Murine Cell Line SX9 Bearing a Mutation in the dna-pkcs Gene Exhibits Aberrant V(D)J Recombination Not Only in the Coding Joint but Also in the Signal Joint*

Ryutaro FukumuraDagger §, Ryoko ArakiDagger , Akira FujimoriDagger , Masahiko MoriDagger , Toshiyuki SaitoDagger , Fumiaki WatanabeDagger , Mika Sarashiparallel , Hiromi ItsukaichiDagger , Kiyomi Eguchi-KasaiDagger , Koki Sato**, Kouichi TatsumiDagger , and Masumi AbeDagger Dagger Dagger

From the Dagger  National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba-shi, Chiba 263-8555, Japan, the § Graduate School of Science and Technology, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba-shi, Chiba 263, Japan, parallel  Perkin-Elmer Japan Co., Ltd., Applied Biosystems Division, Mihama 1-9-2, Urayasu-shi, Chiba 279, Japan, and the ** Faculty of Biology-Oriented Science and Technology, Kinki University, Uchita-cho, Naga-gun, Wakayama 649-64, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide "T" (9572) to "C" transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12.2% as compared with the normal cell line. To confirm the responsibility of the dna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild type dna-pkcs cDNA was observed. SX9 is a novel dna-pkcs-deficient cell line.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The ionizing radiation-sensitive mammalian cell lines reported to date can be divided into at least six complementation groups (1). Thus far, the responsible genes for three of the groups (XRCC4, 5, and 7) have been identified and isolated. A positional candidate for XRCC2 was recently reported (2).

XRCC4 coding products stimulate the activity of DNA ligase IV (3-8). In contrast, it was demonstrated that genes for both XRCC5 and XRCC7 encode the subunits of DNA-PK,1 which was discovered as protein kinase requiring DNA fragments for its activity (9). Extensive studies demonstrated the critical role of DNA-PK in following signal transduction in response to ionizing radiation, through the binding of the DNA-PK complex to the double strand break ends caused by DNA damaging agents including ionizing radiation (10). DNA-PK holoenzyme consists of Ku70 (70 kDa), Ku86 (86 kDa), and DNA-PKcs (465 kDa). The responsible genes for XRCC5 and XRCC7 encode Ku86 and DNA-PKcs molecules, respectively (11-18). The analyses of ku86-deficient cells, ku86 knockout mice, dna-pkcs-deficient cells, and severe combined immune deficiency (scid) mice demonstrated that the DNA-PK affects the machinery of V(D)J recombination, double strand break repair, and the sensitivity to ionizing radiation (19-21). The role of DNA-PK in the above mechanisms remains to be elucidated on the molecular level.

While both ku86 and dna-pkcs-deficient cells are highly sensitive to ionizing radiation, they exhibit different phenotypes in the joint formation of V(D)J recombination. Both joint formation steps, i.e. coding joint and signal joint formation, are defective in ku86-deficient cells (xrs-6 and lymphocytes derived from ku86 knockout mice). In contrast, dna-pkcs-deficient cells (scid, V3 and irs-20) retain the ability to form correct signal joint even though they are inflicted with a severe defect in coding joint formation (22-24). Recently, however, it was suggested in a study on equine scid caused by the aberration of the DNA-PKcs molecule that not only coding joint formation but also signal joint was severely impaired (25, 26). Detailed studies on V(D)J recombination using DNA-PK mutants will help to clarify how DNA-PK holoenzyme and/or DNA-PKcs molecule itself contributes to joint formation of V(D)J recombination and double strand break repair.

We previously established the radiosensitive cell line SX9 from the mouse mammary carcinoma cell line FM3A by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (27). Employing the cell fusion technique, we found that SX9 cells belong to a complementation group different from that of radiosensitive mammalian cell lines irs-1,2,3 and L5178Y-S (28). In this study, we report the identification of the responsible gene and mutation for the SX9 cell line.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture-- SX9, SX10, and SR-1 cells were derived from mouse mammary carcinoma FM3A cells. SR-1 cells were utilized as a wild-type control (27, 28). SR-1, SX9, SX10, CHO-K1, xrs-6, AA8, and V3 cells were cultured in alpha -minimum essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 1% L-glutamine, penicillin (100 units/ml), and streptomycin (0.1 mg/ml). Cells were grown at 37 °C in 5% CO2. Chinese hamster x-ray sensitive mutant cells xrs-6 and V3 cells were kindly provided by Dr. P. Jeggo and Dr. G. F. Whitmore, respectively.

