicing, apoptosis, transcription, DNA replication, and repair pathways. Viruses lacking both E4orf3 and E4orf6 have a severe replication defect, partially characterized by the formation of genome concatemers. Concatemer formation is dependent upon the cellular Mre11 complex and is prevented by both the E4orf3 and E4orf6 proteins. The Mre11/Rad50/Nbs1 proteins are targeted for proteasome-mediated degradation by the Ad5 viral E1b55K/E4orf6 complex. The expression of Ad5 E4orf3 causes a redistribution of Mre11 complex members and results in their exclusion from viral replication centers. For this study, we further analyzed the interactions of E4 proteins from different adenovirus serotypes with the Mre11 complex. Analyses of infections with serotypes Ad4 and Ad12 demonstrated that the degradation of Mre11/Rad50/Nbs1 proteins is a conserved feature of the E1b55K/E4orf6 complex. Surprisingly, Nbs1 and Rad50 were localized to the replication centers of both Ad4 and Ad12 viruses prior to Mre11 complex degradation. The transfection of expression vectors for the E4orf3 proteins of Ad4 and Ad12 did not alter the localization of Mre11 complex members. The E4orf3 proteins of Ad4 and Ad12 also failed to complement defects in both concatemer formation and late protein production of a virus with a deletion of E4. These results reveal surprising differences among the highly conserved E4orf3 proteins from different serotypes in the ability to disrupt the Mre11 complex.

The Mre11 complex (in Saccharomyces cerevisiae: Mre11, Rad50 and Xrs2) influences multiple facets of chromosome break metabolism. A conserved feature of the Mre11 complex is a zinc-coordinating motif in Rad50 called the Rad50 hook. We established a diploid yeast strain, rad50(hook), in which Rad50 is encoded in halves, one from each of the two RAD50 alleles, with the residues constituting the hook deleted. In all respects, rad50(hook) phenocopies complete Rad50 deficiency. Replacing the hook domain with a ligand-inducible FKBP dimerization cassette partially mitigated all phenotypes in a ligand-dependent manner. The data indicate that the Rad50 hook is critical for Mre11 complex-dependent DNA repair, telomere maintenance and meiotic double-strand break formation. Sister chromatid cohesion was unaffected by Rad50 deficiency, suggesting that molecular bridging required for recombinational DNA repair is qualitatively distinct from cohesin-mediated sister chromatid cohesion.

In Saccharomyces cerevisiae, telomere replication occurs in late S phase and is accompanied by dynamic remodeling of its protein components. Here, we show that MRX (Mre11-Rad50-Xrs2), an evolutionarily conserved protein complex involved in DNA double-strand break (DSB) repair, is recruited to the telomeres in late S phase. MRX is required for the late S phase-specific recruitment of ATR-like kinase Mec1 to the telomeres. Mec1, in turn, contributes to the assembly of the telomerase regulators Cdc13 and Est1 at the telomere ends. Our results provide a model for the hierarchical assembly of telomere-replication proteins in late S phase; this involves triggering by the loading of MRX onto the chromosome termini. The recruitment of DNA repair-related proteins to the telomeres at particular times in the cell cycle suggests that the normal terminus of a chromosome is recognized as a DSB during the course of replication.

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

Werner syndrome (WS) is an autosomal recessive disease that predisposes individuals to a wide range of cancers. The gene mutated in WS, WRN, encodes a member of the RecQ family of DNA helicases. The precise DNA metabolic processes in which WRN participates remain to be elucidated. However, it has been proposed that WRN might play an important role in the maintenance of genetic stability during DNA replication, possibly cooperating with other proteins. Here, we show that, following DNA replication arrest, WRN associates and colocalizes with the MRE11 complex at PCNA sites. We also provide evidence that both WRN/MRE11 complex association and proper WRN relocalization after HU treatment require a functional MRE11 complex. We demonstrate that mutations altering the functionality of WRN or that of the MRE11 complex result in chromosomal breakage during DNA replication and enhanced cell death following replication arrest. Finally, we show that the DNA breakage in replicating cells and apoptosis observed in WS are not enhanced by concomitant knock down of MRE11 by RNAi, indicating that WRN and MRE11 complex act in a common pathway. These results suggest a functional relationship between WRN and the MRE11 complex in response to replication fork arrest, disclosing a common action of WRN and the MRE11 complex in the pathway(s) preserving genome stability during DNA replication.

The Werner syndrome and the Nijmegen breakage syndrome are recessive genetic disorders that show increased genomic instability, cancer predisposition, hypersensitivity to mitomycin C and gamma-irradiation, shortened telomeres, and cell cycle defects. The protein mutated in the premature aging disease known as the Werner syndrome is designated WRN and is a member of the RecQ helicase family. The Nbs1 protein is mutated in Nijmegen breakage syndrome individuals and is part of the mammalian Mre11 complex together with the Mre11 and Rad50 proteins. Here, we show that WRN associates with the Mre11 complex via binding to Nbs1 in vitro and in vivo. In response to gamma-irradiation or mitomycin C, WRN leaves the nucleoli and co-localizes with the Mre11 complex in the nucleoplasm. We detect an increased association between WRN and the Mre11 complex after cellular exposure to gamma-irradiation. Small interfering RNA and complementation experiments demonstrated convergence of WRN and Nbs1 in response to gamma-irradiation or mitomycin C. Nbs1 is required for the Mre11 complex promotion of WRN helicase activity. Taken together, these results demonstrate a functional link between the two genetic diseases with partially overlapping phenotypes in a pathway that responds to DNA double strand breaks and interstrand cross-links.

