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Patrick Concannon - One of the best experts on this subject based on the ideXlab platform.
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Nuclear export of NBN is required for normal cellular responses to radiation.
Molecular and cellular biology, 2008Co-Authors: Christine Vissinga, Karen Cerosaletti, Tiong Chia Yeo, Sarah E. Warren, James V. Brawley, Jennifer Phillips, Patrick ConcannonAbstract:Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding Nibrin, a component of the MRE11/RAD50/Nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse Nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, Nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of Nibrin significantly altered the cellular distribution of Nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in Nibrin slowed the turnover of phosphorylated Nibrin after irradiation, indicating that nuclear export of Nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.
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Active role for Nibrin in the kinetics of atm activation.
Molecular and cellular biology, 2006Co-Authors: Karen Cerosaletti, Jocyndra A. Wright, Patrick ConcannonAbstract: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.
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Independent Roles for Nibrin and Mre11-Rad50 in the Activation and Function of Atm
The Journal of biological chemistry, 2004Co-Authors: Karen Cerosaletti, Patrick ConcannonAbstract:Abstract 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.
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Nibrin Forkhead-Associated Domain and Breast Cancer C-terminal Domain Are Both Required for Nuclear Focus Formation and Phosphorylation
The Journal of biological chemistry, 2003Co-Authors: Karen Cerosaletti, Patrick ConcannonAbstract: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.
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Distinct functional domains of Nibrin mediate Mre11 binding, focus formation, and nuclear localization.
Molecular and cellular biology, 2001Co-Authors: Ami Desai-mehta, Karen Cerosaletti, Patrick ConcannonAbstract: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.
Martin Digweed - One of the best experts on this subject based on the ideXlab platform.
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Nijmegen Breakage Syndrome (NBS) is a Telomeropathy: Analysis of Telomere Length in NBS Homo- and Heterozygotes and Humanized Nbs Mice
2019Co-Authors: Raneem Habib, Martin Digweed, Ilja Demuth, Heidemarie Neitzel, Krystyna H. Chrzanowska, Ryong Kim, E. Seemanova, R. Faber, K. Jäger, Karl SperlingAbstract:Abstract The autosomal recessive genetic disorder Nijmegen breakage syndrome (NBS) is characterized by a defect in DNA double-strand break repair protein Nibrin and chromosome instability associated with a high predisposition to cancer. Here we hypothesized that impaired Nibrin/MRE11/RAD50 telomere maintenance complex may also affect telomere length and modulate the cancer phenotype. Telomere length was studied in blood from 38 homozygous and 27 heterozygous individuals, in one homozygous fetus, and in sex NBS lymphoblastoid cell lines (all with the founder mutation c.657_661del5), and in three humanized Nbs mice, using qPCR, TRF and Q-FISH. Telomere lengths were markedly but uniformly reduced to 20-40% of healthy controls. There was no correlation between telomere length and severity of clinical phenotype or age of death. By contrast, individual patients with very short telomeres displayed long survival times after cancer manifestation. Mildly accelerated telomere attrition was found in older NBS heterozygotes. In the NBS-fetus, the spinal cord, brain and heart had the longest telomeres, skin the shortest. Humanized Nbs mice (with much longer telo-meres than those in human beings) did not show accelerated telomere attrition. Our data clearly show that NBS is a secondary telomeropathy with unique features. Te- lomere attrition in NBS may cause genetic instability and contribute to the high cancer incidence in NBS. On the other hand, short telomeres may prevent an even worse pheno-type when a tumor has developed. These data may help to understand the high cancer rate in NBS and also the bifunctional role of telomere shortening in cancerogenesis. Author Summary DNA damage is harmful because it leads to mutations in genes that initiate or accelerate cancerogenesis. The devastating consequences of DNA damage are manifested in diseases with non-functional repair pathways such as Nijmegen breakage syndrome (NBS). A common feature of these diseases is a high tumor incidence. However, cancer incidence varies and is not clear why it is highest for NBS. In a previous study, we have shown that the underlying nebrin mutation not only leads to defective DNA repair but also to higher degree of oxidative stress that generates further DNA lesions. Nibrin may play also an important role in protecting chromosome ends, the telomeres, from inap-propriate DNA repair. Therefore we examined the telomere length in NBS and show markedly reduced values in affected patients but not in NBC mice (with much milder phenotype and longer telomeres). Telomere attrition contributes to genetic instability and may thus contribute to the high cancer incidence in NBS. Individual patients with very short telomeres, however, displayed long survival times after cancer manifestation. Thus, short telomeres may also prevent an even worse phenotype when a tumor has developed. These data are fundamental to understanding the high cancer rate in NBS and also the bifunctional role of telomere shortening in cancer.
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Deficiency of the DNA repair protein Nibrin increases the basal but not the radiation induced mutation frequency in vivo
Mutation research, 2014Co-Authors: Petra Wessendorf, Jan Vijg, André Nussenzweig, Martin DigweedAbstract:Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type Nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with Nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type Nibrin.
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Nijmegen breakage syndrome: the clearance pathway for mutant Nibrin protein is allele specific.
