Double strand break metabolism and cancer susceptibility: lessons from the mre11 complex

Hypomorphic mutants affecting the Mre11 complex components Mre11 (Mre11(ATLD1/ATLD1)) and Nbs1 (Nbs1(DeltaB/DeltaB)) have been established in the mouse. These mutations recapitulate those inherited in human chromosome fragility syndromes, the ataxia-telangiectasia like disorder and Nijmegen breakage syndrome. At the cellular level, the human and murine mutants exhibit defects in the intra S and G2/M checkpoints and marked chromosome instability. Whereas these outcomes are associated with predisposition to malignancy in humans, similar predisposition was not observed in either Mre11(ATLD1/ATLD1) or Nbs1(DeltaB/DeltaB) mice. These data demonstrate that chromosome breakage per se is insufficient to significantly enhance the initiation of tumorigenesis. However, these mutations greatly enhanced the risk of malignancy in p53+/- mice. We propose that proper metabolism of chromosome breaks arising during DNA replication is uniquely important for suppressing loss of heterozygosity and thus the penetrance of recessive oncogenic lesions.     

Chk2 suppresses the oncogenic potential of DNA replication-associated DNA damage

The Mre11 complex (Mre11, Rad50, and Nbs1) and Chk2 have been implicated in the DNA-damage response, an inducible process required for the suppression of malignancy. The Mre11 complex is predominantly required for repair and checkpoint activation in S phase, whereas Chk2 governs apoptosis. We examined the relationship between the Mre11 complex and Chk2 in the DNA-damage response via the establishment of Nbs1(DeltaB/DeltaB) Chk2(-/-) and Mre11(ATLD1/ATLD1) Chk2(-/-) mice. Chk2 deficiency did not modify the checkpoint defects or chromosomal instability of Mre11 complex mutants; however, the double-mutant mice exhibited synergistic defects in DNA-damage-induced p53 regulation and apoptosis. Nbs1(DeltaB/DeltaB) Chk2(-/-) and Mre11(ATLD1/ATLD1) Chk2(-/-) mice were also predisposed to tumors. In contrast, DNA-PKcs-deficient mice, in which G1-specific chromosome breaks are present, did not exhibit synergy with Chk2(-/-) mutants. These data suggest that Chk2 suppresses the oncogenic potential of DNA damage arising during S and G2 phases of the cell cycle.     

Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes

The Mre11/Rad50 complex is a critical component of the cellular response to DNA double-strand breaks, in organisms ranging from archaebacteria to humans. In mammalian cells, Mre11/Rad50 (M/R) associates with a third component, Nbs1, that regulates its activities and is targeted by signaling pathways that initiate DNA damage-induced checkpoint responses. Mutations in the genes that encode Nbs1 and Mre11 are responsible for the human radiation sensitivity disorders Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, which are characterized by defective checkpoint responses and high levels of chromosomal abnormalities. Here we demonstrate nucleotide-dependent DNA binding by the human M/R complex that requires the Nbs1 protein and is specific for double-strand DNA duplexes. Efficient DNA binding is only observed with non-hydrolyzable analogs of ATP, suggesting that ATP hydrolysis normally effects DNA release. The alleles of MRE11 associated with ATLD and the C-terminal Nbs1 polypeptide associated with NBS were expressed with the other components and found to form triple complexes except in the case of ATLD 3/4, which exhibits variability in Nbs1 association. The ATLD 1/2, ATLD 3/4, and p70 M/R/N complexes exhibit nucleotide-dependent DNA binding and exonuclease activity equivalent to the wild-type enzyme, although the ATLD complexes both show reduced activity in endonuclease assays. Sedimentation equilibrium analysis of the recombinant human complexes indicates that Mre11 is a stable dimer, Mre11 and Nbs1 form a 1:1 complex, and both M/R and M/R/N form large multimeric assemblies of approximately 1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.     

Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template.

