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1.
Lee JH  Lim DS 《The FEBS journal》2006,273(8):1630-1636
The Nbs1 protein associates with Mre11 and Rad50 proteins to form the Mre11-Rad50-Nbs1 complex, which plays an important role in the intracellular signaling pathway activated in response to DNA damage. Mutations in the genes for each of these three components of the Mre11-Rad50-Nbs1 complex result in human diseases characterized by genomic instability. Insight into the functions of Nbs1 in the DNA damage response mediated by the protein kinase, ataxia telangiectasia mutated, has been provided by recent studies. Nbs1 acts both as a downstream target of ataxia telangiectasia mutated in the S-phase checkpoint of the cell cycle as well as an upstream modulator or activator of ataxia telangiectasia mutated in the DNA damage response.  相似文献   

2.
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.  相似文献   

3.
The intra-S-phase checkpoint is a signaling pathway that induces slow DNA replication in the presence of DNA damage. In humans, defects in this checkpoint pathway might account for phenotypes seen in autosomal recessive diseases including ataxia telangiectasia-like disorder and Nijmegen breakage syndrome, where MRN complex components, Mre11 and Nbs1, are mutated. Here we provide evidence that the equivalent budding yeast complex, MRX (Mre11/Rad50/Xrs2), is not required for the intra-S-phase checkpoint in response to DNA alkylation damage, but is required in the presence of double-stranded DNA breaks. These data indicate, at least in budding yeast, that alternate pathways enforce replication slowing depending on the particular DNA lesion.  相似文献   

4.
The Mre11-Rad50-Nbs1 (MRN) complex has many biological functions: processing of double-strand breaks in meiosis, homologous recombination, telomere maintenance, S-phase checkpoint, and genome stability during replication. In the S-phase DNA damage checkpoint, MRN acts both in activation of checkpoint signaling and downstream of the checkpoint kinases to slow DNA replication. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5′ resection to create single-stranded DNA that is required for both signaling and homologous recombination. However, it is unclear whether resection is essential for all of the cellular functions of MRN. To dissect the various roles of MRN, we performed a structure–function analysis of nuclease dead alleles and potential separation-of-function alleles analogous to those found in the human disease ataxia telangiectasia-like disorder, which is caused by mutations in Mre11. We find that several alleles of rad32 (the fission yeast homologue of mre11), along with ctp1Δ, are defective in double-strand break repair and most other functions of the complex, but they maintain an intact S phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads us to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.  相似文献   

5.
Studies of human Nijmegen breakage syndrome (NBS) cells have led to the proposal that the Mre11/Rad50/ NBS1 complex, which is involved in the repair of DNA double-strand breaks (DSBs), might also function in activating the DNA damage checkpoint pathways after DSBs occur. We have studied the role of the homologous budding yeast complex, Mre11/Rad50/Xrs2, in checkpoint activation in response to DSB-inducing agents. Here we show that this complex is required for phosphorylation and activation of the Rad53 and Chk1 checkpoint kinases specifically in response to DSBs. Consistent with defective Rad53 activation, we observed defective cell-cycle delays after induction of DSBs in the absence of Mre11. Furthermore, after gamma-irradiation phosphorylation of Rad9, which is an early event in checkpoint activation, is also dependent on Mre11. All three components of the Mre11/Rad50/Xrs2 complex are required for activation of Rad53, however, the Ku80, Rad51 or Rad52 proteins, which are also involved in DSB repair, are not. Thus, the integrity of the Mre11/Rad50/Xrs2 complex is specifically required for checkpoint activation after the formation of DSBs.  相似文献   

6.
The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.  相似文献   

7.
Together with the Tel1 PI3 kinase, the Mre11/Rad50/Xrs2 (MRX) complex is involved in checkpoint activation in response to double-strand breaks (DSBs), a function also conserved in human cells by Mre11/Rad50/Nbs1 acting with ATM. It has been proposed that the yeast Tel1/MRX pathway is activated in the presence of DSBs that cannot be resected. The Mec1 PI3 kinase, by contrast, would be involved in detecting breaks that can be processed. The significance of a Mec1/MRX DSB-activated DNA damage checkpoint has yet to be reported. To understand whether the MRX complex works specifically with Tel1 or Mec1, we investigated MRX function in checkpoint activation in response to endonuclease-induced DSBs in synchronized cells. We found that the expression of EcoRI activated the G1 and intra-S phase checkpoints in a MRX- and Mec1-dependent, but Tel1-independent manner. The pathways identified here are therefore different from the Tel1/MRX pathway that was previously reported. Thus, our results demonstrate that MRX can function in concert with both Mec1 and Tel1 PI3K-like kinases to trigger checkpoint activation in response to DSBs. Importantly, we also describe a novel MRX-independent checkpoint that is activated in late S-phase when cells replicate their DNA in the presence of DSBs. The existence of this novel mode of checkpoint activation explains why several previous studies had reported that mutations in the MRX complex did not abrogate DSB-induced checkpoint activation in asynchronous cells.  相似文献   