Cell Fusion and Hybrid Selection-- 106 cells of each of two mutants were harvested, mixed, and centrifuged. The pellet was washed once with serum-free medium. The pellet was loosened by brief vibration. To this suspension, 0.5 ml of PEG1500 (50% in 75 mM Hepes buffer) was added. After 1 min at room temperature, 10 ml of serum-free medium was slowly added. The mixture was left for 10 min and spun down with added serum. The pellet was dispersed, plated in 10-cm dishes, and placed in a CO2 incubator. Selection started on the following day in medium containing HAT/ouabain (50 µM hypoxanthine, 20 µM azaserine, 7.5 µM thymidine, 2 mM ouabain). Hybrid cells grown in suspension were plated in 0.3% soft agar medium containing 10% fetal bovine serum and HAT/ouabain. Several hybrid clones were isolated and examined. Then only the typical dose-response curves were presented.

Gamma Ray Survival-- Radiation-sensitive mutants were irradiated with 60Co gamma rays at room temperature at a dose rate of 81 gray/min, and surviving fractions were determined.

Semiquantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)-- Total RNAs were prepared from 1 × 107 cells, and one-tenth volume of each sample was used for first-strand synthesis with 5'-CCTTTATCTGACCATCTCGCCAGCACCATA-3' primer. One-twentieth of the first strand was used for long and accuracy (LA)-PCR. One-fifth of the LA-PCR products was subjected to electrophoresis. The primers 5'-GGTGATGGCGGAGGAGGGAACCGGCGTACG-3' and 5'-AGGAGGTCCTCTCGGAGACAGAATGCTTTA-3' were used for LA-PCR. The protocol was described in detail previously (11).

Sequence of the Whole Region of dna-pkcs and Ku70 cDNA-- RT-PCR and sequence of dna-pkcs and Ku70 cDNA were performed as described previously (11). Briefly, both cDNAs were amplified by the RT-PCR technique and the amplified products were sequenced by the dye terminator cycle-sequencing procedure.

Confirmation of the Mutation by DdeI Digestion and Sequence-- The following primers were used for genomic PCR to confirm the mutation: INT30-3-M13F (5'-TGTAAACGACGGCCAGTAATCAGGTAGCTTTCATGACTCTGACATTG-3') and EXO30-2-M13Rv (5'-CAGGAAAACAGCTATGACCTCTTCCTTTGGCTCGTACACTTCCCTGTCA-3'). We employed the LA-PCR Kit version 2 (Takara Shuzo, Kyoto). DNA prepared from SR-1 and SX9 cells with SepaGene Kit (Sanko-jyunyaku, Tokyo) were amplified by LA-PCR. The conditions of genomic PCR were as follows: 94 °C, 1 min; 98 °C, 20 s, 68 °C, 3 min, 40 cycles; 72 °C, 10 min. Five microliters of each product was digested with restriction enzyme DdeI.

The following primers were used for the cDNA PCR: 9395+mDNA-PK-M13F(5'-TGTAAACGACGGCCAGTCTGTACAGACTTTAGCAGA- AATTGAGGAGT-3') and EXO30-2-M13Rv (5'-CAGGAAAACAGCTATGACCTCTTCCTTTGGCTCGTACACTTCCCTGTCA-3'). First strand synthesis of dna-pkcs from each cell line was performed as described previously (11). The condition of cDNA PCR was the same as that of genomic PCR. The genomic and cDNA PCR products were sequenced. Because we designed the PCR primers including the sequence for standard dye primers (M13-21 forward and reverse), the DNA analysis of the PCR products was performed directly with the standard primers using an Applied Biosystems PRISM Dye Primer Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer).

V(D)J Recombination Assay-- V(D)J recombination assay is reported elsewhere (29). Briefly, eukaryotic expression vector BCMGSNeo with cytomegalovirus promotor bearing recombination activating gene (rag)-1 or rag-2 (BCMGSNeo-TAG1, 17.8 µg and BCMGSNeo-TAG2, 22.2 µg), which were generously provided by Dr. Y. Shinkai, were co-transfected into 5 × 106 cells (SR-1 and SX9) with either pJH200 (2 µg) or pJH290 (2 µg) by electroporation. pJH200 and pJH290 are used for signal joint and coding joint formation assay, respectively. Furthermore, to test the responsibility of DNA-PKcs to SX9, the construct bearing full-length cDNA (pME-PK7, 17.5 µg) was additionally co-transfected with the above. After 48 h, the transfectants were harvested and the assay vectors were recovered. Then the recovered DNA was digested with restriction enzyme DpnI to eliminate unreplicated plasmids. Digested DNA solution was transfected into Escherichia coli DH10B strain (Life Technologies, Inc.). The accuracy of joint formation was assessed by digestion with restriction enzyme or DNA sequencing.

