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V(D)J rearrangement in Nijmegen breakage syndrome
Molecular Immunology 37 (2000) 1131– 1139 www.elsevier.com/locate/molimm
V(D)J rearrangement in Nijmegen breakage syndrome Tiong Chia Yeo a,b, Dong Xia b,c, Samar Hassouneh b,c, Xuexian O. Yang b,c, Daniel E. Sabath c,d, Karl Sperling e, Richard A. Gatti f, Patrick Concannon a,b, Dennis M. Willerford b,c,* a
Molecular Genetics Program, Virginia Mason Research Center, 1201 Ninth A6enue, Seattle, WA 98101 -2795, USA b Department of Immunology, Uni6ersity of Washington School of Medicine, Seattle, WA 98195, USA c Di6ision of Hematology, Department of Medicine, Uni6ersity of Washington School of Medicine, Campus Box 357710, Seattle, WA 98195, USA d Department of Laboratory Medicine, Uni6ersity of Washington School of Medicine, Seattle, WA 98195, USA e Institute of Human Genetics, Charite´, Humboldt Uni6ersity, Augustenburger Platz 1, D-13353 Berlin, Germany f Department of Pathology, UCLA School of Medicine, Los Angeles, CA 90095 -1732, USA Received 11 August 2000; received in revised form 14 February 2001; accepted 5 April 2001
Abstract Repair of DNA double-strand breaks is essential for maintenance of genomic stability, and is specifically required for rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) loci during development of the immune system. Abnormalities in these repair processes also contribute to oncogenic chromosomal rearrangements that underlie many lymphoid malignancies. Nijmegen breakage syndrome (NBS) is a rare autosomal recessive condition characterized by immunodeficiency, radiation sensitivity, and increased predisposition to lymphoid cancers bearing oncogenic Ig and TCR locus translocations. NBS patients fail to produce nibrin, a protein required for the nuclear localization and function of a DNA repair complex that includes Mre11 and Rad50. Mre11 has biochemical properties that suggest a potential role in V(D)J recombination. We studied V(D)J recombination in NBS cells in vitro and in vivo, using cell lines and peripheral blood leukocyte DNA from NBS patients. We found that NBS cells were competent to rejoin signal substrates with normal efficiency and high fidelity. Coding substrates were similarly rejoined efficiently, and coding end structures appeared normal. In B cells from NBS patients, the spectrums of IgH CDR3 regions were diverse and normally distributed. Moreover, the lengths and composition of Igk VJ joins and IgH VDJ joins derived from NBS and normal subjects were indistinguishable. Our data indicate that nibrin plays no essential role in V(D)J recombination and is not required for the generation of an apparently diverse B cell repertoire. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: CDR3; Mre11; Nibrin; Nijmegen breakage syndrome; V(D)J recombination
1. Introduction During the course of their development, lymphocytes undergo V(D)J rearrangement of antigen receptor loci, a process that compromises genomic integrity in order to create the vast diversity of antigen specificities that forms the basis for adaptive immunity (Alt et al., 1992; Roth et al., 1995; Grawunder et al., 1998; Vanasse et al., 1999a). V(D)J recombination is required for T and * Corresponding author. Tel.: + 1-206-2217714; fax: +1-2065433560. E-mail address: [email protected] (D.M. Willerford).
B cell development, and defects at any step in the reaction lead to severe immune deficiency in vivo (Taccioli and Alt, 1992; Roth et al., 1995; Schwarz et al., 1996; Vanasse et al., 1999a). V(D)J rearrangement is initiated by the Rag1/Rag2 endonuclease complex, which creates DNA double-strand breaks (DSB) adjacent to recombination signal sequences (RSS) that flank variable region gene segments (McBlane et al., 1995; Grawunder et al., 1998; Fugmann et al., 2000). These DSB are rejoined by ubiquitously expressed proteins involved in non-homologous end joining (NHEJ), a pathway that is also involved in repairing radiation damage (Taccioli et al., 1993; Grawunder et al., 1998;
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Vanasse et al., 1999a). Rejoining of DSB induced by radiation or by the Rag proteins during V(D)J recombination is impaired in both cell lines and mice with mutations affecting any of the NHEJ components, including the DNA-PK catalytic subunit (DNA-PKcs), Ku70, Ku80, XRCC4 and DNA ligase IV (Grawunder et al., 1998; Vanasse et al., 1999a). Genomic instability in lymphocytes is implicated in the pathogenesis of many lymphoid malignancies, which characteristically bear oncogenic chromosomal translocations involving antigen receptor loci (Tycko et al., 1991; Korsmeyer, 1992; Danska and Guidos, 1997; Vanasse et al., 1999a). In mice, B-lineage tumors bearing Ig-locus translocations occur at a high frequency when a genetic defect in one of the NHEJ components, including DNA-PKcs, Ku80 and XRCC4, is coupled with a p53-null mutation. (Bogue et al., 1996; Guidos et al., 1996; Nacht et al., 1996; Vanasse et al., 1999a,b; Difilippantonio et al., 2000; Gao et al., 2000). In mice with combined DNA-PKcs and p53 mutations, development of translocation-associated pro-B cell tumors requires Rag-2, indicating that initiation of V(D)J recombination forms part of the oncogenic pathway (Vanasse et al., 1999b). These observations suggest that suppression of aberrant rearrangements during V(D)J recombination requires both efficient rejoining of DNA ends by the NHEJ pathway and an intact cellular response to unresolved DNA ends. In humans, genetic susceptibility to antigen receptor translocations is found in three recessive syndromes characterized by defective cellular responses to DSB: ataxia– telangiectasia (A–T), Bloom’s syndrome, and NBS (Vanasse et al., 1999a). Hence, the corresponding ATM, BLM and nibrin proteins may suppress oncogenic translocations that might otherwise arise during the V(D)J reaction. NBS is characterized by microcephaly, growth retardation, and mild to moderate mental retardation, in addition to hypersensitivity to ionizing radiation (Weemaes et al., 1994; van der Burgt et al., 1996; International Nijmegen Breakage Syndrome Study Group, 2000). NBS patients suffer from recurrent infections and display immune system abnormalities, including poorly differentiated thymi, reduction in specific T and B cell subsets, and deficits in specific immunoglobulin subclasses. There is an extraordinary incidence of hematologic malignancies, predominantly non-Hodgkin’s lymphomas and lymphoid leukemias. In addition, PHA-stimulated lymphoblasts from NBS patients frequently exhibit translocations involving TCR genes (Weemaes et al., 1994). In normal cells, the product of the NBS1 gene, nibrin, forms a complex with two other proteins, Rad50 and Mre11, which participate in DNA repair (Carney et al., 1998). Mre11 has been demonstrated to have both endo- and exonuclease activities that are increased by addition of Rad50 in vitro, and these nuclease activities are significantly enhanced in
the presence of nibrin (Paull and Gellert, 1998). Among the substrates that the complex of nibrin, Mre11 and Rad50 is able to cleave in vitro are DNA hairpins resembling intermediates formed at the coding joint ends following Rag-mediated DNA cleavage in V(D)J recombination (Paull and Gellert, 1998). The Nibrin/ Mre11/Rad 50 complex is recruited to macromolecular nuclear foci in response to DNA damage, and nibrin is required for both the nuclear localization of the complex and its recruitment to these structures (Carney et al., 1998; Nelms et al., 1998; Ito et al., 1999; Cerosaletti et al., 2000; Desai-Mehta et al., 2001). Recently, it was shown that nibrin-containing nuclear foci are formed in immature thymocytes and are spatially coincident with the TCR alpha locus, which undergoes V(D)J reacombination at this developmental stage (Chen et al., 2000). Taken together, the defects in immune system development and susceptibility to antigen receptor translocations observed in NBS patients, along with the growing body of literature regarding nibrin function in DNA damage responses, raise the question of whether nibrin plays a role in V(D)J recombination in vivo. In the current study, we have examined the requirement for nibrin in the execution of the V(D)J reaction, using extrachromosomal recombination assays in NBS cells, as well as analysis of V(D)J joints in B cells from NBS patients.