8.
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.  相似文献   

9.
The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.  相似文献   

10.
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.  相似文献   

11.
The role of Mre11 phosphorylation in the cellular response to DNA double-strand breaks (DSBs) is not well understood. Here, we show that phosphorylation of Mre11 at SQ/TQ motifs by PIKKs (PI3 Kinase-related Kinases) induces MRN (Mre11–Rad50–Nbs1) complex dissociation from chromatin by reducing Mre11 affinity for DNA. Whereas phosphorylation of Mre11 at these residues is not required for DSB-induced ATM (Ataxia-Telangiectasia mutated) activation, abrogation of Mre11 dephosphorylation impairs ATM signaling. Our study provides a functional characterization of the DNA damage-induced Mre11 phosphorylation, and suggests that MRN inactivation participates in the down-regulation of damage signaling during checkpoint recovery following DSB repair.  相似文献   

12.
The Mre11 complex is a multisubunit nuclease that is composed of Mre11, Rad50 and Nbs1/Xrs2. Mutations in the genes that encode components of this complex result in DNA- damage sensitivity, genomic instability, telomere shortening and aberrant meiosis. The molecular defect that underlies these phenotypes has long been thought to be related to a DNA repair deficiency. However, recent studies have uncovered functions for the Mre11 complex in checkpoint signalling and DNA replication.  相似文献   

13.
Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.  相似文献   

14.
The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.  相似文献   

15.
Defining the factors that lead to genomic instability is one of the most important fields in cancer biology. DNA damage can arise from exogenous sources or as a result of normal cellular metabolism. Regardless of the cause, when damaged DNA is not properly repaired the genome acquires mutation(s). Under normal circumstances, to prevent such chromosome instability the cell activates the checkpoint response, which inhibits cell cycle progression until DNA repair is complete. The Mre11 complex is formed by three components: Mre11, Rad50, and Nbs1/Xrs2 and is involved in the signaling pathways that lead to both checkpoint activation and DNA repair. In response to DNA damage two functions of the complex will be discussed, one involves its role in initiating kinase activation and the second involves its ability to tether and link DNA strands. This review will highlight the functions of the Mre11 complex during the process of DNA double strand break recognition and repair, and during the process of replication. Understanding how the Mre11 complex is working at the molecular level is important for understanding why disruptions in components of the complex lead to genomic instability and cancer predisposition syndromes in humans.  相似文献   

16.
The Mre11-Rad50-Nbs1 (MRN) complex is required for mediating the S-phase checkpoint following UV treatment, but the underlying mechanism is not clear. Here we demonstrate that at least two mechanisms are involved in regulating the S-phase checkpoint in an MRN-dependent manner following UV treatment. First, when replication forks are stalled, MRN is required upstream of ataxia telangiectasia mutated and Rad3-related protein (ATR) to facilitate ATR activation in a substrate and dosage-dependent manner. In particular, MRN is required for ATR-directed phosphorylation of RPA2, a critical event in mediating the S-phase checkpoint following UV treatment. Second, MRN is a downstream substrate of ATR. Nbs1 is phosphorylated by ATR at Ser-343 when replication forks are stalled, and this phosphorylation event is also important for down-regulating DNA replication following UV treatment. Moreover, we demonstrate that MRN and ATR/ATR-interacting protein (TRIP) interact with each other, and the forkhead-associated/breast cancer C-terminal domains (FHA/BRCT) of Nbs1 play a significant role in mediating this interaction. Mutations in the FHA/BRCT domains do not prevent ATR activation but specifically impair ATR-mediated Nbs1 phosphorylation at Ser-343, which results in a defect in the S-phase checkpoint. These data suggest that MRN plays critical roles both upstream and downstream of ATR to regulate the S-phase checkpoint when replication forks are stalled.  相似文献   

17.
The plant Rad50-Mre11 protein complex   总被引:4,自引:0,他引:4  
The Rad50-Mre11-Xrs2/Nbs1 protein complex plays critical roles in cellular processes involving DNA ends. This complex is implicated in DNA recombination and replication, meiosis, telomere maintenance and cellular DNA damage responses. The Rad50 and Mre11 proteins are essential for viability in animals, although not in yeast. We have prepared antibodies to the Rad50 protein of the model plant Arabidopsis thaliana which recognize a 175 kDa protein in wild-type Arabidopsis protein extracts. Furthermore, we report here demonstration of the existence of the Rad50-Mre11 complex by co-immunoprecipitation of the Rad50 and Mre11 proteins from the plant cell extracts.  相似文献   

18.
Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5 degrees C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37 degrees C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair.  相似文献   

19.
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.  相似文献   

20.
The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the cellular DNA damage response. Mutations in scaANBS1, 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 scaANBS1 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 uvsBATR gene, but also on the mre11 complex. scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra-S-phase checkpoint. ScaANBS1 also participates in G2-M checkpoint control upon DNA damage caused by MMS. In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A. nidulans. Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development.  相似文献   

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