Mutagenesis, Vol. 15, No. 3, 281-286,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Retroviral expression of the NBS1 gene in cultured Nijmegen breakage syndrome cells restores normal radiation sensitivity and nuclear focus formation
Molecular Genetics Program, Virginia Mason Research Center, Seattle, WA 98101 and Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA and 1 Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, The Netherlands
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The majority of cases of the autosomal recessive disorder Nijmegen breakage syndrome (NBS) are associated with null mutations in the NBS1 gene, which encodes a 95 kDa protein, nibrin. Cell lines established from NBS patients fail to express nibrin and display hypersensitivity to ionizing radiation and dysregulation of the nuclear localization of two key proteins involved in DNA repair, Mre11 and Rad50. Conclusive proof that mutations in the NBS1 gene are responsible for NBS requires that re-expression of normal nibrin in NBS cells complements these phenotypes. In the current study, retroviral expression vectors containing a normal copy of the NBS1 gene or a mutated form derived from a NBS patient were introduced into a well- characterized NBS cell line. Introduction of a normal copy of the NBS1 gene, but not the mutant form, resulted in robust expression of nibrin that displayed correct nuclear localization. Expression of nibrin also restored the ability of nibrin, Mre11 and Rad50 to complex and to redistribute within the nucleus in response to ionizing radiation. Radiation sensitivity of NBS cells expressing wild-type nibrin was restored to normal levels. Hence, introduction of the NBS1 gene can correct the phenotypes observed in NBS cells.
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Introduction |
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Nijmegen breakage syndrome (NBS) (OMIM no. 251260) is an autosomal recessive disorder characterized by microcephaly, growth retardation, mild to moderate mental retardation, cellular and humoral immunodeficiency, chromosomal instability, hypersensitivity to ionizing radiation and an increased incidence of malignancies, particularly B cell lymphomas (van der Burgt et al., 1996
). NBS is very rare, with only 70 cases currently recorded in the NBS registry maintained at the University Hospital in Nijmegen (International Nijmegen Breakage Syndrome Study Group, 2000). Although the original reported case was of Dutch origin, the vast majority of NBS patients are of Eastern European origin.
Genetic linkage studies in NBS families revealed a common marker haplotype on chromosome 8q21 in the majority of patients (Saar et al., 1997; Cerosaletti et al., 1998). Ancestral recombination events could be inferred that limited the region likely to contain an NBS gene to ~400 kb (Varon et al., 1998). Within this region, a gene, NBS1, was identified that contained a truncating mutation, 657del5, present on all NBS chromosomes with the common shared haplotype, and additional truncating or nonsense mutations in other NBS patients (Varon et al., 1998).
NBS1 encodes a 95 kDa protein called nibrin or p95. The N-terminus of the protein contains adjacent forkhead-associated and breast cancer C-terminal domains characteristic of DNA repair and/or cell cycle checkpoint control proteins (Carney et al., 1998; Matsuura et al., 1998; Varon et al., 1998). In normal fibroblasts, nibrin is localized in the nucleus and interacts with at least two additional proteins involved in the repair of DNA double-strand breaks, Rad50 and Mre11 (Carney et al., 1998). Upon exposure to ionizing radiation, complexes of these proteins redistribute, forming foci in the nucleus that stain brightly with antibodies to Rad50, Mre11 or nibrin (Maser et al., 1997; Carney et al., 1998). These foci are hypothesized to be sites of DNA damage and repair. In NBS cells with truncating or nonsense mutations in the NBS1 gene, nibrin is absent. Although Mre11 and Rad50 still complex with each other in the absence of nibrin, foci do not form in response to irradiation and the majority of Mre11 and Rad50 complexes are found in the cytoplasm (Carney et al., 1998).
Cultured cells derived from NBS patients have a phenotype consistent with the disorder. NBS cells display increased sensitivity to the lethal effects of ionizing radiation and radiomimetic compounds in colony survival assays (Taalman et al., 1983; Jaspers et al., 1988). This phenotype, in conjunction with the inability to form Rad50/Mre11/nibrin nuclear foci after irradiation, is uniquely diagnostic for NBS. In the current study, we demonstrate that introduction and expression of a normal, exogenous copy of the NBS1 gene in NBS cells fully complements these specific defects. These results confirm the role of nibrin in NBS.