The Mre11-Rad50-Nbs1 (MRN) complex is providing paradigm-shifting results of exceptional biomedical interest. MRN is among the earliest respondents to DNA double-strand breaks (DSBs), and MRN mutations cause the human cancer predisposition diseases Nijmegen breakage syndrome and ataxia telangiectasia-like disorder (ATLD). MRN's 3-protein multidomain composition promotes its central architectural, structural, enzymatic, sensing, and signaling functions in DSB responses. To organize the MRN complex, the Mre11 exonuclease directly binds Nbs1, DNA, and Rad50. Rad50, a structural maintenance of chromosome (SMC) related protein, employs its ATP-binding cassette (ABC) ATPase, Zn hook, and coiled coils to bridge DSBs and facilitate DNA end processing by Mre11. Contributing to MRN regulatory roles, Nbs1 harbors N-terminal phosphopeptide interacting FHA and BRCT domains, as well as C-terminal ataxia telangiectasia mutated (ATM) kinase and Mre11 interaction domains. Current emerging structural and biological evidence suggests that MRN has 3 coupled critical roles in DSB sensing, stabilization, signaling, and effector scaffolding: (1) expeditious establishment of protein--nucleic acid tethering scaffolds for the recognition and stabilization of DSBs; (2) initiation of DSB sensing, cell-cycle checkpoint signaling cascades, and establishment of epigenetic marks via the ATM kinase; and (3) functional regulation of chromatin remodeling in the vicinity of a DSB.     

The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.     

The Mre11 complex and the metabolism of chromosome breaks: the importance of communicating and holding things together

The conserved Mre11 complex (Mre11, Rad50, and Nbs1) plays a role in each aspect of chromosome break metabolism. The complex acts as a break sensor and functions in the activation and propagation of signaling pathways that govern cell cycle checkpoint functions in response to DNA damage. In addition, the Mre11 complex influences recombinational DNA repair through promoting recombination between sister chromatids. The Mre11 complex is required for mammalian cell viability but hypomorphic mutants of Mre11 and Nbs1 have been identified in human genetic instability disorders. These hypomorphic mutations, as well as those identified in yeast, have provided a benchmark for establishing mouse models of Mre11 complex deficiency. In addition to consideration of Mre11 complex functions in human cells and yeast, this review will discuss the characterization of mouse models and insight gleaned from those models regarding the metabolism of chromosome breaks. The current picture of break metabolism supports a central role for the Mre11 complex at the interface of chromosome stability and the regulation of cell growth. Further genetic analysis of the Mre11 complex will be an invaluable tool for dissecting its function on an organismal level and determining its role in the prevention of malignancy.     

Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice

In this study, mice expressing one of the two Mre11 alleles inherited in the human ataxia-telangiectasia like disorder (A-TLD) were derived. The mutation had a profound maternal effect on embryonic viability, revealing an acute requirement for Mre11 complex function in early embryogenesis. Mre11(ATLD1/ATLD1) mice exhibited several indices of impaired ATM function. The mice also exhibited pronounced chromosomal instability. Despite this phenotypic spectrum, the animals were not prone to malignancy. These data indicate that defective cell cycle checkpoints and chromosomal instability are insufficient to significantly enhance the initiation of tumorigenesis. In contrast, the latency of malignancy in p53(+/-) mice was dramatically reduced. We propose that in Mre11(ATLD1/ATLD1) mice, genome instability and cell cycle checkpoint defects reduce viability in early embryos and in proliferating cells, while promoting malignancy in the context of an initiating lesion.     

Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.     

Linkage between Werner syndrome protein and the Mre11 complex via Nbs1

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.     

Ataxia-telangiectasia-like disorder (ATLD)-its clinical presentation and molecular basis

Comparison of the clinical and cellular phenotypes of different genomic instability syndromes provides new insights into functional links in the complex network of the DNA damage response. A prominent example of this principle is provided by examination of three such disorders: ataxia-telangiectasia (A-T) caused by lack or inactivation of the ATM protein kinase, which mobilises the cellular response to double strand breaks in the DNA; ataxia-telangiectasia-like disease (ATLD), a result of deficiency of the human Mre11 protein; and the Nijmegen breakage syndrome (NBS), which represents defective Nbs1 protein. Mre11 and Nbs1 are members of the Mre11/Rad50/Nbs1 (MRN) protein complex. MRN and its individual components are involved in different responses to cellular damage induced by ionising radiation and radiomimetic chemicals, including complexing with chromatin and with other damage response proteins, formation of radiation-induced foci, and the induction of different cell cycle checkpoints. The phosphorylation of Nbs1 by ATM would indicate that ATM acts upstream of the MRN complex. Consistent with this were the suggestions that ATM could be activated in the absence of fully functional Nbs1 protein. In contrast, the regulation of some ATM target proteins, e.g. Smc1 requires the MRN complex as well as ATM. Nbs1 may, therefore, be both a substrate for ATM and a mediator of ATM function. Recent studies that indicate a requirement of the MRN complex for proper ATM activation suggest that the relationship between ATM and the MRN complex in the DNA damage response is yet to be fully determined. Despite the fact that both Mre11 and Nbs1 are part of the same MRN complex, deficiency in either protein in humans does not lead to the same clinical picture. This suggests that components of the complex may also act separately.     