Construction of Full-length Murine dna-pkcs Expression Vector-- The full-length cDNA of murine dna-pkcs was amplified from first strand DNA prepared from SR-1 total RNA with primers AatII-(-4)MuDNAPK38(+) (5'-AAGACGTCGGTGATGGCGGAGGAGGGAACCGGCGTACG-3') and SpeI-12438-MuDNAPK37(-) (5'-GACTAGTAGGAGGTCCTCTCGGAGACAGAATGCTTTA-3'), using LA-PCR kit version 2 (Takara). The EcoRI site of mammalian expression vector pME18S possessing SRalpha promotor was digested with restriction enzyme EcoRI, blunted with Klenow fragment (Takara), and ligated with AatII linker with T4 ligase. The PCR product digested with AatII and SpeI was cloned into the AatII-SpeI digested pME18S modified vector. DNA sequence analysis showed five nucleotide substitutions in this construct as compared with SR-1 cDNA. Three of these substitutions did not alter the amino acid: C1371 right-arrow T (Cys right-arrow Cys); A2439 right-arrow G (Gly right-arrow Gly); C7434 right-arrow T (Asp right-arrow Asp). However, two of the substitutions, T270 right-arrow G (Cys right-arrow Trp), and C9899 right-arrow T (Thr right-arrow Met), changed the amino acid residue and so were returned to the original nucleotide using a QuickChange site-directed mutagenesis kit (Stratagene).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

dna-pkcs Deficient Cell Line V3 (XRCC7) Does Not Complement the High Radiosensitivity of SX9 Cells, whereas ku86-deficient Cell Line xrs-6 (XRCC5) Does-- Previously, the SX9 cell line was found to express low levels of DNA-PK protein capable of binding to damaged DNA but lacking any detectable kinase activity (30). Our in vitro assay system also suggested the defect of DNA-PK activity in SX9. We could not, however, arrive at a final conclusion on this matter, because DNA-PK activity in mice is extremely low compared with that in human cells for the purpose of obtaining the rigid confidence (data not shown). Therefore, we employed a cell-cell fusion technique to test whether the ku86-deficient or dna-pkcs-deficient cell lines exhibit the ability to complement the SX9 phenotype. Consequently, it was demonstrated that the xrs-6 cells had the ability to complement the SX9 cells while V3 cells did not (Fig. 1, A and B).


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Fig. 1.   Gamma ray survival curves of hybrid cells and their parental cell line. Cell survival after ionizing radiation is shown. Error bars represent standard errors derived from triplicate experiments. A: square , SR-1; Delta , CHO-K1; open circle , SX9-xrs-6 hybrid cells; black-square, SX9; black-triangle, xrs-6. B: square , SR-1; Delta , AA8; open circle , SX9-V3 hybrid cells; black-square, SX9; black-triangle, V3. SR-1, CHO-K1, and AA8 are parental cell lines for SX9, xrs-6, and V3, respectively.

As shown in Fig. 1B, however, while the sensitivity of V3-SX9 hybrid cells decreases compared with their parent cell lines SX9 and V3, the survival curve of hybrid cells is not entirely equal to that of parent cells. Therefore, it was impossible to conclude solely on the basis of our complementation test that the SX9 should be assigned to the XRCC7 group.

Sequence of ku70 cDNA and Its Expression in SX9 Is Equal to Parent Cell SR-1-- The defect of DNA-PK activity in SX9 suggests the defect of Ku70 as well as Ku80 and DNA-PKcs. Therefore, we analyzed the DNA sequence and gene expression of Ku70. ku70 cDNA was amplified from total RNA of SX9 and parent SR-1. DNA sequence was determined with the appropriate primer set (the sequence of primers is available upon request) by the dye-terminator method. As a result, the DNA sequence of ku70 of SX9 was identical to that of SR-1 (DDBJ accession number: AB010282). The expression of the ku70 gene in SX9 and SR-1 was compared by the RT-PCR technique. No difference was observed.