2. Materials and methods
2.1. Cell lines SV40-transformed fibroblast lines included the control GM637 line (Coriell Institute, Camden, NJ) and the NBS line NBS-ILB1, which is homozygous for the common 657del5 NBS1 mutation (Kraakman-van der Zwet et al., 1999). The primary fibroblast line, 82-6, derived from normal foreskin, was provided by Dr. Thomas Norwood (University of Washington). Primary fibroblasts from NBS patients included WG1799 (Der Kaloustian et al., 1996), 780816 and 880823 (kindly provided by Dr. Dominique Smeets). Control B lymphoblastoid cell lines (B-LCL) Sweig, HOM-1 and MAT were provided by Dr. Gerald Nepom (Virginia Mason Research Center, Seattle, WA). The NBS BLCL, NBS03LA, is also homozygous for the common NBS founder mutation 657del5. None of the NBS cell lines utilized in this study make levels of nibrin detectable by Western blotting. Fibroblasts were grown in Dulbecco’s Modified Eagle Medium (Life Technologies, Rockville, MD) supplemented with 10% fetal calf serum, glutamine, penicillin and streptomycin. B-LCL lines were grown in RPMI (Life Technologies, Rockville, MD) with the same supplements.
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2.2. V(D)J recombination assays Extrachromosomal plasmid recombination substrates were used to assay V(D)J coding (pGG51) and signal (pGG49) joins (Gauss et al., 1998). Substrates were co-transfected with Rag-1 and Rag-2 expression plasmids and recombination frequency assayed after 24 h by comparing recovery of recombinant (ampicillin+ chloramphenicol resistant) versus total (ampicillin resistant) plasmids as described (Hesse et al., 1987; Taccioli et al., 1993). Fibroblasts were transfected using the CaPO4 method, while B-LCLs were electroporated. The structures of signal and coding joints were analyzed by DNA sequencing of individual recombinant plasmids.
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framework 3 consensus primer 5%-ACA CGG C(C/T)G T(G/A)T ATT ACT GT-3%. The framework 3 primer was labeled at the 5% end with 6-hexachloroflourescein. PCR products were sized using an ABI 310 Analyzer with a capillary containing POP4 polymer (PerkinElmer, Foster City, CA). Carboxy-X-rhodamine size standards and GeneScan 3.1 software (Perkin-Elmer) were used for PCR product size determination. Igk VJ sequences were amplified as described previously (Gong et al., 1999), cloned into the pGEM-T vector (Promega, Madison, WI), and plasmid inserts amplified from individual colonies sequenced. IgH VDJ sequences were analyzed in a similar fashion, using the same PCR method described above for the CDR3 length analysis, except that non-labeled primers were used.
2.3. Analysis of Ig coding regions in NBS Lymphocytes Peripheral blood lymphocyte (PBL) DNA from seven control individuals and 9 NBS patients was used in the IgH CDR3 analysis. The latter included eight patients who were homozygous for the 657del5 mutation and one patient homozygous for the 842insT mutation (Perez-Vera et al., 1997). Patient materials were used according to protocols approved by the Institutional Review Board of Virginia Mason Research Center. PCR amplification of the IgH CDR3 region of lymphocytes was done according to methods previously described (Segal et al., 1992). Briefly, 500 ng of genomic DNA were amplified with the JH consensus primer 5%-ACC TGA GGA GAC GGT GAC C-3% and the VH
3. Results
3.1. V(D)J recombination in NBS cell lines To determine whether nibrin might play a role in the rejoining of DNA cleaved during the V(D)J reaction, we assessed the ability of NBS cells to carry out V(D)J recombination of extrachromosomal plasmid substrates, which assay either coding or signal end joining. We applied these assays to three different cell types: primary fibroblasts, SV40-transformed fibroblasts, and B-LCLs (Table 1). No significant differences were observed in the efficiency of coding or signal joint forma-
Table 1 V(D)J recombination in control (WT) and NBS cells Cell line
Cell type
Genotype
pGG51 (coding)
pGG49 (Signal)
Fraction Correct
AmpRCamR/AmpR
Percentage
AmpRCamR/AmpR
Percentage
5.7 3.3 6.0 11 5.7 6.1 2.5 0.025 0.039 0.025
Experiment I GM637 SV40-F 1LB1 SV40-F 82-6 Fibroblast WG1799 Fibroblast 780816 Fibroblast 880823 Fibroblast Sweig B-LCL Hom1 B-LCL MAT B-LCL NBS03LA B-LCL
WT NBS WT NBS NBS NBS WT WT WT NBS
199/10560 173/3824 168/6286 149/5894 680/28000 211/7777 216/17600 13/139500 10/197500 25/127500
1.9 4.5 2.7 2.5 2.4 2.7 1.2 0.01 0.005 0.020
1360/23880 91/2728 636/10661 365/3325 76/1332 1552/25340 637/25400 39/155500 68/174500 29/114000
Experiment II GM637 SV40-F 1LB1 SV40-F 82-6 Fibroblast WG1799 Fibroblast 780816 Fibroblast
WT NBS WT NBS NBS
642/45600 189/12650 114/4480 94/3311 79/9464
1.4 1.5 2.5 2.8 0.83
145/5520 343/7150 289/5334 426/3500 392/18620
18/18 17/17 17/17 15/16 15/15 12/12
2.6 4.6 5.4 12.2 2.1
The recombination frequency is the fraction AmpRCamR/AmpR colonies recovered 24 h after transfection of the indicated cell line with Rag-1 and Rag-2 expression plasmids. No recombinant colonies were recovered in the absence of Rag-1 and Rag-2 (not shown). Fraction correct for signal joints was calculated as the number of signal sequences that were precisely joined divided by the number of sequences analyzed. SV40-F denotes SV40-transformed fibroblast line.
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CDR3 lengths in PBL from nine NBS patients and seven controls using PCR. Generic VH and JH primers were used to amplify genomic DNA prepared from PBL. The bulk PCR product, representing a mixture of different CDR3 regions was fractionated on an automated sequencer to generate a profile of CDR3 lengths for each subject. Representative profiles from two patients and two controls are shown in Fig. 2. CDR3 lengths from both the control and NBS samples were normally distributed; there were no significant differences in the median CDR3 lengths. Two patient and two control samples did exhibit non-normal distributions and irregularities in the heights of the peaks (not shown), most likely reflecting a lower quantity of amplifiable B cell-derived DNA in these samples. These findings indicate that the IgH V(D)J junctions in NBS B cells are diverse and that V(D)J assembly generates VH genes exhibiting the appropriate spectrum of lengths. Fig. 1. Coding sequences from recombined extrachromosomal recombination substrates. The unmodified coding sequence from the pGG51 coding substrate is shown at the top. Sequences of individual plasmids recovered from the V(D)J recombination assay in control (GM637) and NBS (ILB1) cells are depicted below, with solid lines denoting nucleotide losses. Asterisks indicate coding sequences with no nucleotide loss. Nucleotide additions (representing P nucleotides) are shown in parentheses.
tion in fibroblast lines when comparing NBS and control cells. V(D)J recombination efficiency was consistently lower in B-LCL than in fibroblasts and varied widely even among different control cell lines, thereby limiting the value of comparisons among B-LCLs of different genotypes. However, the one NBS B-LCL that was examined fell within the range of rearrangement efficiencies observed in the controls. The quality of the signal and coding joints recovered from extrachromosomal substrates was assessed by DNA sequencing of individual recombinant plasmids. In both SV40-transformed and primary fibroblasts from NBS patients and from normal control individuals, most signal ends were accurately rejoined without the gain or loss of nucleotides (Table 1). For coding joints, 31 and 26 rearranged plasmids were sequenced from the normal GM637 fibroblast and the NBS-ILB1 cell lines, respectively (Fig. 1). The extent of deletions at coding ends did not differ significantly between NBS and control cells, suggesting that coding end processing was not affected by the NBS mutation. These data demonstrate that nibrin is not required for efficient completion of the V(D)J reaction and that the products of recombination in these assays appear qualitatively normal.