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Cell lines
Complementation experiments were performed using the SV40-transformed NBS fibroblast cell line NBS-ILB1 (Kraakman-van der Zwet et al., 1999
). NBS-ILB1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Rockville, MD) supplemented with 15% fetal calf serum (Hyclone Laboratories, Logan, UT), 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies). NBS-ILB1 cells expressing NBS1 transgenes were maintained in the above medium supplemented with 500 µg/ml G418 (Life Technologies). The Phoenix A packaging cell line (Achacoso et al., in preparation) was grown in DMEM medium with high glucose, 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. The SV40-transformed fibroblast cell line MRC5 (Huschtscha and Holliday, 1983) was used as a normal control and was maintained in DMEM/Ham's F12 medium supplemented with 15% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. The AT3BI cell line is a SV40-transformed ataxia telangiectasia (A-T) fibroblast line and was grown in DMEM with 15% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were grown at 37°C with 5% CO2.
Vectors and gene expression
A full-length human NBS1 cDNA was isolated from an EBV-transformed B cell cDNA library cloned into the EcoRI site of the pBK-CMV vector (Stratagene, La Jolla, CA). The 4455 bp cDNA includes 62 bp of sequence 5' of the ATG and 2128 bp of 3'-untranslated region. For further manipulations, the NBS1 cDNA was cloned into the BamHI and XhoI sites of pBluescript SK (Stratagene). Nibrin was expressed in NBS fibroblasts using the retroviral vector pLXIN (Clontech, Palo Alto, CA) that contains the MoMuLV 5'- and 3'-long terminal repeats (LTRs) flanking an internal ribosome entry site (IRES)–neomycin resistance gene cassette. A BamHI–NcoI fragment of the NBS1 cDNA, which extends 21 bp 3' of the stop codon but ends upstream of the first polyadenylation site, was subcloned into the HpaI site of pLXIN.
As a negative control, a mutant version of nibrin was generated containing the 1142delC mutation previously reported in a NBS patient (Varon et al., 1998). For this construct, cDNA was synthesized from the NBS primary fibroblast line WG1799 (Der Kaloustian et al., 1996) and a fragment from 433 to 1440 bp was amplified by PCR. This fragment was digested with AatII and AccI and cloned into AatII + AccI-digested wild-type NBS1 cDNA in pBluescript. Clones containing the 1142delC mutation were identified by sequencing. For expression in NBS fibroblasts, the 1142delC cDNA was subcloned into pLXIN as described above for wild-type NBS1.
Retroviral vectors were packaged in Phoenix A cells. For transfection, 1.75x106 cells were plated in 60 mm tissue culture dishes and grown for 24 h. An aliquot of 15 µg of retroviral plasmid DNA was used in calcium phosphate-mediated transfection as described (Pear et al., 1996; www.stanford.edu/group/nolan). Ten hours later the tissue culture medium was replaced with 3 ml of fresh medium. Retroviral supernatants were collected 48 h post-transfection and centrifuged for 5 min at 1500 r.p.m. NBS-ILB1 cells were plated at 2x105 cells in 100 mm tissue culture dishes 2 days prior to infection. Cells were infected with 2 ml fresh retroviral supernatant in a total volume of 6 ml DMEM supplemented with 10% heat-inactivated fetal calf serum. Polybrene was added to a final concentration of 5 µg/ml and cells were incubated at 37°C. The growth medium was replaced at 24 h. NBS-ILB1 cells were placed under selection 48 h post-infection using 1 mg/ml G418. Bulk transformed cell lines were established within 2 weeks.
Western blotting
Transgene protein expression was assessed by western blot analysis. Total cell lysates were prepared from trypsinized cells resuspended at 2.5x104/µl in SDS lysis buffer (2% SDS, 20% glycerol, 62.5 mMTris, pH6.8, 1% ß-mercaptoethanol, 5 µg/ml bromophenol blue). Lysates were sonicated for 3 min on ice and boiled for 5 min. Aliquots of 20 µl of protein lysate (5x105 cell equiv.) were analyzed per lane on a discontinuous 7.5% SDS–polyacrylamide gel. Gels were prepared using a Bio-Rad mini-PROTEAN II apparatus according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Electrophoresis was carried out in 25 mM Tris, 192 mM glycine, 0.1% SDS at 100 V for 2 h. Proteins were transferred to Immobilon P membranes (Millipore Corp., Bedford, MA) in 25 mM Tris, 192 mM glycine, 20% methanol at 30 V overnight.