"T" (9572) to "C" Transition in the dna-pkcs Gene of SX9 Cells Results in Substitution of Coded Amino Acid Residues from Leucine to Proline-- Next, in order to achieve a definite conclusion, we isolated the whole region of dna-pkcs cDNA from SX9 and SR-1 cells by the LA-PCR technique and compared them at the DNA sequence level (Fig. 2A). Consequently, point mutation T to C (9572) (Fig. 2B) was discovered in dna-pkcs cDNA of SX9 cells. The mutation is located in exon 69 (31) and causes an amino acid substitution from leucine (3191) to proline. No other mutations could be identified in the open reading frame (DDBJ accession number: AB007544).


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Fig. 2.   Mutation in dna-pkcs gene of SX9. A, LA-PCR of dna-pkcs transcript in SX9. lambda HindIII and 1-kilobase pair DNA Ladder (Life Technologies, Inc.) were used for molecular weight markers. RNA for 5 × 105 cells were used for each lane. B, mutation in dna-pkcs of SX9. The solid bar and rectangle indicate the intron and the exon 69, respectively. Only the mutated region is indicated above. The arrows indicate the primers used for genomic PCR. "CTCAG" is a recognition site of restriction enzyme DdeI. By DdeI digestion, the PCR products amplified from mutated allele are divided into 85- and 125-base pair (bp) fragments as shown. Amino acid residues are noted by three-letter symbols. C, confirmation of the mutation in genomic DNA. "M" indicates the molecular weight marker (100-base pair DNA Ladder, Life Technologies, Inc.). Explanations for the fragments indicated by arrows are presented in B.

To verify our findings, we assessed the mutation with genomic DNA prepared from SR-1, SX9, and SX10 cells. We also used the SX10 cell line as a normal control, because SX10 and SX9 cells were established from FM3A cells coincidentally and the SX10 cells exhibit a very high radiosensitivity while exhibiting normal DNA-PK activity as reported previously (30). Since the mutated nucleotide T (9572) is included in the restriction enzyme DdeI recognition sequence, the DdeI site disappears when the mutation occurs (Fig. 2B). Interestingly, it was demonstrated that SX9 cells have wild-type allele as well as mutated allele (Fig. 2C, DdeI+, SX9). We discuss this observation below. The absence of the mutation in SR-1 and SX10 cells was confirmed by DdeI digestion (Fig. 2C), and the findings were also confirmed by DNA sequence analysis (Fig. 3).


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Fig. 3.   Functional hemizygosity of prkdc locus in SX9. Products amplified from cDNA and genomic DNA of SR-1 and SX9 were directly sequenced by the dye primer method. Each color bar indicates a nucleotide as follows: black, G; blue, C; green, A; and red, T. The arrowheads indicate the mutated and corresponding nucleotide.

dna-pkcs (Prkdc) Locus in SX9 Cells Is Functionally Hemizygous-- Interestingly, analysis of genomic DNA revealed the presence of two kinds of chromosomes related to the mutated region in SX9 cells. One allele has the DdeI recognition site while the other does not (Fig. 2C). This means that the mutation took place in only one of the two alleles. Indeed, only C peak appeared at nucleotide number 9572 in the cDNA sequence pattern, while an equal intensity of C and T peaks clearly appeared at the corresponding nucleotide in the genomic DNA sequence pattern (Fig. 3). We employed the dye primer sequencing procedure because the intensity of peaks obtained by this procedure reflects the original copy number more accurately than the dye terminator sequencing procedure. The findings indicated that the messenger RNA of dna-pkcs in SX9 cells is transcribed exclusively from the mutated allele.

SX9 Has a Severe Defect in Signal Joint Formation as Well as Coding Joint Formation of V(D)J Recombination-- For further characterization of defects in SX9 cells, we assessed the V(D)J recombination in SX9 cells with extrachromosomal assay vectors (pJH200 and pJH290) (29). Because SX9 cells and syngeneic SR-1 cells as a control are not lymphoid cells, rag-1 and rag-2 genes were co-transfected with the assay vector. The frequency and fidelity of coding joint and signal joint formation in SX9 cells and SR-1 are shown in Table I. The fidelity of signal joint formation was assessed by digestion with the restriction enzyme ApaLI, the recognition site of which should be generated when the precise joint is formed. The results showed that SX9 cells exhibit a severe defect in signal joint formation as well as in coding joint formation. The frequency of coding joint decreased to approximately 1/30 that of normal control. The fidelity also drastically decreased. It is particularly intriguing that the fidelity of signal joint decreased to 12.2%.