3.2. IgH CDR3 length analysis in NBS B cells To investigate the role of nibrin in the processing of coding ends in vivo, we examined the spectrum of IgH
3.3. Ig V(D)J sequences in NBS B cells To further determine the in-vivo characteristics of V(D)J coding joins in NBS, we determined the Igk VJ and IgH VDJ sequences from control and NBS patients. PCR was used to amplify rearranged Igk or IgH alleles, products were cloned and plasmid inserts from individual bacterial colonies were sequenced. Igk VJ sequences from NBS patients were diverse and exhibited the expected degree of nucleotide loss (Table 2). There were occasional complementary nucleotides consistent with P elements, as well as infrequent N nucleotides. Overall, there were no discernable differences when compared with sequences from control PBL. The fraction of Igk sequences that were in frame was similar in control subjects and NBS patients (14/21 and 16/21, respectively). Examination of IgH VDJ sequences revealed the expected high degree of diversity, with contributions from different V, D and J segments, as well as nucleotide losses and additions (Table 3). There were no significant differences in the overall lengths of the sequences corresponding to the N–D–N regions when the samples from the control and NBS individuals were compared, consistent with results from the bulk CDR3 length analysis. As with Igk coding joins, the fraction of in-frame junctions was similar in control subjects and NBS patients patients and controls (12/15 and 20/25, respectively).
4. Discussion The clinical characteristics of NBS include frequent pulmonary infections, reduced numbers of T and B cells and susceptibility to chromosomal translocations involving antigen receptor loci (Weemaes et al., 1994;
T.C. Yeo et al. / Molecular Immunology 37 (2000) 1131–1139
van der Burgt et al., 1996; International Nijmegen Breakage Syndrome Study Group, 2000). Since V(D)J recombination plays a critical role in both lymphoid development and probably also in many of the oncogenic antigen receptor translocations that underlie lymphoid malignancies (Vanasse et al., 1999a), the phenotype of NBS patients raises the possibility that nibrin could play a role in physiologic antigen receptor gene
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rearrangement. Our data indicate that efficient V(D)J recombination of extrachromosomal substrates does not require nibrin (Table 1). Similar results were obtained in SV40-transformed fibroblasts, primary fibroblasts and, at a lower efficiency, in B-LCLs. Because we studied cell lines established from five different NBS patients with defined clinical diagnoses and NBS1 mutations, our results are not likely due to the patients
Fig. 2. Analysis of IgH CDR3 length in PBL from control individuals and NBS patients. IgH CDR3 regions were amplified using PCR, and products analyzed by electrophoresis using ABI 310 analyzer. The lengths of amplified products are denoted by the number of bases as indicated above the graph. The intensities of the peaks are expressed as arbitrary units on the y-axis. 7033 and 7041 are DNAs derived from PBL of control donors, while patients 5450 and 8165 represent PBL samples from two NBS patients.
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Table 2 Igk VJ junction sequences in control individuals and NBS patients V segment Control GTACTCCTCC GTACTCCTA ACTT GTACTCCTCC GTACTCC GTACTCCTCC GTACTCCTC GTACTCC GTACTC GTTATTC GTACTCC GTACTCCTC ACTGGCCTC ACTG GCTCAC GTATTCCTC GTACTCCTC GTACTCCTCC GTACTCC GTACTCCTC NBS GTACTCC GCTCACC GTACTCC GTACTCCTC GTACTCC GTACTCCT GTACTCCTCC GCT GTACTCCTC GTACTCCT GTACTCCTCC GTACTCCTC GTACTCCT GTACTCCTC GTACTCCTC TTACTCCT GTACTCCTCC GTACTCC GTACTCC ACTTACCTCC GTACTCCTC
P+N
GC GTAC
C
AGA
A C
CAC GA AAA T T TC
TC CC GC G6 A
J segment
GTGGACGTTC ACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GACGTTC GTGGACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GTGGACGTTC ACGTTC GGACGTTC GTGGACGTTC GTGGACGTTC GGACGTTC GGACGTTC ACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GTGGACGTTC GTGGACGTTC ACGTTC GACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GGACGTTC GGACGTTC GTGGACGTTC GGACGTTC TGGACGTTC GGACGTTC GGACGTTC GACGTTC GTGGACGTTC GGACGTTC GGACGTTC GACGTTC GGACGTTC
After assignment to known V and J segments, residual bases were characterized as P or N nucleotides. Potential P elements are underlined.
having alleles with residual nibrin function. All of the patient cell lines studied here have truncating mutations on both alleles of NBS1 (Varon et al., 1998) and produce no detectable nibrin by Western blotting. These results suggest that the immune developmental defects in NBS patients are unlikely to be due to impaired V(D)J recombination. Recent data comparing the immunological phenotypes among a large number of NBS patients homozygous for the common 657del5 mutation in NBS1 reveal a substantial variation, further suggesting that this phenotype does not reflect a
fundamental block in developmental processes (International Nijmegen Breakage Syndrome Study Group, 2000). Biochemical data indicate that nibrin is part of a DNA repair complex that includes Mre11 and Rad50. In NBS cells, where nibrin is absent, Mre11 and Rad50 fail to localize to the nucleus, and although they complex with each other, they remain largely cytoplasmic (Carney et al., 1998). In reconstitution experiments using NBS cells, a wild-type NBS1 construct, but not a construct bearing a mutation in the Mre11 binding site, redirected Mre11 to the nucleus, demonstrating that nibrin plays an essential role in the nuclear localization of Mre11 (Ito et al., 1999; Cerosaletti et al., 2000; Desai-Mehta et al., 2001). In vitro, nibrin potentiates the endonuclease function of the complex of Mre11 and Rad50, allowing it to efficiently cleave substrates modeled on coding end hairpins (Paull and Gellert, 1998). This is an enzymatic activity necessary for V(D)J recombination for which the source in vivo has not been clearly identified, although the Rag-1/Rag-2 complex has recently been shown to have this function (Besmer et al., 1998; Shockett and Schatz, 1999; Fugmann et al., 2000; Kale et al., 2001). The apparent dispensability of nibrin for V(D)J recombination indirectly suggests that Mre11 does not play a critical role in this process. It remains possible, however, that the effects of nibrin on Mre11 localization and nuclease function are not essential for any V(D)J-related activity. V(D)J recombination in vivo is more complex than is reflected in extrachromosomal plasmid assays, in that it requires target loci to assume an appropriate chromatin configuration, and because coding ends are in some cases further modified by TdT-mediated nucleotide addition (Sleckman et al., 1996; Grawunder et al., 1998; Vanasse et al., 1999a; Fugmann et al., 2000). We therefore employed an assay for IgH CDR3 length as a screen for abnormalities in coding end processing (Fig. 2). This assay enabled us to examine hundreds of individual V(D)J coding joins at the IgH locus in PBL from nine NBS patients with defined mutations and from seven control individuals. We observed that the CDR3 profiles of NBS patients and controls were broadly similar, providing no suggestion of a severe defect in coding joint formation, as might be expected in the absence of a crucial component of the re-joining machinery, as in the SCID or Ku-deficient mouse strains. However, since this parameter is also determined by usage of individual V, D, and J elements, non-templated nucleotide additions and by selection events, this analysis does not directly address the precise structure of coding ends. Direct examination of V(D)J coding junctions in peripheral B cells from NBS patients showed no significant differences in either Igk VJ joins or IgH V(D)J joins. This finding is consistent with the coding end sequences recovered from extra-