To detect nibrin expression, membranes were blocked with 10% non-fat milk in Tris-buffered saline, pH 7.6 (20 mM Tris, 137 mM NaCl, 0.1% Tween-20) for 1 h, washed and probed with rabbit anti-p95 polyclonal antisera (Novus Biologicals, Littleton, CO) diluted 1:10 000 in Tris-buffered saline with 1% BSA. After washing, the blots were probed with horseradish peroxidase-conjugated goat anti-rabbit IgG (PharMingen, San Diego, CA) diluted 1:5000 in Tris-buffered saline with 1% BSA. Western blots were developed using the Amersham ECL system (Amersham Pharmacia Biotech, Piscataway, NJ). Human Mre11 expression was detected as described above but using rabbit anti-hMre11 polyclonal antiserum (provided by John Petrini, University of Wisconsin) at a 1:5000 dilution. Blots were stripped by incubating in 2% SDS, 62.5 mM Tris, pH 6.8, 100 mM ß-mercaptoethanol at 50°C for 30 min.
Colony survival assay
To test radiation sensitivity, cell lines were plated in quadruplicate at 600 and 1200 cells/100 mm tissue culture dish following treatment with 0, 1, 2 or 3 Gy from a JL Shepherd Mark I Model 22 cesium source (J.L. Shepherd and Associates, San Fernando, CA). After 10 days at 37°C, the tissue culture dishes were washed with phosphate-buffered saline (PBS) and stained with Coomassie blue stain (Bio-Rad) for 5 min, then washed again with PBS. Colonies per plate were quantitated and expressed as a percentage of the unirradiated control. The means and standard deviations of quadruplicate colony survival values at the various radiation doses were determined and graphed using GraphPad Prism v.3.00 for Windows (GraphPad Software, San Diego, CA).
Nuclear focus formation
To assess cellular localization and nuclear focus formation following irradiation, fibroblast cell lines were plated at 1–2x105 cells on coverslips in glass vials (Viromed, Minneapolis, MN). Cells were grown for 24 h and then irradiated with 12 Gy. The medium was replaced and cells were incubated for an additional 8 h. Cells were then fixed and permeabilized in 4% paraformaldehyde with 0.1% Triton-X 100 for 10 min.
Immunofluorescence staining was performed as described (Ziv et al., 1997) with a few modifications. After washing with PBS, coverslips were blocked in PBS with 10% fetal calf serum (FCS) and incubated at 4°C overnight. Cells were washed and incubated with rabbit anti-p95 polyclonal antiserum (Novus Biologicals) at 1:1500 dilution and a monoclonal anti-Mre11 antibody (provided by Tony DeMaggio, ICOS Corp., Bothell, WA) at 0.6 mg/ml for 1 h at room temperature. After washing with PBS, cells were incubated with Alexa Fluor 568 goat anti-rabbit IgG conjugate (Molecular Probes, Eugene, OR) and Alexa Fluor 488 goat anti-mouse IgG conjugate (Molecular Probes), both at 1:150 dilution, for 1 h at room temperature. Coverslips were washed and nuclei were counterstained with TOTO-3 iodide (Molecular Probes) at 1 µM for 30 min. After a final wash, coverslips were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
Cellular immunofluorescence was analyzed using a Nikon fluorescence microscope and a Bio-Rad confocal imaging system at 488 and 568 nm. For visualizing foci, individual cells were z planed (18–20 sections) and images of individual sections were stacked to produce a final image of the cell. The percentage of cells with nuclear foci was determined by counting a minimum of 50 cells using the Nikon fluorescent microscope. Cells were considered positive if there were 
5 foci in the cell.