Mutation Inactivation of Nijmegen Breakage Syndrome Gene (NBS1) in Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma

Nijmegen breakage syndrome (NBS) with NBS1 germ-line mutation is a human autosomal recessive disease characterized by genomic instability and enhanced cancer predisposition. The NBS1 gene codes for a protein, Nbs1(p95/Nibrin), involved in the processing/repair of DNA double-strand breaks. Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with several genomic alterations. Recent studies have shown that heterozygous NBS1 mice exhibited a higher incidence of HCC than did wild-type mice. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of human primary liver cancer, including HBV-associated HCC and intrahepatic cholangiocarcinoma (ICC). Eight missense NBS1 mutations were identified in six of 64 (9.4%) HCCs and two of 18 (11.1%) ICCs, whereas only one synonymous mutation was found in 89 control cases of cirrhosis and chronic hepatitis B. Analysis of the functional consequences of the identified NBS1 mutations in Mre11-binding domain showed loss of nuclear localization of Nbs1 partner Mre11, one of the hallmarks for Nbs1 deficiency, in one HCC and two ICCs with NBS1 mutations. Moreover, seven of the eight tumors with NBS1 mutations had at least one genetic alteration in the TP53 pathway, including TP53 mutation, MDM2 amplification, p14ARF homozygous deletion and promoter methylation, implying a synergistic effect of Nbs1 disruption and p53 inactivation. Our findings provide novel insight on the molecular pathogenesis of primary liver cancer characterized by mutation inactivation of NBS1, a DNA repair associated gene.     