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Table 3 Sequences of the IgH VDJ junctions from control subjects and NBS patients V segment Control GCGAGAGG GCGAGAG GCGAGAGA GCGAGAGA GCGAGAGG GCGAAAGA GCGAGAGG GTGA GTGC GTGCGAGAG GTGCGAG GCGAGAGA GCA GCGAGA GCGAGAGA NBS GCGA GCGAGAGAT GCGAAAGA GCGAGAGA GCGAGAGC GCGAGAGG GCGAGAGA GT GCGAGA GCAAAAGAT GCGAGAGA GCGAGAGG GTGCGAGA GTGCGAGA GTGCGAGA GCGAAAGA GTGCGA GTGCG GCGAAAGG GCGAGAGT GCGAAAGA GCGAGACA GCGAGAGA GCGAGA GCGAGAG
P+N
D
TCC TCGGGTGC CGCTCCC ACCAACC TAAATTA AAACAGTGGGACGAGCACCTC AACCCC GGTTTACTGC T TG CTGG AGAA TCGCGGTG C CGGAGTG GCGCGAGTC TGGAC ACCTGTCGA GGG GGAAG CCA CTTC TTGGCTAAATGGGG GGCACG ATCTGTT GAGTGGGGCGTGC TCCCGGTC TGGA TTCTATCCAAGGAGACAA TTCAGGGCA GACCTTTTA TGG GGTT TCCCTT T CCGGGG TTGG
P+N
J segment
GGTG GATATTGTAGTGGTGGTAGCTGCTACT ATAGCAGCAGCTGGTACGTGGGGGGAG GACTACGGTGACTAC GCAGTGGCTGGTA GGGAGAGGTAGGAATAGCAGCAGCTGGTAC GGTG GCTACTT TATTGTAGTGGTGGTAGCTGCTAC TGGCTGGTA TGACTAC TTATAA TTATAAC ATATTGTGGTGGTGA GCAGTGGCTGG
C TT GGA GG TTTTGGG TTCTCCT C T CCGGATTCGTCGGACG TGACT GCTT TTACGACC TCCTTTC TCAAACCCC CGA
ACTACTGGGGCCAGGG GATGCTTTTGATATCT ACTACTACTACTA CTGGTTCGACCCC CTTTGACTACTGG CTACTGGG ACTACTGGG GACTCCT TTTGACTACTG TCTG ATGCTTTTGATATCT GATGCTTTTGATATCT GAGTACTTCCATC GGTACTTCGATCTC TTTGACTACTG
GGATACAGCTATGGTTAC GCAGCAGCTGGTAC AGTACCAGCTGCTAT ATAGTAGTGGTTATTACTAC ATTACTATGGTTCGGGGAGTTATT TTGGGG ATTACTATGATAGTAGTGGTTAT TATAGCAGCAGCTGGTA CTACGGTGACTAC TTACTATGATAGTAGTGG TTACTATGATGGCACTGGCT TATAGCAGTGGCTGGTAC TATAGCAGTGGCTGGT ATAGTAGTA GTATTACGATTTTTGGAGTGGTTA GATAGCAGTGGCT CTATGG TATAGTGGGAGCT TGACTACGGAGA TAGCAGTGGCTGG CCGGA GTATAGTGGGAG AGT GTGGGAGCTAC TAGCAGTGGCTGG
GCTTCTTCG T GATC GACTTAC TGGCCC CGACCTA GTCCCCTT AT GCC CCC CTTACCAGGCCCGTC ATGGAAGG CAT GCGGA GGG GGGCTTATTAT CTCCGTATTATTTTGC CCAGGTTC TTCCGAACTTCCC CAT GAGGGGGTA
CTACTG ATGCTTTTGATATC TACTACTACTACTA ACTGGTACTTCGAT TTACTACTACTACTA GATGCTTTTGAT ATACTTCCAATA TTTGACTACTGGG TGACTACTGG CTTTGACTACTGG ACTTTGACTACTG CTG ACTACTTTGACTACTG TTGACTACTG ACTTTGACTACTGGGG TACTACTACG CACTG GACTACTGGGG GATGCTTTTGATATCT GATGCTTTTGATATCT TGACTACTG CTACTTTGACTACTG GATGCTTTTGATTTCT CTTTGACTACTG GATGCTTTTGATATCT
CGT TTCCCC CAT
The region was amplified from PBL DNA, cloned, and individual colonies sequenced. Nucleotides were assigned to known V, D, and J elements, and residual bases classified as P or N nucleotides.
chromosomal plasmid substrates undergoing V(D)J rearrangement in NBS cells. Because of the limited number of sequences examined and potential biases introduced by the PCR primers, we cannot rule out differences in the usage of individual variable region components. Recent experiments that place nibrin-containing nuclear bodies near the site of V(D)J recombination in the thymus raise the question of what role nibrin may play in the V(D)J process (Chen et al., 2000). While our data indicate that nibrin is not fundamentally required for the V(D)J reaction or for Ig gene asembly, it may be that nibrin-containing nuclear bodies play a supportive role, for example in bringing distant rearranging elements into position or sequestering DNA breaks from inappropriate DNA repair processes.
The participation of nibrin in the cellular response to DNA damage is linked with ATM function, as it was recently shown that ATM phosphorylates nibrin on at least four serine residues in response to ionizing radiation (Gatei et al., 2000; Lim et al., 2000a; Wu et al., 2000; Zhao et al., 2000). This biochemical link correlates with several similarities between A–T and NBS cells, including radiation sensitivity and radioresistant DNA synthesis (Carney et al., 1998; Shiloh, 1997). Moreover, both A–T and NBS patients exhibit immune deficiency and susceptibility to tumors bearing antigen receptor translocations. As we have shown here for NBS cells, V(D)J recombination of extrachromosomal substrates is also normal in A–T cells (Hsieh et al., 1993). The immune deficiencies associated with A– T and NBS may arise from defects in cell-cycle regulation
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that impair proliferation during lymphocyte development or antigen responses. The tumor suppressor function of ATM and nibrin, while not directly linked to the V(D)J reaction, may involve a response to DNA ends that are left unresolved by the normally efficient V(D)J reaction, perhaps analogous to the role postulated for p53 in suppressing Ig translocations that occur in mice in the setting of impaired V(D)J rejoining (Vanasse et al., 1999a,b; Difilippantonio et al., 2000; Gao et al., 2000; Lim et al., 2000b).
Acknowledgements The authors are grateful to Dr. Michael Lieber for providing plasmid recombination substrates. This work was supported by grants from the National Cancer Institute to P.C. (CA57569) and to D.M.W (CA88075). R.A.G. is supported by grants from the US Department of Energy (87ER60548), the National Institutes of Health (NS35322), the Ataxia– Telangiectasia Medical Research Foundation, and the Joseph Drown Foundation.
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T.C. Yeo et al. / Molecular Immunology 37 (2000) 1131–1139 Nacht, M., Strasser, A., Chan, Y.R., Harris, A.W., Schlissel, M., Bronson, R.T., Jacks, T., 1996. Mutations in the p53 and SCID genes cooperate in tumorigenesis. Genes Dev. 10, 2055 –2066. Nelms, B.E., Maser, R.S., MacKay, J.F., Lagally, M.G., Petrini, J.H., 1998. In situ visualization of DNA double-strand break repair in human fibroblasts. Science 280, 590 –592. Paull, T.T., Gellert, M., 1998. The 3% to 5% exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. Mol. Cell 1, 969 – 979. Perez-Vera, P., Gonzalez-del Angel, A., Molina, B., Gomez, L., Frias, S., Gatti, R.A., Carnevale, A., 1997. Chromosome instability with bleomycin and X-ray hypersensitivity in a boy with Nijmegen breakage syndrome. Am. J. Med. Genet. 70, 24 – 27. Roth, D.B., Lindahl, T., Gellert, M., 1995. Repair and recombination. How to make ends meet. Curr. Biol. 5, 496 –499. Schwarz, K., Gauss, G.H., Ludwig, L., Pannicke, U., Li, Z., Lindner, D., Friedrich, W., Seger, R.A., Hansen-Hagge, T.E., Desiderio, S., Lieber, M.R., Bartram, C.R., 1996. RAG mutations in human B cell-negative SCID. Science 274, 97 –99. Segal, G.H., Wittwer, C.T., Fishleder, A.J., Stoler, M.H., Tubbs, R.R., Kjeldsberg, C.R., 1992. Identification of monoclonal B-cell populations by rapid cycle polymerase chain reaction. A practical screening method for the detection of immunoglobulin gene rearrangements. Am. J. Pathol. 141, 1291 –1297. Shiloh, Y., 1997. Ataxia –telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. Annu. Rev. Genet. 31, 635 – 662. Shockett, P.E., Schatz, D.G., 1999. DNA hairpin opening mediated by the RAG1 and RAG2 proteins. Mol. Cell. Biol. 19, 4159 – 4166. Sleckman, B.P., Gorman, J., Alt, F.W., 1996. Accessibility control of antigen-receptor variable-region gene assembly: role of cis-acting elements. Annu. Rev. Immunol. 14, 459 –481. Taccioli, G.E., Alt, F.W., 1992. Potential targets for autosomal SCID mutations. Curr. Opin. Immunol. 7, 436 –440.