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Expression of nibrin
In order to express nibrin in NBS cells, a normal copy of the NBS1 gene was cloned upstream of the IRES–neomycin resistance gene cassette in the retroviral vector pLXIN, generating a bicistronic mRNA. As a negative control for expression of NBS1, a mutant form of the gene, 1142delC, from a known NBS patient, was cloned into the pLXIN vector. The 1142delC mutation results in a frameshift and premature truncation of the nibrin protein at amino acid 403. These retroviral constructs, as well as the empty pLXIN vector, were packaged in the Phoenix A packaging cell line and used to infect NBS-ILB1, an SV40-transformed NBS fibroblast cell line (Kraakman-van der Zwet et al., 1999
). This cell line is homozygous for the 657del5 mutation and produces no detectable nibrin (Figure 1). A previous report showed that NBS-ILB1 cells display radioresistant DNA synthesis and have an intermediate radiation sensitivity phenotype relative to normal and A-T cells (Kraakman-van der Zwet et al., 1999). Following selection with G418, bulk lines infected with each of the expression constructs were established.
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Western blot analysis of the NBS-ILB1 cell lines with a polyclonal antibody against nibrin revealed consistent expression of a 95 kDa band corresponding to full-length nibrin in the line infected with the wild-type NBS1 retrovirus (Figure 1
, lane 3). The level of nibrin expressed was similar to that observed in the normal control MRC5 (lane 1). No full-length nibrin was detected in NBS-ILB1 cells infected with vector alone or in cells infected with the mutant 1142delC virus (lanes 2 and 7). In 1142delC cells, a lighter band was detected at 45 kDa, consistent with the predicted 48 kDa size of the truncated protein (data not shown). As a control for loading, the western blot was stripped and reprobed with a polyclonal antibody against human Mre11. All cell lines displayed similar levels of the 85 kDa Mre11 protein.
An additional minor band of ~79 kDa was detected in the wild-type NBS1-transfected cell line with the nibrin antiserum (lane 3). Characterization of NBS1 cDNA in this line revealed the presence of a minor transcript expressed from the retroviral construct, but containing a novel frameshift mutation, 1958insA. This mutation would be predicted to generate a product with a molecular weight consistent with the minor band observed on the western blot. Subsequent analysis of individual bacterial preparations of the wild-type NBS1 cDNA revealed that 1958insA is a common spontaneously occurring mutation that arises during passage of the NBS1 gene sequence in bacteria. Clonal analysis of the bulk NBS1 cell line showed that only 6% of the cells in the line expressed the 1958insA mutation. However, as a control for genetic heterogeneity, subclones of the bulk NBS1 cell line were isolated. Three subclones, NBS1.6, NBS1.14 and NBS1.17, which expressed only full-length nibrin are shown in Figure 1 (lanes 4–6) and were included in all analyses.
Complementation of radiation sensitivity
Only cells from NBS and A-T patients display selective sensitivity to the killing effects of ionizing radiation in colony-forming assays. Therefore, such assays are an important tool for diagnosis of these disorders. In order to measure the effect of nibrin expression on the sensitivity of NBS-ILB1 cells to ionizing radiation, the various NBS-ILB1 infected lines were irradiated with increasing doses of X-rays and plated at clonal cell concentrations. Colonies were counted after 10 days and survival relative to untreated cells was determined. For comparison, the MRC5 fibroblast cell line was included as a normal control and an A-T cell line, AT3BI, was included as a radiation-sensitive control. Figure 2 shows the results of this colony survival assay.
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NBS-ILB1 cells infected with the pLXIN vector alone displayed a radiation sensitivity that was intermediate between that of AT3BI cells and the normal control, MRC5, consistent with previous observations (Figure 2A
; Kraakman-van der Zwet et al., 1999). Introduction of the retrovirus expressing the 1142delC mutant did not significantly alter the radiosensitivity of NBS-ILB1 cells. However, introduction of the full-length NBS1 construct restored radiation resistance to normal levels in the bulk line. The individual subclones expressing full-length nibrin also displayed complementation of radiosensitivity (Figure 2B). NBS1.14 and NBS1.17 were as radiation resistant as MRC5, similar to the bulk NBS1 cell line. The subclone NBS1.6 was less radiation sensitive than NBS-ILB1 cells infected with vector alone or infected with the 1142delC mutant, but did not display full complementation relative to the NBS1 bulk line or the MRC5 control.
