Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells

Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB). On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers. To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins. Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM. Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells. Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage.     

Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization

The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.     

Analysis of ionizing radiation-induced foci of DNA damage repair proteins

Repair of DNA double-strand breaks by homologous recombination requires an extensive set of proteins. Among these proteins are Rad51 and Mre11, which are known to re-localize to sites of DNA damage into nuclear foci. Ionizing radiation-induced foci can be visualized by immuno-staining. Published data show a large variation in the number of foci-positive cells and number of foci per nucleus for specific DNA repair proteins. The experiments described here demonstrate that the time after induction of DNA damage influenced not only the number of foci-positive cells, but also the size of the individual foci. The dose of ionizing radiation influenced both the number of foci-positive cells and the number of foci per nucleus. Furthermore, ionizing radiation-induced foci formation depended on the cell cycle stage of the cells and the protein of interest that was investigated. Rad51 and Mre11 foci seemed to be mutually exclusive, though a small subset of cells did show co-localization of these proteins, which suggests a possible cooperation between the proteins at a specific moment during DNA repair.     

Nibrin forkhead-associated domain and breast cancer C-terminal domain are both required for nuclear focus formation and phosphorylation

The Mre11.Rad50.nibrin protein complex plays an essential role in the mammalian cellular response to DNA double-strand breaks. The disorder Nijmegen breakage syndrome (NBS) results from mutations in the NBS1 gene that encodes nibrin, and NBS cells are radiosensitive and defective in S-phase checkpoint activation following irradiation. In response to radiation, nibrin is phosphorylated by Atm, and the Mre11.Rad50.nibrin complex relocalizes to form punctate nuclear foci. The N terminus of nibrin contains a forkhead-associated (FHA) domain and a breast cancer C-terminal (BRCT) domain, the functions of which are unclear. To determine the role of the FHA and BRCT domains in nibrin function, we have performed site-directed mutagenesis of conserved residues in these motifs. Mutations in the nibrin FHA and BRCT domains did not affect interaction with Mre11.Rad50 or nuclear localization of the complex. However, mutation of conserved residues in either domain disrupted nuclear focus formation and blocked nibrin phosphorylation after irradiation, suggesting that these events may be functionally interdependent. Despite an effect on nibrin phosphorylation, expression of the FHA or BRCT mutants in NBS cells restored the downstream phosphorylation of Chk2 and Smc1, necessary for S-phase checkpoint activation. None of the mutations revealed separate functions for the FHA or BRCT domains, suggesting they do not function independently.     

Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses

Rad50/Mre11/NBS1 (R/M/N) is a multi-functional protein complex involved in DNA repair, cell cycle checkpoint activation, DNA replication and replication block-induced responses. Ionizing radiation (IR) induces the phosphorylation of NBS1 and nuclear foci formation of the complex. Although it has been suggested that the R/M/N complex is associated with DNA damage sites, we present here biochemical evidence for chromatin association of the complex. We show that the chromatin association of R/M/N is independent of IR and ataxia telangiectasia mutated (ATM). We also demonstrate that optimal chromatin association of the Rad50/Mre11/NBS1 proteins requires both the conserved forkhead-associated (FHA) and breast cancer C-terminus (BRCT) domains of NBS1. Moreover, both these domains of NBS1 are required for its phosphorylation on Ser343 but not on Ser278. Importantly, both the FHA and BRCT domains are essential for IR-induced foci (IRIF) formation of R/M/N and S phase checkpoint activation, but only the BRCT domain is needed for cell survival after IR. These data demonstrate that the FHA and BRCT domains of NBS1 are crucial for the functions of the R/M/N complex.     

Rapid activation of ATR by ionizing radiation requires ATM and Mre11

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.     

Active role for nibrin in the kinetics of atm activation

The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. Previously, we showed that a C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, was sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, we expressed an Mre11 transgene with a C-terminal NLS sequence in NBS fibroblasts. The Mre11-NLS protein complexed with Rad50, localized to the nucleus in NBS fibroblasts, and associated with chromatin. However, Atm autophosphorylation was not stimulated in cells expressing Mre11-NLS, nor were downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, we expressed nibrin transgenes lacking the Atm binding domain in NBS fibroblasts. The nibrin DeltaAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation was dramatically reduced after irradiation in NBS cells expressing the nibrin DeltaAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction.     