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Taccioli, G.E., Rathbun, G., Oltz, E., Stamato, T., Jeggo, P.A., Alt, F.W., 1993. Impairment of V(D)J recombination in double-strand break repair mutants. Science 260, 207 – 210. Tycko, B., Smith, S.D., Sklar, J., 1991. Chromosomal translocations joining LCK and TCRB loci in human T cell leukemia. J. Exp. Med. 174, 867 – 873. van der Burgt, I., Chrzanowska, K.H., Smeets, D., Weemaes, C., 1996. Nijmegen breakage syndrome. J. Med. Genet. 33, 153 –156. Vanasse, G., Concannon, P., Willerford, D.M., 1999a. Regulated genomic instability and neoplasia in the lymphoid lineage. Blood 94, 3997 – 4010. Vanasse, G.J., Halbrook, J., Thomas, S., Burgess, A., Hoekstra, M., Disteche, C.M., Willerford, D.M., 1999b. Genetic pathway to recurrent chromosome translocations in murine lymphoma involves V(D)J recombinase. J. Clin. Invest. 103, 1669 – 1675. Varon, R., Vissinga, C., Platzer, M., Cerosaletti, K.M., Chrzanowska, K.H., Saar, K., Beckmann, G., Seemanova, E., Cooper, P.R., Nowak, N.J., Stumm, M., Weemaes, C.M., Gatti, R.A., Wilson, R.K., Digweed, M., Rosenthal, A., Sperling, K., Concannon, P., Reis, A., 1998. Nibrin, a novel DNA doublestrand break repair protein is mutated in Nijmegen breakage syndrome. Cell 93, 467 – 476. Weemaes, C.M., Smeets, D.F., van der Burgt, C.J., 1994. Nijmegen breakage syndrome: a progress report. Int. J. Radiat. Biol. 66, S185 – 188. Wu, X., Ranganathan, V., Weisman, D.S., Heine, W.F., Ciccone, D.N., O’Neill, T.B., Crick, K.E., Pierce, K.A., Lane, W.S., Rathbun, G., Livingston, D.M., Weaver, D.T., 2000. ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature 405, 477 – 482. Zhao, S., Weng, Y.C., Yuan, S.S., Lin, Y.T., Hsu, H.C., Lin, S.C., Gerbino, E., Song, M.H., Zdzienicka, M.Z., Gatti, R.A., Shay, J.W., Ziv, Y., Shiloh, Y., Lee, E.Y., 2000. Functional link between ataxia – telangiectasia and Nijmegen breakage syndrome gene products. Nature 405, 473 – 477.
V(D)J rearrangement in Nijmegen breakage syndrome Tiong Chia Yeo a,b, Dong Xia b,c, Samar Hassouneh b,c, Xuexian O. Yang b,c, Daniel E. Sabath c,d, Karl Sperling e, Richard A. Gatti f, Patrick Concannon a,b, Dennis M. Willerford b,c,* a
Molecular Genetics Program, Virginia Mason Research Center, 1201 Ninth A6enue, Seattle, WA 98101 -2795, USA b Department of Immunology, Uni6ersity of Washington School of Medicine, Seattle, WA 98195, USA c Di6ision of Hematology, Department of Medicine, Uni6ersity of Washington School of Medicine, Campus Box 357710, Seattle, WA 98195, USA d Department of Laboratory Medicine, Uni6ersity of Washington School of Medicine, Seattle, WA 98195, USA e Institute of Human Genetics, Charite´, Humboldt Uni6ersity, Augustenburger Platz 1, D-13353 Berlin, Germany f Department of Pathology, UCLA School of Medicine, Los Angeles, CA 90095 -1732, USA Received 11 August 2000; received in revised form 14 February 2001; accepted 5 April 2001
Abstract Repair of DNA double-strand breaks is essential for maintenance of genomic stability, and is specifically required for rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) loci during development of the immune system. Abnormalities in these repair processes also contribute to oncogenic chromosomal rearrangements that underlie many lymphoid malignancies. Nijmegen breakage syndrome (NBS) is a rare autosomal recessive condition characterized by immunodeficiency, radiation sensitivity, and increased predisposition to lymphoid cancers bearing oncogenic Ig and TCR locus translocations. NBS patients fail to produce nibrin, a protein required for the nuclear localization and function of a DNA repair complex that includes Mre11 and Rad50. Mre11 has biochemical properties that suggest a potential role in V(D)J recombination. We studied V(D)J recombination in NBS cells in vitro and in vivo, using cell lines and peripheral blood leukocyte DNA from NBS patients. We found that NBS cells were competent to rejoin signal substrates with normal efficiency and high fidelity. Coding substrates were similarly rejoined efficiently, and coding end structures appeared normal. In B cells from NBS patients, the spectrums of IgH CDR3 regions were diverse and normally distributed. Moreover, the lengths and composition of Igk VJ joins and IgH VDJ joins derived from NBS and normal subjects were indistinguishable. Our data indicate that nibrin plays no essential role in V(D)J recombination and is not required for the generation of an apparently diverse B cell repertoire. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: CDR3; Mre11; Nibrin; Nijmegen breakage syndrome; V(D)J recombination
1. Introduction During the course of their development, lymphocytes undergo V(D)J rearrangement of antigen receptor loci, a process that compromises genomic integrity in order to create the vast diversity of antigen specificities that forms the basis for adaptive immunity (Alt et al., 1992; Roth et al., 1995; Grawunder et al., 1998; Vanasse et al., 1999a). V(D)J recombination is required for T and * Corresponding author. Tel.: + 1-206-2217714; fax: +1-2065433560. E-mail address: [email protected] (D.M. Willerford).
B cell development, and defects at any step in the reaction lead to severe immune deficiency in vivo (Taccioli and Alt, 1992; Roth et al., 1995; Schwarz et al., 1996; Vanasse et al., 1999a). V(D)J rearrangement is initiated by the Rag1/Rag2 endonuclease complex, which creates DNA double-strand breaks (DSB) adjacent to recombination signal sequences (RSS) that flank variable region gene segments (McBlane et al., 1995; Grawunder et al., 1998; Fugmann et al., 2000). These DSB are rejoined by ubiquitously expressed proteins involved in non-homologous end joining (NHEJ), a pathway that is also involved in repairing radiation damage (Taccioli et al., 1993; Grawunder et al., 1998;
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Vanasse et al., 1999a). Rejoining of DSB induced by radiation or by the Rag proteins during V(D)J recombination is impaired in both cell lines and mice with mutations affecting any of the NHEJ components, including the DNA-PK catalytic subunit (DNA-PKcs), Ku70, Ku80, XRCC4 and DNA ligase IV (Grawunder et al., 1998; Vanasse et al., 1999a). Genomic instability in lymphocytes is implicated in the pathogenesis of many lymphoid malignancies, which characteristically bear oncogenic chromosomal translocations involving antigen receptor loci (Tycko et al., 1991; Korsmeyer, 1992; Danska and Guidos, 1997; Vanasse et al., 1999a). In mice, B-lineage tumors bearing Ig-locus translocations occur at a high frequency when a genetic defect in one of the NHEJ components, including DNA-PKcs, Ku80 and XRCC4, is coupled with a p53-null mutation. (Bogue et al., 1996; Guidos et al., 1996; Nacht et al., 1996; Vanasse et al., 1999a,b; Difilippantonio et al., 2000; Gao et al., 2000). In mice with combined DNA-PKcs and p53 mutations, development of translocation-associated pro-B cell tumors requires Rag-2, indicating that initiation of V(D)J recombination forms part of the oncogenic pathway (Vanasse et al., 1999b). These observations suggest that suppression of aberrant rearrangements during V(D)J recombination requires both efficient rejoining of DNA ends by the NHEJ pathway and an intact cellular response to unresolved DNA ends. In humans, genetic susceptibility to antigen receptor translocations is found in three recessive syndromes characterized by defective cellular responses to DSB: ataxia– telangiectasia (A–T), Bloom’s syndrome, and NBS (Vanasse et al., 1999a). Hence, the corresponding ATM, BLM and nibrin proteins may suppress oncogenic translocations that might otherwise arise during the V(D)J reaction. NBS is characterized by microcephaly, growth retardation, and mild to moderate mental retardation, in addition to hypersensitivity to ionizing radiation (Weemaes et al., 1994; van der Burgt et al., 1996; International Nijmegen Breakage Syndrome Study Group, 2000). NBS patients suffer from recurrent infections and display immune system abnormalities, including poorly differentiated thymi, reduction in specific T and B cell subsets, and deficits in specific immunoglobulin subclasses. There is an extraordinary incidence of hematologic malignancies, predominantly non-Hodgkin’s lymphomas and lymphoid leukemias. In addition, PHA-stimulated lymphoblasts from NBS patients frequently exhibit translocations involving TCR genes (Weemaes et al., 1994). In normal cells, the product of the NBS1 gene, nibrin, forms a complex with two other proteins, Rad50 and Mre11, which participate in DNA repair (Carney et al., 1998). Mre11 has been demonstrated to have both endo- and exonuclease activities that are increased by addition of Rad50 in vitro, and these nuclease activities are significantly enhanced in
the presence of nibrin (Paull and Gellert, 1998). Among the substrates that the complex of nibrin, Mre11 and Rad50 is able to cleave in vitro are DNA hairpins resembling intermediates formed at the coding joint ends following Rag-mediated DNA cleavage in V(D)J recombination (Paull and Gellert, 1998). The Nibrin/ Mre11/Rad 50 complex is recruited to macromolecular nuclear foci in response to DNA damage, and nibrin is required for both the nuclear localization of the complex and its recruitment to these structures (Carney et al., 1998; Nelms et al., 1998; Ito et al., 1999; Cerosaletti et al., 2000; Desai-Mehta et al., 2001). Recently, it was shown that nibrin-containing nuclear foci are formed in immature thymocytes and are spatially coincident with the TCR alpha locus, which undergoes V(D)J reacombination at this developmental stage (Chen et al., 2000). Taken together, the defects in immune system development and susceptibility to antigen receptor translocations observed in NBS patients, along with the growing body of literature regarding nibrin function in DNA damage responses, raise the question of whether nibrin plays a role in V(D)J recombination in vivo. In the current study, we have examined the requirement for nibrin in the execution of the V(D)J reaction, using extrachromosomal recombination assays in NBS cells, as well as analysis of V(D)J joints in B cells from NBS patients.