Subcellular localization
In normal fibroblasts, nibrin is confined to the nucleus, where it interacts with two proteins involved in DNA repair, Mre11 and Rad50 (Carney et al., 1998). Upon exposure to ionizing radiation, complexes of nibrin, Mre11 and Rad50 coalesce into punctate nuclear foci that stain brightly with antibodies to any of the three components (Carney et al., 1998). In NBS cells that lack nibrin, Mre11 and Rad50 lose their nuclear localization and are broadly distributed throughout the nucleus and cytoplasm. The irradiation-induced ability to form nuclear foci is also lost in NBS cells (Carney et al., 1998).
Indirect immunofluorescence using polyclonal anti-nibrin antiserum was performed to assess the subcellular localization of nibrin and the ability to form nuclear foci after exposure to 12 Gy ionizing radiation. As observed in other NBS cell lines (Carney et al., 1998), NBS-ILB1 cells infected with the pLXIN vector alone showed nearly undetectable nibrin staining (Figure 3A). In the bulk NBS1-infected cell line, robust nibrin expression was detected, with most cells expressing nibrin at levels greater than or equal to that in MRC5 cells. Nibrin was confined entirely to the nucleus in the NBS1-infected cells and nuclear localization of Mre11 was also restored (Figure 3B). Similar results were observed with the individual subclones expressing NBS1 (data not shown). NBS-ILB1 cells expressing the 1142delC mutant showed dull nuclear staining, consistent with the lower levels of truncated protein observed by western blot analysis, and Mre11 remained in the cytoplasm (data not shown).
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In irradiated cells, nuclear foci were detectable by staining with antibodies to either nibrin or Mre11. Figure 3B
shows a representative cell from the bulk NBS1 line either unirradiated or after exposure to 12 Gy ionizing radiation. In untreated cells, both nibrin and Mre11 were localized in the nucleus. Following irradiation, bright punctate nuclear foci were evident with both nibrin and Mre11 antiserum. When the nibrin and Mre11 images were superimposed, the foci appeared yellow, indicating that nibrin and Mre11 co-localized within the foci, as observed in normal cells. One or two additional foci were detected per cell that stained with the nibrin antibody alone. These structures were observed in most cells both before and after irradiation and appear to be the result of retroviral expression of NBS1.
To obtain a quantitative measure of the complementation of nuclear focus formation in NBS-ILB1 cells, a sample of cells from the MRC5 control and the NBS-ILB1 cell lines was counted to determine the percentage of cells with nibrin foci before and after irradiation (Table 1). In the MRC5 line, 33% of cells displayed nibrin foci prior to radiation. After exposure to 12 Gy ionizing radiation, 98% of MRC5 cells contained nibrin foci. The number of foci per cell did not change dramatically following irradiation, however. Untreated MRC5 cells contained an average of 8.2 ± 2.6 nibrin foci/cell and irradiated MRC5 cells contained 12.4 ± 3.4 foci. Similar results were obtained with NBS-ILB1 cells infected with the NBS1-expressing retrovirus. Prior to irradiation, 31% of cells in the bulk NBS1 line contained nibrin foci, increasing to 63% after irradiation. Like MRC5 cells, the average number of foci per cell did not differ before (9.7 ± 4.2) and after (10.6 ± 5.1) radiation exposure. Complementation of nibrin focus formation was also observed in the NBS1 subclones. In contrast, NBS-ILB1 cells expressing the 1142delC mutant did not contain nuclear foci before or after irradiation.
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The results reported here provide definitive proof, beyond the circumstantial evidence from mutational analysis, that nibrin expression is sufficient to correct the pathological phenotype of NBS. Introduction of the wild-type NBS1 gene into a well-characterized NBS fibroblast cell line restored nibrin expression and localization to the nucleus. Nibrin expression also restored the radiation sensitivity of the NBS-ILB1 cell line to normal levels. Furthermore, expression of full-length nibrin was necessary and sufficient to restore formation of irradiation-induced nuclear foci containing both nibrin and Mre11. Immunoprecipitation of nibrin from NBS-ILB1 cells transfected with NBS1 also precipitates Mre11 and Rad50 (unpublished observations), providing further proof that expression of nibrin in NBS-ILB1 cells complements nibrin–Mre11–Rad50 complex formation. In contrast, expression of a mutant form of nibrin, 1142delC, did not result in even modest complementation of radiation sensitivity or induction of nibrin foci following irradiation.