The Mre11 Complex and the Response to Dysfunctional Telomeres

In this study, we examine the telomeric functions of the mammalian Mre11 complex by using hypomorphic Mre11 and Nbs1 mutants (Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB, respectively). No telomere shortening was observed in Mre11^ATLD1/ATLD1 cells after extensive passage through culture, and the rate of telomere shortening in telomerase-deficient (Tert^Δ/Δ) Mre11^ATLD1/ATLD1 cells was the same as that in Tert^Δ/Δ alone. Although telomeres from late-passage Mre11^ATLD1/ATLD1 Tert^Δ/Δ cells were as short as those from Tert^Δ/Δ, the incidence of telomere fusions was reduced. This effect on fusions was also evident upon acute telomere dysfunction in Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB cells rendered Trf2 deficient by cre-mediated TRF2 inactivation than in wild-type cells. The residual fusions formed in Mre11 complex mutant cells exhibited a strong tendency toward chromatid fusions, with an almost complete bias for fusion of telomeres replicated by the leading strand. Finally, the response to acute telomere dysfunction was strongly impaired by Mre11 complex hypomorphism, as the formation of telomere dysfunction-induced DNA damage foci was reduced in both cre-infected Mre11^ATLD1/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^F/F cells. These data indicate that the Mre11 complex influences the cellular response to telomere dysfunction, reminiscent of its influence on the response to interstitial DNA breaks, and suggest that it may promote telomeric DNA end processing during DNA replication.The Mre11 complex (in mammals, Mre11, Rad50, and Nbs1) plays a central role in the cellular response to DNA double-strand breaks (DSBs). The Mre11 complex acts as a DSB sensor, promoting the activation of ATM-dependent DNA damage signaling pathways, DNA repair, and apoptosis. In addition, the complex plays a direct role in recombinational DNA repair, influencing both homologous recombination and nonhomologous end joining (NHEJ) (39). The Mre11 complex''s diverse functions in the DNA damage response are likely predicated on its physical association with chromatin. In this regard, one of the least-understood roles of the Mre11 complex in mammals is its association with telomeres.In mammals, telomeric DNA consists of double-stranded TTAGGG repeats ending in a single-stranded 3′ G overhang, and an array of telomere binding proteins called the shelterin complex that function to prevent telomeres from being recognized as DNA breaks (33). DNA of the overhang invades the double-stranded telomeric repeat sequence to form a t-loop structure (14, 32). The formation of the t-loop requires the telomere protection and remodeling proteins that make up the shelterin complex (7), and these may also contribute to telomere length regulation by preventing telomerase access to chromosomal ends.Data regarding the role of the Mre11 complex at the telomere have implicated the Mre11 complex in several aspects of telomere maintenance and function. For example, it has been suggested that the Mre11 complex may promote formation of the 3′ telomeric overhang by influencing 5′-to-3′ resection of newly replicated chromosome ends (6). In Saccharomyces cerevisiae, the Mre11 complex recruits the ATM orthologue, Tel1, which is in turn required to recruit telomerase (12, 45). Consequently, Mre11 complex deficiency results in telomere shortening. In mammals, recruitment of telomerase is thought to be regulated primarily by the telomeric protein components TRF1, TPP1, and POT1 (24, 46, 53). However, telomere shortening has also been noted to occur in cell lines from Nijmegen breakage syndrome (NBS) patients in which a hypomorphic Nbs1 allele is expressed, leading to the suggestion that the Mre11 complex may also promote telomerase function in mammals (36). The Mre11 complex associates with telomeres through its interaction with the shelterin component Trf2, apparently in a cell cycle-dependent manner (47, 54). The significance of this physical association is unclear, as genetic depletion of Rad50, a component of the Mre11 complex, does not phenocopy depletion of Trf2 in most respects (1).To examine the function of the Mre11 complex at mammalian telomeres, we established mouse embryonic fibroblasts (MEFs) derived from a mouse expressing the hypomorphic Mre11^ATLD1 allele, crossed to telomerase deficient Tert^Δ/Δ mice (23, 42), and assessed the rate of telomere shortening. Mre11 complex hypomorphism in MEFs did not affect telomere length, irrespective of telomerase status. In Mre11^ATLD1/ATLD1 Tert^Δ/Δ cells, the fusion of eroded telomeres was reduced compared to Tert^Δ/Δ cells with telomeres shortened to the same extent, suggesting that the Mre11 complex is involved in the response to critically short telomeres. This interpretation was supported by data obtained using a conditional Trf2 allele to generate acute telomere dysfunction in Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB cells. Collectively the data support a role for the Mre11 complex in the recognition and signaling of dysfunctional telomeres. The character of fusions arising in cre-infected Mre11^ATLD1/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^F/F cells further suggests that the Mre11 complex may influence the processing of chromosome ends following DNA replication en route to t-loop formation.     

Mre11 assembles linear DNA fragments into DNA damage signaling complexes

Mre11/Rad50/Nbs1 complex (MRN) is essential to suppress the generation of double-strand breaks (DSBs) during DNA replication. MRN also plays a role in the response to DSBs created by DNA damage. Hypomorphic mutations in Mre11 (which causes an ataxia-telangiectasia-like disease [ATLD]) and mutations in the ataxia-telangiectasia-mutated (ATM) gene lead to defects in handling damaged DNA and to similar clinical and cellular phenotypes. Using Xenopus egg extracts, we have designed a simple assay to define the biochemistry of Mre11. MRN is required for efficient activation of the DNA damage response induced by DSBs. We isolated a high molecular weight DNA damage signaling complex that includes MRN, damaged DNA molecules, and activated ATM. Complex formation is partially dependent upon Zn²⁺ and requires an intact Mre11 C-terminal domain that is deleted in some ATLD patients. The ATLD truncation can still perform the role of Mre11 during replication. Our work demonstrates the role of Mre11 in assembling DNA damage signaling centers that are reminiscent of irradiation-induced foci. It also provides a molecular explanation for the similarities between ataxia-telangiectasia (A-T) and ATLD.     

The Mre11 complex and ATM: collaborating to navigate S phase

Recently, findings regarding a group of cancer predisposition and chromosome instability syndromes, Nijmegen breakage syndrome (NBS), the ataxia-telangiectasia-like disorder (A-TLD) and ataxia telangiectasia have shed light on the unexpected role of recombinational DNA repair proteins in DNA-damage-dependent cell-cycle regulation. Mutations in the Mre11 complex cause A-TLD and NBS. In addition, functions of the Mre11 complex have been biochemically linked to ATM, the large protein kinase that is defective in ataxia-telangiectasia cells by the observation that Nbs1 is a bona fide substrate of the ATM kinase.     

The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.     

A murine model of Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.     