2. Materials and methods
2.1. Cell lines SV40-transformed fibroblast lines included the control GM637 line (Coriell Institute, Camden, NJ) and the NBS line NBS-ILB1, which is homozygous for the common 657del5 NBS1 mutation (Kraakman-van der Zwet et al., 1999). The primary fibroblast line, 82-6, derived from normal foreskin, was provided by Dr. Thomas Norwood (University of Washington). Primary fibroblasts from NBS patients included WG1799 (Der Kaloustian et al., 1996), 780816 and 880823 (kindly provided by Dr. Dominique Smeets). Control B lymphoblastoid cell lines (B-LCL) Sweig, HOM-1 and MAT were provided by Dr. Gerald Nepom (Virginia Mason Research Center, Seattle, WA). The NBS BLCL, NBS03LA, is also homozygous for the common NBS founder mutation 657del5. None of the NBS cell lines utilized in this study make levels of nibrin detectable by Western blotting. Fibroblasts were grown in Dulbecco’s Modified Eagle Medium (Life Technologies, Rockville, MD) supplemented with 10% fetal calf serum, glutamine, penicillin and streptomycin. B-LCL lines were grown in RPMI (Life Technologies, Rockville, MD) with the same supplements.
T.C. Yeo et al. / Molecular Immunology 37 (2000) 1131–1139
2.2. V(D)J recombination assays Extrachromosomal plasmid recombination substrates were used to assay V(D)J coding (pGG51) and signal (pGG49) joins (Gauss et al., 1998). Substrates were co-transfected with Rag-1 and Rag-2 expression plasmids and recombination frequency assayed after 24 h by comparing recovery of recombinant (ampicillin+ chloramphenicol resistant) versus total (ampicillin resistant) plasmids as described (Hesse et al., 1987; Taccioli et al., 1993). Fibroblasts were transfected using the CaPO4 method, while B-LCLs were electroporated. The structures of signal and coding joints were analyzed by DNA sequencing of individual recombinant plasmids.
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framework 3 consensus primer 5%-ACA CGG C(C/T)G T(G/A)T ATT ACT GT-3%. The framework 3 primer was labeled at the 5% end with 6-hexachloroflourescein. PCR products were sized using an ABI 310 Analyzer with a capillary containing POP4 polymer (PerkinElmer, Foster City, CA). Carboxy-X-rhodamine size standards and GeneScan 3.1 software (Perkin-Elmer) were used for PCR product size determination. Igk VJ sequences were amplified as described previously (Gong et al., 1999), cloned into the pGEM-T vector (Promega, Madison, WI), and plasmid inserts amplified from individual colonies sequenced. IgH VDJ sequences were analyzed in a similar fashion, using the same PCR method described above for the CDR3 length analysis, except that non-labeled primers were used.
2.3. Analysis of Ig coding regions in NBS Lymphocytes Peripheral blood lymphocyte (PBL) DNA from seven control individuals and 9 NBS patients was used in the IgH CDR3 analysis. The latter included eight patients who were homozygous for the 657del5 mutation and one patient homozygous for the 842insT mutation (Perez-Vera et al., 1997). Patient materials were used according to protocols approved by the Institutional Review Board of Virginia Mason Research Center. PCR amplification of the IgH CDR3 region of lymphocytes was done according to methods previously described (Segal et al., 1992). Briefly, 500 ng of genomic DNA were amplified with the JH consensus primer 5%-ACC TGA GGA GAC GGT GAC C-3% and the VH
3. Results
3.1. V(D)J recombination in NBS cell lines To determine whether nibrin might play a role in the rejoining of DNA cleaved during the V(D)J reaction, we assessed the ability of NBS cells to carry out V(D)J recombination of extrachromosomal plasmid substrates, which assay either coding or signal end joining. We applied these assays to three different cell types: primary fibroblasts, SV40-transformed fibroblasts, and B-LCLs (Table 1). No significant differences were observed in the efficiency of coding or signal joint forma-
Table 1 V(D)J recombination in control (WT) and NBS cells Cell line
Cell type
Genotype
pGG51 (coding)
pGG49 (Signal)
Fraction Correct
AmpRCamR/AmpR
Percentage
AmpRCamR/AmpR
Percentage
5.7 3.3 6.0 11 5.7 6.1 2.5 0.025 0.039 0.025
Experiment I GM637 SV40-F 1LB1 SV40-F 82-6 Fibroblast WG1799 Fibroblast 780816 Fibroblast 880823 Fibroblast Sweig B-LCL Hom1 B-LCL MAT B-LCL NBS03LA B-LCL
WT NBS WT NBS NBS NBS WT WT WT NBS
199/10560 173/3824 168/6286 149/5894 680/28000 211/7777 216/17600 13/139500 10/197500 25/127500
1.9 4.5 2.7 2.5 2.4 2.7 1.2 0.01 0.005 0.020
1360/23880 91/2728 636/10661 365/3325 76/1332 1552/25340 637/25400 39/155500 68/174500 29/114000
Experiment II GM637 SV40-F 1LB1 SV40-F 82-6 Fibroblast WG1799 Fibroblast 780816 Fibroblast
WT NBS WT NBS NBS
642/45600 189/12650 114/4480 94/3311 79/9464
1.4 1.5 2.5 2.8 0.83
145/5520 343/7150 289/5334 426/3500 392/18620
18/18 17/17 17/17 15/16 15/15 12/12
2.6 4.6 5.4 12.2 2.1
The recombination frequency is the fraction AmpRCamR/AmpR colonies recovered 24 h after transfection of the indicated cell line with Rag-1 and Rag-2 expression plasmids. No recombinant colonies were recovered in the absence of Rag-1 and Rag-2 (not shown). Fraction correct for signal joints was calculated as the number of signal sequences that were precisely joined divided by the number of sequences analyzed. SV40-F denotes SV40-transformed fibroblast line.