Class switch recombination (CSR) is a region-specific, transcriptionally regulated, nonhomologous recombinational process that is initiated by activation-induced cytidine deaminase (AID). The initial lesions in the switch (S) regions are processed and resolved, leading to a recombination of the two S regions involved. The mechanism involved in the repair and ligation of the broken DNA ends is however still unclear. Here, we describe that switching is less efficient in cells from patients with Mre11 deficiency (Ataxia-Telangiectasia-like disorder, ATLD) and, more importantly, that the switch recombination junctions resulting from the in vivo switching events are aberrant. There was a trend toward an increased usage of microhomology (≥4 bp) at the switch junctions in both ATLD and Nijmegen breakage syndrome (NBS) patients. However, the DNA ends were not joined as “perfectly” as those from Ataxia-Telangiectasia (A-T) patients and 1–2 bp mutations or insertions were often observed. In switch junctions from ATLD patients, there were fewer base substitutions due to transitions and, most strikingly, the substitutions that occurred most often in controls, C → T transitions, never occurred at, or close to, the junctions derived from the ATLD patients. In switch junctions from NBS patients, all base substitutions were observed at the G/C nucleotides, and transitions were preferred. These data suggest that the Mre11-Rad50-Nbs1 complex (Mre11 complex) is involved in the nonhomologous end joining pathway in CSR and that Mre11, Nbs1, and protein mutated in ataxia-telangiectasia (ATM) might have both common and independent roles in this process.

NFBD1/MDC1 (mediator of DNA damage checkpoint 1) is a nuclear factor with an amino-terminal FHA (forkhead-associated) domain and a tandem repeat of BRCT (breast cancer susceptibility gene-1 carboxyl terminus) domains. We have previously shown that NFBD1 is an early participant in DNA damage signaling pathways and that ionizing radiation-induced nuclear foci (IRIF) of NFBD1 colocalize with several DNA checkpoint signaling and repair factors. We report here that NFBD1 physically associates with ATM, p53, components of the MRE11-RAD50-NBS1 (MRN) complex, and gamma-H2AX. An overexpressed FHA domain-containing fragment of NFBD1 binds to endogenous NFBD1 and components of the MRN complex, but not to gamma-H2AX. This fragment interferes with IRIF formation by endogenous NFBD1, MRE11, or NBS1. A BRCT domain-containing fragment of NFBD1 binds to gamma-H2AX and 53BP1, but not to components of the MRN complex, and abolishes IRIF formation by NFBD1, MRE11, NBS1, 53BP1, CHK2 phospho-T68, gamma-H2AX, and possible ATM/ATR substrates recognized by anti-phospho-SQ/TQ antibody. These results suggest that NFBD1 is an ATM/ATR-dependent organizer that recruits DNA checkpoint signaling and repair proteins to the sites of DNA damage.

The induction of DNA double-strand breaks (DSBs) culminates in the activation of cell cycle checkpoint responses and DNA repair machinery. The mechanism of DSB detection remains unclear although many candidate sensor proteins have been identified through cytologic, biochemical and genetic studies. In light of recent advances in our understanding of the cellular response to DSBs, we have proposed criteria for defining sensor proteins. We discuss the possible role of the Mre11 complex as a primary damage sensor and the complex relationship between DNA damage sensors, transducers and mediators.

The Mre11-Rad50-Nbs1 protein complex has emerged as a central player In the cellular DNA damage response. Mutations In scaA N B S 1 , which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti-topoisomerase I drug camptothecin. We have used the scaA N B S 1 cDNA as a bait in a yeast two-hybrid screening and report the Identification of the A. nidulans Mre11 homologue (mreA). The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents. Septation in A. nidulans is dependent not only on the uvsB A T R gene, but also on the mre11 complex. scaA N B S 1 and mreA genes are both Involved in the DNA replication checkpoint whereas mreA Is specifically involved in the intra-S-phase checkpoint. ScaA N B S 1 also participates In G2-M checkpoint control upon DNA damage caused by MMS. In addition, the scaA N B S 1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A. nldulans. Consistent with this view, the Mre11 complex and the uvsC R A D 5 1 gene are highly expressed at the mRNA level during the sexual development.

Inverted repeats (IRs) that can form a hairpin or cruciform structure are common in the human genome and may be sources of instability. An IR involving the human Alu sequence (Alu-IR) has been studied as a model of such structures in yeast. We found that an Alu-IR is a mitotic recombination hotspot requiring MRE11/RAD50/XRS2 and SAE2. Using a newly developed approach for mapping rare double-strand breaks (DSBs), we established that induction of recombination results from breaks that are terminated by hairpins. Failure of the mre11, rad50, xrs2, and sae2 mutants to process the hairpins blocks recombinational repair of the DSBs and leads to generation of chromosome inverted duplications. Our results suggest an additional role for the Mre11 complex in maintaining genome stability.