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.     

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.     

Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex

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.     

NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres

Nijmegen breakage syndrome, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. The NBS1 gene product, p95 (NBS1 or nibrin) forms a complex with Rad50 and Mre11. Cells deficient in the formation of this complex are defective in DNA double-strand break repair, cell cycle checkpoint control, and telomere length maintenance. How the NBS1 complex is involved in telomere length maintenance remains unclear. Here we show that the C-terminal region of NBS1 interacts directly with a telomere repeat binding factor, TRF1, by both yeast two-hybrid and in vivo DNA-coimmunoprecipitation assays. NBS1 and Mre11 colocalize with TRF1 at promyelocytic leukemia (PML) nuclear bodies in immortalized telomerase-negative cell lines, but rarely in telomerase-positive cell lines. The translocation of NBS1 to PML bodies occurs specifically during late S to G(2) phases of the cell cycle and coincides with active DNA synthesis in these NBS1-containing PML bodies. These results suggest that NBS1 may be involved in alternative lengthening of telomeres in telomerase-negative immortalized cells.     

DNA damage-dependent nuclear dynamics of the Mre11 complex

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.     

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.     

A mutant allele of MRE11 found in mismatch repair-deficient tumor cells suppresses the cellular response to DNA replication fork stress in a dominant negative manner

The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3'-5' exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3' single-strand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time- and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner.     

Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.     

Replication Protein A is Required for Etoposide-Induced Assembly of MRE11/RAD50/NBS1 Complex Repair Foci

The presence of DNA damage activates a specific response cascade culminating in DNA repair activity and cell cycle checkpoints. Although the type of lesion dictates what proteins are involved in the response, replication protein A (RPA) and the Mre11/Rad50/Nbs1 complex (MRN) respond to most types of lesions. To examine the relationship of RPA and the MRN complex in DNA damage responses, we used siRNA-mediated protein depletion of RPA-p70 and Mre11. Depletion of RPA-p70 decreased the ability of cells to form phospho-Nbs1 foci and increased levels of DNA double-strand breaks (DSBs) following treatment with etoposide (ETOP). In contrast, depletion of Mre11 led to increased levels of RPA-p34 foci formation, but abrogated phospho-RPA-p34 foci formation. These data support a role for RPA as an initial signal/sensor for DNA damage that facilitates recruitment of MRN and ATM/ATR to sites of damage, where they then work together to fully activate the DNA damage response.     

Independent roles for nibrin and Mre11-Rad50 in the activation and function of Atm

The Atm protein kinase and Mre11-Rad50-nibrin (MRN) complex play an integral role in the cellular response to DNA double-strand breaks. Mutations in Mre11 and nibrin result in the radiosensitivity disorders ataxia-telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS), respectively. Cells from ATLD and NBS patients are deficient in activation of the Atm protein kinase and phosphorylation of downstream Atm targets following irradiation. However, the roles of individual MRN complex proteins in Atm function are not clear, because the mutations in NBS and ATLD cells result in global effects on the MRN complex. Previously we showed that the C-terminal 100 amino acids of nibrin were necessary and sufficient to translocate the MRN complex to the nucleus. Here we have taken advantage of this feature of nibrin to create isogenic cell lines lacking either nibrin or Mre11-Rad50 in the nucleus. We found that nuclear expression of Mre11-Rad50, but not nibrin, stimulated Atm activation at early times after low doses of radiation. At later times or higher doses of irradiation, Atm activation was independent of Mre11-Rad50 or nibrin. The requirement of MRN complex proteins for downstream Atm phosphorylation events following irradiation was more complex. Phosphorylation of nibrin and Chk2 by Atm required Mre11-Rad50 expression in the nucleus at early times after irradiation, reflecting the stimulation of Atm activation by Mre11-Rad50. By contrast, autophosphorylation of Chk2 and phosphorylation of Smc1 at Ser-957 was dependent on the MRN complex 60 min after irradiation, even though Atm was activated at that time point. These results indicate an independent role for Mre11-Rad50 in the activation of Atm and suggest nibrin and/or Mre11-Rad50 also act as adaptors for some downstream Atm phosphorylation events.     

Chk2 Activation Dependence on Nbs1 after DNA Damage

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.