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CDR3 lengths in PBL from nine NBS patients and seven controls using PCR. Generic VH and JH primers were used to amplify genomic DNA prepared from PBL. The bulk PCR product, representing a mixture of different CDR3 regions was fractionated on an automated sequencer to generate a profile of CDR3 lengths for each subject. Representative profiles from two patients and two controls are shown in Fig. 2. CDR3 lengths from both the control and NBS samples were normally distributed; there were no significant differences in the median CDR3 lengths. Two patient and two control samples did exhibit non-normal distributions and irregularities in the heights of the peaks (not shown), most likely reflecting a lower quantity of amplifiable B cell-derived DNA in these samples. These findings indicate that the IgH V(D)J junctions in NBS B cells are diverse and that V(D)J assembly generates VH genes exhibiting the appropriate spectrum of lengths. Fig. 1. Coding sequences from recombined extrachromosomal recombination substrates. The unmodified coding sequence from the pGG51 coding substrate is shown at the top. Sequences of individual plasmids recovered from the V(D)J recombination assay in control (GM637) and NBS (ILB1) cells are depicted below, with solid lines denoting nucleotide losses. Asterisks indicate coding sequences with no nucleotide loss. Nucleotide additions (representing P nucleotides) are shown in parentheses.
tion in fibroblast lines when comparing NBS and control cells. V(D)J recombination efficiency was consistently lower in B-LCL than in fibroblasts and varied widely even among different control cell lines, thereby limiting the value of comparisons among B-LCLs of different genotypes. However, the one NBS B-LCL that was examined fell within the range of rearrangement efficiencies observed in the controls. The quality of the signal and coding joints recovered from extrachromosomal substrates was assessed by DNA sequencing of individual recombinant plasmids. In both SV40-transformed and primary fibroblasts from NBS patients and from normal control individuals, most signal ends were accurately rejoined without the gain or loss of nucleotides (Table 1). For coding joints, 31 and 26 rearranged plasmids were sequenced from the normal GM637 fibroblast and the NBS-ILB1 cell lines, respectively (Fig. 1). The extent of deletions at coding ends did not differ significantly between NBS and control cells, suggesting that coding end processing was not affected by the NBS mutation. These data demonstrate that nibrin is not required for efficient completion of the V(D)J reaction and that the products of recombination in these assays appear qualitatively normal.
3.2. IgH CDR3 length analysis in NBS B cells To investigate the role of nibrin in the processing of coding ends in vivo, we examined the spectrum of IgH
3.3. Ig V(D)J sequences in NBS B cells To further determine the in-vivo characteristics of V(D)J coding joins in NBS, we determined the Igk VJ and IgH VDJ sequences from control and NBS patients. PCR was used to amplify rearranged Igk or IgH alleles, products were cloned and plasmid inserts from individual bacterial colonies were sequenced. Igk VJ sequences from NBS patients were diverse and exhibited the expected degree of nucleotide loss (Table 2). There were occasional complementary nucleotides consistent with P elements, as well as infrequent N nucleotides. Overall, there were no discernable differences when compared with sequences from control PBL. The fraction of Igk sequences that were in frame was similar in control subjects and NBS patients (14/21 and 16/21, respectively). Examination of IgH VDJ sequences revealed the expected high degree of diversity, with contributions from different V, D and J segments, as well as nucleotide losses and additions (Table 3). There were no significant differences in the overall lengths of the sequences corresponding to the N–D–N regions when the samples from the control and NBS individuals were compared, consistent with results from the bulk CDR3 length analysis. As with Igk coding joins, the fraction of in-frame junctions was similar in control subjects and NBS patients patients and controls (12/15 and 20/25, respectively).
4. Discussion The clinical characteristics of NBS include frequent pulmonary infections, reduced numbers of T and B cells and susceptibility to chromosomal translocations involving antigen receptor loci (Weemaes et al., 1994;
T.C. Yeo et al. / Molecular Immunology 37 (2000) 1131–1139
van der Burgt et al., 1996; International Nijmegen Breakage Syndrome Study Group, 2000). Since V(D)J recombination plays a critical role in both lymphoid development and probably also in many of the oncogenic antigen receptor translocations that underlie lymphoid malignancies (Vanasse et al., 1999a), the phenotype of NBS patients raises the possibility that nibrin could play a role in physiologic antigen receptor gene
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rearrangement. Our data indicate that efficient V(D)J recombination of extrachromosomal substrates does not require nibrin (Table 1). Similar results were obtained in SV40-transformed fibroblasts, primary fibroblasts and, at a lower efficiency, in B-LCLs. Because we studied cell lines established from five different NBS patients with defined clinical diagnoses and NBS1 mutations, our results are not likely due to the patients
Fig. 2. Analysis of IgH CDR3 length in PBL from control individuals and NBS patients. IgH CDR3 regions were amplified using PCR, and products analyzed by electrophoresis using ABI 310 analyzer. The lengths of amplified products are denoted by the number of bases as indicated above the graph. The intensities of the peaks are expressed as arbitrary units on the y-axis. 7033 and 7041 are DNAs derived from PBL of control donors, while patients 5450 and 8165 represent PBL samples from two NBS patients.
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Table 2 Igk VJ junction sequences in control individuals and NBS patients V segment Control GTACTCCTCC GTACTCCTA ACTT GTACTCCTCC GTACTCC GTACTCCTCC GTACTCCTC GTACTCC GTACTC GTTATTC GTACTCC GTACTCCTC ACTGGCCTC ACTG GCTCAC GTATTCCTC GTACTCCTC GTACTCCTCC GTACTCC GTACTCCTC NBS GTACTCC GCTCACC GTACTCC GTACTCCTC GTACTCC GTACTCCT GTACTCCTCC GCT GTACTCCTC GTACTCCT GTACTCCTCC GTACTCCTC GTACTCCT GTACTCCTC GTACTCCTC TTACTCCT GTACTCCTCC GTACTCC GTACTCC ACTTACCTCC GTACTCCTC
P+N
GC GTAC
C
AGA
A C
CAC GA AAA T T TC
TC CC GC G6 A
J segment
GTGGACGTTC ACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GACGTTC GTGGACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GTGGACGTTC ACGTTC GGACGTTC GTGGACGTTC GTGGACGTTC GGACGTTC GGACGTTC ACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GTGGACGTTC GTGGACGTTC ACGTTC GACGTTC GTGGACGTTC GGACGTTC GTGGACGTTC GGACGTTC GGACGTTC GTGGACGTTC GGACGTTC TGGACGTTC GGACGTTC GGACGTTC GACGTTC GTGGACGTTC GGACGTTC GGACGTTC GACGTTC GGACGTTC
After assignment to known V and J segments, residual bases were characterized as P or N nucleotides. Potential P elements are underlined.