Dual functions of Nbs1 in the repair of DNA breaks and proliferation ensure proper V(D)J recombination and T-cell development

Immunodeficiency and lymphoid malignancy are hallmarks of the human disease Nijmegen breakage syndrome (NBS; OMIM 251260), which is caused by NBS1 mutations. Although NBS1 has been shown to bind to the T-cell receptor alpha (TCRα) locus, its role in TCRβ rearrangement is unclear. Hypomorphic mutations of Nbs1 in mice and patients result in relatively mild T-cell deficiencies, raising the question of whether the truncated Nbs1 protein might have clouded a certain function of NBS1 in T-cell development. Here we show that the deletion of the entire Nbs1 protein in T-cell precursors (Nbs1(T-del)) results in severe lymphopenia and a hindrance to the double-negative 3 (DN3)-to-DN4 transition in early T-cell development, due to abnormal TCRβ coding and signal joints as well as the functions of Nbs1 in T-cell expansion. Chromatin immunoprecipitation (ChIP) analysis of the TCR loci reveals that Nbs1 depletion compromises the loading of Mre11/Rad50 to V(D)J-generated DNA double-strand breaks (DSBs) and thereby affects resection of DNA termini and chromatin conformation of the postcleavage complex. Although a p53 deficiency relieves the DN3→DN4 transition block, neither a p53 deficiency nor ectopic expression of TCRαβ rescues the major T-cell loss in Nbs1(T-del) mice. All together, these results demonstrate that Nbs1's functions in both repair of V(D)J-generated DSBs and proliferation are essential for T-cell development.     

Molecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere maintenance

The human MRN complex is a multisubunit nuclease that is composed of Mre11, Rad50, and Nbs1 and is involved in homologous recombination and DNA damage checkpoints. Mutations of the MRN genes cause genetic disorders such as Nijmegen breakage syndrome. Here we identified a Schizosaccharomyces pombe nbs1(+) homologue by screening for mutants with mutations that caused methyl methanesulfonate (MMS) sensitivity and were synthetically lethal with the rad2Delta mutation. Nbs1 physically interacts with the C-terminal half of Rad32, the Schizosaccharomyces pombe Mre11 homologue, in a yeast two-hybrid assay. nbs1 mutants showed sensitivities to gamma-rays, UV, MMS, and hydroxyurea and displayed telomere shortening similar to the characteristics of rad32 and rad50 mutants. nbs1, rad32, and rad50 mutant cells were elongated and exhibited abnormal nuclear morphology. These findings indicate that S. pombe Nbs1 forms a complex with Rad32-Rad50 and is required for homologous recombination repair, telomere length regulation, and the maintenance of chromatin structure. Amino acid sequence features and some characteristics of the DNA repair function suggest that the S. pombe Rad32-Rad50-Nbs1 complex has functional similarity to the corresponding MRN complexes of higher eukaryotes. Therefore, S. pombe Nbs1 will provide an additional model system for studying the molecular function of the MRN complex associated with genetic diseases.     

Rad50S alleles of the Mre11 complex: questions answered and questions raised

We find that Rad50S mutations in yeast and mammals exhibit constitutive PIKK (PI3-kinase like kinase)-dependent signaling [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-4354.]. The signaling depends on Mre11 complex functions, consistent with its role as a DNA damage sensor. Rad50S is distinct from hypomorphic mutations of Mre11 and Nbs1 in mammals [M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-3054.; J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, Le M. Beau, J.R. Yates, III, L. Hays, W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93 (1998) 477-486.; G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers, A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99 (1999) 577-587.; B.R. Williams, O.K. Mirzoeva, W.F. Morgan, J. Lin, W. Dunnick, J.H. Petrini, A murine model of nijmegen breakage syndrome. Curr. Biol. 12 (2002) 648-653.; J.W. Theunissen, M.I. Kaplan, P.A. Hunt, B.R. Williams, D.O. Ferguson, F.W. Alt, J.H. Petrini, Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol. Cell 12 (2003) 1511-1523.] and the Mre11 complex deficiency in yeast [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; D'D. Amours, S.P. Jackson, The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 15 (2001) 2238-49. ; M. Grenon, C. Gilbert, N.F. Lowndes, Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3 (2001) 844-847. ] where the signaling is compromised. Herein, we describe evidence for chronic signaling by Rad50S and discuss possible mechanisms.