Gene, 2013Co-Authors: Bastian Salewsky, Petra Wessendorf, Daniel Hirsch, Harald Krenzlin, Martin DigweedAbstract:Abstract The autosomal recessive disorder Nijmegen breakage syndrome (NBS) is caused by mutations in the NBN gene which codes for the protein Nibrin (NBS1; p95). In the majority of cases, a 5 bp deletion, a founder mutation, leads to a hypomorphic 70 kD protein, p70-Nibrin, after alternative initiation of translation. Protein levels are of relevance for the clinical course of the disease, particularly with regard to malignancy. Here, mechanisms and efficiency of mutant protein clearance were examined in order to establish whether these have an impact on Nibrin abundance. Cell lines from NBS patients and retroviral transductants were treated with proteasome and lysosome inhibitors and examined by semi-quantitative immunoblotting for p70-Nibrin and p95-Nibrin levels. The results show that p70-Nibrin is degraded by the proteasome with varying efficiency in cell lines from different NBS patients leading to lower or higher steady state levels of this partially active protein fragment. In contrast, a previously described NBN missense mutation, which disturbs protein folding due to the substitution of a critical arginine by tryptophan, was found to be cleared by lysosomal microautophagy leading also to lower cellular levels. The data show that truncated Nibrin and misfolded Nibrin have different clearance pathways.
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DNA damage in Nijmegen Breakage Syndrome cells leads to PARP hyperactivation and increased oxidative stress.
PLoS genetics, 2012Co-Authors: Harald Krenzlin, Bastian Salewsky, Petra Wessendorf, Ilja Demuth, Alexander Bürkle, Kathrin Weidele, Martin DigweedAbstract:Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the NBN gene, which codes for the protein Nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of Nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated Nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage.
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Clinical variability and expression of the NBN c.657del5 allele in Nijmegen Breakage Syndrome.
Gene, 2009Co-Authors: Stephan Lins, Lars Krüger, Krystyna H. Chrzanowska, Eva Seemanova, Ryong Kim, Martin DigweedAbstract:Patients affected by the autosomal recessive Nijmegen Breakage Syndrome (NBS [MIM 251260]) have possibly the highest risk for developing a malignancy of all the chromosomal instability syndromes. This reflects the profound disturbance to genomic integrity and cellular homeostasis that is caused by the mutation of the essential mammalian gene, NBN. Whilst null-mutation of Nbn is lethal in the mouse, NBS patients survive due to the fact that the common human founder mutation, found in over 90% of patients, is in fact hypomorphic and leads, by alternative translation, to varying amounts of a partially functional carboxy-terminal protein fragment, p70-Nibrin. The expression level of p70-Nibrin correlates with cancer incidence amongst patients. Using real-time PCR we have now found that the variation in p70-Nibrin expression cannot be attributed to differences in mRNA quantity and that nonsense-mediated mRNA decay is not responsible for the observed variation. We discuss an alternative explanation for p70-Nibrin expression variation.
J. Pincheira - One of the best experts on this subject based on the ideXlab platform.
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Roles of Nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage.
Biological research, 2005Co-Authors: Katherine Marcelain, Consuelo De La Torre, Patricio González, J. PincheiraAbstract:Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of Nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, Nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with Nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of Nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.
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roles of Nibrin and atm atr kinases on the g2 checkpoint under endogenous or radio induced dna damage
Biological Research, 2005Co-Authors: Katherine Marcelain, Consuelo De La Torre, Patricio González, J. PincheiraAbstract:Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of Nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, Nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with Nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of Nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.
Karen Cerosaletti - One of the best experts on this subject based on the ideXlab platform.
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Nuclear export of NBN is required for normal cellular responses to radiation.
Molecular and cellular biology, 2008Co-Authors: Christine Vissinga, Karen Cerosaletti, Tiong Chia Yeo, Sarah E. Warren, James V. Brawley, Jennifer Phillips, Patrick ConcannonAbstract:Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding Nibrin, a component of the MRE11/RAD50/Nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse Nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, Nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of Nibrin significantly altered the cellular distribution of Nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in Nibrin slowed the turnover of phosphorylated Nibrin after irradiation, indicating that nuclear export of Nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.
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Active role for Nibrin in the kinetics of atm activation.
Molecular and cellular biology, 2006Co-Authors: Karen Cerosaletti, Jocyndra A. Wright, Patrick ConcannonAbstract: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.
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Independent Roles for Nibrin and Mre11-Rad50 in the Activation and Function of Atm
The Journal of biological chemistry, 2004Co-Authors: Karen Cerosaletti, Patrick ConcannonAbstract:Abstract 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.
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Nibrin Forkhead-Associated Domain and Breast Cancer C-terminal Domain Are Both Required for Nuclear Focus Formation and Phosphorylation
The Journal of biological chemistry, 2003Co-Authors: Karen Cerosaletti, Patrick ConcannonAbstract: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.
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Distinct functional domains of Nibrin mediate Mre11 binding, focus formation, and nuclear localization.
Molecular and cellular biology, 2001Co-Authors: Ami Desai-mehta, Karen Cerosaletti, Patrick ConcannonAbstract: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.
Katherine Marcelain - One of the best experts on this subject based on the ideXlab platform.
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Roles of Nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage.
Biological research, 2005Co-Authors: Katherine Marcelain, Consuelo De La Torre, Patricio González, J. PincheiraAbstract:Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of Nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, Nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with Nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of Nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.
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roles of Nibrin and atm atr kinases on the g2 checkpoint under endogenous or radio induced dna damage
Biological Research, 2005Co-Authors: Katherine Marcelain, Consuelo De La Torre, Patricio González, J. PincheiraAbstract:Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of Nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, Nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with Nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of Nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.