having alleles with residual nibrin function. All of the patient cell lines studied here have truncating mutations on both alleles of NBS1 (Varon et al., 1998) and produce no detectable nibrin by Western blotting. These results suggest that the immune developmental defects in NBS patients are unlikely to be due to impaired V(D)J recombination. Recent data comparing the immunological phenotypes among a large number of NBS patients homozygous for the common 657del5 mutation in NBS1 reveal a substantial variation, further suggesting that this phenotype does not reflect a
fundamental block in developmental processes (International Nijmegen Breakage Syndrome Study Group, 2000). Biochemical data indicate that nibrin is part of a DNA repair complex that includes Mre11 and Rad50. In NBS cells, where nibrin is absent, Mre11 and Rad50 fail to localize to the nucleus, and although they complex with each other, they remain largely cytoplasmic (Carney et al., 1998). In reconstitution experiments using NBS cells, a wild-type NBS1 construct, but not a construct bearing a mutation in the Mre11 binding site, redirected Mre11 to the nucleus, demonstrating that nibrin plays an essential role in the nuclear localization of Mre11 (Ito et al., 1999; Cerosaletti et al., 2000; Desai-Mehta et al., 2001). In vitro, nibrin potentiates the endonuclease function of the complex of Mre11 and Rad50, allowing it to efficiently cleave substrates modeled on coding end hairpins (Paull and Gellert, 1998). This is an enzymatic activity necessary for V(D)J recombination for which the source in vivo has not been clearly identified, although the Rag-1/Rag-2 complex has recently been shown to have this function (Besmer et al., 1998; Shockett and Schatz, 1999; Fugmann et al., 2000; Kale et al., 2001). The apparent dispensability of nibrin for V(D)J recombination indirectly suggests that Mre11 does not play a critical role in this process. It remains possible, however, that the effects of nibrin on Mre11 localization and nuclease function are not essential for any V(D)J-related activity. V(D)J recombination in vivo is more complex than is reflected in extrachromosomal plasmid assays, in that it requires target loci to assume an appropriate chromatin configuration, and because coding ends are in some cases further modified by TdT-mediated nucleotide addition (Sleckman et al., 1996; Grawunder et al., 1998; Vanasse et al., 1999a; Fugmann et al., 2000). We therefore employed an assay for IgH CDR3 length as a screen for abnormalities in coding end processing (Fig. 2). This assay enabled us to examine hundreds of individual V(D)J coding joins at the IgH locus in PBL from nine NBS patients with defined mutations and from seven control individuals. We observed that the CDR3 profiles of NBS patients and controls were broadly similar, providing no suggestion of a severe defect in coding joint formation, as might be expected in the absence of a crucial component of the re-joining machinery, as in the SCID or Ku-deficient mouse strains. However, since this parameter is also determined by usage of individual V, D, and J elements, non-templated nucleotide additions and by selection events, this analysis does not directly address the precise structure of coding ends. Direct examination of V(D)J coding junctions in peripheral B cells from NBS patients showed no significant differences in either Igk VJ joins or IgH V(D)J joins. This finding is consistent with the coding end sequences recovered from extra-
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Table 3 Sequences of the IgH VDJ junctions from control subjects and NBS patients V segment Control GCGAGAGG GCGAGAG GCGAGAGA GCGAGAGA GCGAGAGG GCGAAAGA GCGAGAGG GTGA GTGC GTGCGAGAG GTGCGAG GCGAGAGA GCA GCGAGA GCGAGAGA NBS GCGA GCGAGAGAT GCGAAAGA GCGAGAGA GCGAGAGC GCGAGAGG GCGAGAGA GT GCGAGA GCAAAAGAT GCGAGAGA GCGAGAGG GTGCGAGA GTGCGAGA GTGCGAGA GCGAAAGA GTGCGA GTGCG GCGAAAGG GCGAGAGT GCGAAAGA GCGAGACA GCGAGAGA GCGAGA GCGAGAG
P+N
D
TCC TCGGGTGC CGCTCCC ACCAACC TAAATTA AAACAGTGGGACGAGCACCTC AACCCC GGTTTACTGC T TG CTGG AGAA TCGCGGTG C CGGAGTG GCGCGAGTC TGGAC ACCTGTCGA GGG GGAAG CCA CTTC TTGGCTAAATGGGG GGCACG ATCTGTT GAGTGGGGCGTGC TCCCGGTC TGGA TTCTATCCAAGGAGACAA TTCAGGGCA GACCTTTTA TGG GGTT TCCCTT T CCGGGG TTGG
P+N
J segment
GGTG GATATTGTAGTGGTGGTAGCTGCTACT ATAGCAGCAGCTGGTACGTGGGGGGAG GACTACGGTGACTAC GCAGTGGCTGGTA GGGAGAGGTAGGAATAGCAGCAGCTGGTAC GGTG GCTACTT TATTGTAGTGGTGGTAGCTGCTAC TGGCTGGTA TGACTAC TTATAA TTATAAC ATATTGTGGTGGTGA GCAGTGGCTGG
C TT GGA GG TTTTGGG TTCTCCT C T CCGGATTCGTCGGACG TGACT GCTT TTACGACC TCCTTTC TCAAACCCC CGA
ACTACTGGGGCCAGGG GATGCTTTTGATATCT ACTACTACTACTA CTGGTTCGACCCC CTTTGACTACTGG CTACTGGG ACTACTGGG GACTCCT TTTGACTACTG TCTG ATGCTTTTGATATCT GATGCTTTTGATATCT GAGTACTTCCATC GGTACTTCGATCTC TTTGACTACTG
GGATACAGCTATGGTTAC GCAGCAGCTGGTAC AGTACCAGCTGCTAT ATAGTAGTGGTTATTACTAC ATTACTATGGTTCGGGGAGTTATT TTGGGG ATTACTATGATAGTAGTGGTTAT TATAGCAGCAGCTGGTA CTACGGTGACTAC TTACTATGATAGTAGTGG TTACTATGATGGCACTGGCT TATAGCAGTGGCTGGTAC TATAGCAGTGGCTGGT ATAGTAGTA GTATTACGATTTTTGGAGTGGTTA GATAGCAGTGGCT CTATGG TATAGTGGGAGCT TGACTACGGAGA TAGCAGTGGCTGG CCGGA GTATAGTGGGAG AGT GTGGGAGCTAC TAGCAGTGGCTGG
GCTTCTTCG T GATC GACTTAC TGGCCC CGACCTA GTCCCCTT AT GCC CCC CTTACCAGGCCCGTC ATGGAAGG CAT GCGGA GGG GGGCTTATTAT CTCCGTATTATTTTGC CCAGGTTC TTCCGAACTTCCC CAT GAGGGGGTA
CTACTG ATGCTTTTGATATC TACTACTACTACTA ACTGGTACTTCGAT TTACTACTACTACTA GATGCTTTTGAT ATACTTCCAATA TTTGACTACTGGG TGACTACTGG CTTTGACTACTGG ACTTTGACTACTG CTG ACTACTTTGACTACTG TTGACTACTG ACTTTGACTACTGGGG TACTACTACG CACTG GACTACTGGGG GATGCTTTTGATATCT GATGCTTTTGATATCT TGACTACTG CTACTTTGACTACTG GATGCTTTTGATTTCT CTTTGACTACTG GATGCTTTTGATATCT
CGT TTCCCC CAT
The region was amplified from PBL DNA, cloned, and individual colonies sequenced. Nucleotides were assigned to known V, D, and J elements, and residual bases classified as P or N nucleotides.
chromosomal plasmid substrates undergoing V(D)J rearrangement in NBS cells. Because of the limited number of sequences examined and potential biases introduced by the PCR primers, we cannot rule out differences in the usage of individual variable region components. Recent experiments that place nibrin-containing nuclear bodies near the site of V(D)J recombination in the thymus raise the question of what role nibrin may play in the V(D)J process (Chen et al., 2000). While our data indicate that nibrin is not fundamentally required for the V(D)J reaction or for Ig gene asembly, it may be that nibrin-containing nuclear bodies play a supportive role, for example in bringing distant rearranging elements into position or sequestering DNA breaks from inappropriate DNA repair processes.
The participation of nibrin in the cellular response to DNA damage is linked with ATM function, as it was recently shown that ATM phosphorylates nibrin on at least four serine residues in response to ionizing radiation (Gatei et al., 2000; Lim et al., 2000a; Wu et al., 2000; Zhao et al., 2000). This biochemical link correlates with several similarities between A–T and NBS cells, including radiation sensitivity and radioresistant DNA synthesis (Carney et al., 1998; Shiloh, 1997). Moreover, both A–T and NBS patients exhibit immune deficiency and susceptibility to tumors bearing antigen receptor translocations. As we have shown here for NBS cells, V(D)J recombination of extrachromosomal substrates is also normal in A–T cells (Hsieh et al., 1993). The immune deficiencies associated with A– T and NBS may arise from defects in cell-cycle regulation
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that impair proliferation during lymphocyte development or antigen responses. The tumor suppressor function of ATM and nibrin, while not directly linked to the V(D)J reaction, may involve a response to DNA ends that are left unresolved by the normally efficient V(D)J reaction, perhaps analogous to the role postulated for p53 in suppressing Ig translocations that occur in mice in the setting of impaired V(D)J rejoining (Vanasse et al., 1999a,b; Difilippantonio et al., 2000; Gao et al., 2000; Lim et al., 2000b).
Acknowledgements The authors are grateful to Dr. Michael Lieber for providing plasmid recombination substrates. This work was supported by grants from the National Cancer Institute to P.C. (CA57569) and to D.M.W (CA88075). R.A.G. is supported by grants from the US Department of Energy (87ER60548), the National Institutes of Health (NS35322), the Ataxia– Telangiectasia Medical Research Foundation, and the Joseph Drown Foundation.
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