Unlike complementation experiments in A-T cells, where even slight overexpression of the ATM gene appears to be toxic (Shiloh et al., 1998), the high level of expression seen in the infected NBS fibroblasts cells does not result in hyposensitivity to X-rays or to toxic consequences. The only difference observed between normal cells and NBS-ILB1 cells complemented with wild-type nibrin was the presence of one or two sites of nuclear accumulation of nibrin apparent by immunofluorescence. These bodies were present even in the absence of radiation and may be related to the relatively high level of nibrin expression achieved in the retroviral system.
Robust nibrin expression was achieved with relative ease in the NBS-ILB1 fibroblast cell line. However, other cell types proved more difficult to complement. Attempts to express nibrin in several different NBS B cell lines using episomal vectors were unsuccessful and transformation of NBS B cells with the NBS1-expressing retrovirus yielded only drug-resistant cells that failed to express nibrin. Nibrin may have additional unanticipated functions in B cells or B cells may be more sensitive to the steady-state levels of nibrin. We also observed a novel frameshift mutation in exon 13 of nibrin, 1958insA, that arose spontaneously during propagation of the NBS1 cDNA in bacteria. Interestingly, other investigators (Paull and Gellert, 1999) have also observed exon 13 mutations in nibrin, suggesting that this region may be unstable in bacteria.
These experiments establish a system for performing detailed mutagenesis studies of the domains of nibrin. In the current study, NBS-ILB1 cells infected with the 1142delC form of nibrin make a low level of truncated nibrin protein. Interestingly, this truncated form of nibrin still localizes to the nucleus, despite lacking obvious nuclear localization signals. Furthermore, Mre11 remains cytoplasmic in these cells, suggesting that interaction with Mre11 requires the C-teminus of nibrin. Mutagenesis of the FHA and BRCT domains of nibrin is under way to elucidate their role in nibrin function. It may also be possible to express a dominant negative form of nibrin in normal cells to test for induction of hypersensitivity to radiation or other radiomimetic compounds. Such a scheme may have utility as a radiosensitizing technique.
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Acknowledgments |
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We are grateful to John Petrini for the gift of human Mre11 antiserum and Tony DeMaggio for kindly providing the Mre11 monoclonal antibody. This work was supported by grants from the National Cancer Institute (CA57569) and the A-T Medical Research Foundation to P.C.
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2 To whom correspondence should be addressed at: Molecular Genetics Program, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle, WA 98101, USA. Tel: +1 206 223 6476; Fax: +1 206 625 7213; Email: patcon{at}u.washington.edu
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Received on January 20, 2000; accepted on January 24, 2000.
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R. A. Schwartz, J. A. Palacios, G. D. Cassell, S. Adam, M. Giacca, and M. D. Weitzman The Mre11/Rad50/Nbs1 Complex Limits Adeno-Associated Virus Transduction and Replication J. Virol., December 1, 2007; 81(23): 12936 - 12945. [Abstract] [Full Text] [PDF]
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K. C. Manthey, S. Opiyo, J. G. Glanzer, D. Dimitrova, J. Elliott, and G. G. Oakley NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse J. Cell Sci., December 1, 2007; 120(23): 4221 - 4229. [Abstract] [Full Text] [PDF]
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I. Demuth, P.-O. Frappart, G. Hildebrand, A. Melchers, S. Lobitz, L. Stockl, R. Varon, Z. Herceg, K. Sperling, Z.-Q. Wang, et al. An inducible null mutant murine model of Nijmegen breakage syndrome proves the essential function of NBS1 in chromosomal stability and cell viability Hum. Mol. Genet., October 1, 2004; 13(20): 2385 - 2397. [Abstract] [Full Text] [PDF]
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K. M. Cerosaletti and P. Concannon Nibrin Forkhead-associated Domain and Breast Cancer C-terminal Domain Are Both Required for Nuclear Focus Formation and Phosphorylation J. Biol. Chem., June 6, 2003; 278(24): 21944 - 21951. [Abstract] [Full Text] [PDF]
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A. Desai-Mehta, K. M. Cerosaletti, and P. Concannon Distinct Functional Domains of Nibrin Mediate Mre11 Binding, Focus Formation, and Nuclear Localization Mol. Cell. Biol., March 15, 2001; 21(6): 2184 - 2191. [Abstract] [Full Text]
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