                              
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Table I
Analysis of V(D)J recombination
The results show the sum of four independent transfection experiments.

In SX9 Cells, the Majority of Signal Joint Products Have a Short Deletion-- It was demonstrated by electrophoresis that while the majority of background recombination in pJH200 prepared from SX9 cells occurred with large deletions (data not shown), the majority of the signal joint clones recovered from RAG-expressed SX9 cells have short deletions. We determined the DNA sequences of the break sites in the rearranged clones to obtain more detailed information. The results are summarized in Fig. 4, A and B. Approximately 90% of the recombined clones are ApaLI negative (Table I), and the break point regions in these clones are clearly different from those in the background. The 86 clones analyzed in this study consist of 55 kinds of signal joints. The signal joint in 30 out of 86 clones occurs with short stretches of homologous sequence (Fig. 4B). The break sites in most of the recovered clones exist within the recombination signal sequence (RSS). In fact, only four out of 86 clones had a recombination point outside RSS. In addition, it is highly remarkable that the length of deletions in both directions from putative RAG cleavage site is similar in each recombined product recovered from SX9 cells (32, 33). This symmetrical deletion was frequently observed in the signal joint region (Fig. 4B). Neither hot spot for joining nor N sequence in the break site (34) was observed.


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Fig. 4.   Deletions in pJH200 recovered from rag-introduced SX9. A, sequence of signal joints recovered from SX9 cells. The putative precise signal joint sequence on pJH200 is shown at the top. Signal sequences (heptamer and nonamer) are underlined. Twenty-four representative cases are shown. The broken lines and numbers indicate deleted nucleotides and deleted nucleotide numbers, respectively. The vertical bar indicates the break site of precise joint. B, relation of deleted nucleotide numbers from the border of heptamer edge. Eighty-six ApaLI negative clones were sequenced, and the number of deleted nucleotides was plotted on the graph. The deletion numbers of 12-spacer side and 23-spacer side were plotted on the x and y axis, respectively. The diagonal line is the line of best fit obtained from linear regression analysis. The open and closed circles indicate clones possessing and not possessing short homologous stretch in the recombination site, respectively. We defined them as short stretch homologous regions except when there was 1 base pair of homology of a short stretch homologous sequence in the recombined sequence. We plotted the 23 spacer-side border of short stretch homologous sequence in recombination site as the recombination point.

Full-length dna-pkcs cDNA Complements Abnormal V(D)J Recombination in SX9-- To confirm our findings, full-length dna-pkcs cDNA was transfected into SX9. Full-length dna-pkcs was prepared from SR-1, the parent cell line of SX9, with RT-PCR and cloned into eukaryote expression vector pME18S (DDBJ accession number: AB011543 for dna-pkcs open reading frame of the construct). V(D)J recombination was assessed by extrachromosomal vectors in the presence and absence of full-length dna-pkcs gene. The frequency of signal joint and coding joint is restored to 42 and 32%, respectively, as compared with the parent cell line SR-1. The fidelity of signal joints recovered to 85%. Joint structure in the coding joint which is formed under the presence of dna-pkcs cDNA is shown in Fig. 5.


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Fig. 5.   Sequence of coding joints from SR-1 and dna-pkcs-introduced SX9. The putative precise coding joint sequence on pJH290 is shown at the top. Three clones of SR-1 and 10 clones of dna-pkcs-introduced SX9 are shown. The number and gap indicates deleted nucleotide in coding joint. The vertical bar indicates the break site of precise joint.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The defect of DNA-PK activity in SX9 cells was suggested. Furthermore, our complementation test using DNA-PK defective cell lines by the cell fusion technique indicated that cell lines SX9 and xrs-6 (XRCC5) belong to different complementation groups and that cell lines SX9 and V3 (XRCC7) are in the same group. Recently, a similar observation was reported on the basis of in vitro assay (30).

To verify the fact that SX9 belongs to XRCC7, we compared the dna-pkcs cDNA sequence isolated from SX9 and the control cell line SR-1 and found a T to C transition in the SX9 dna-pkcs gene at nucleotide number 9572 (Fig. 2B). None of the other mutations was found in the open reading frame. The mutation could be identified in the dna-pkcs cDNA of SX9 cells but in that of neither SR-1 cells nor SX10 cells (Fig. 2C). Our investigation indicates the responsibility of identified T to C transition for SX9 abnormal phenotype.

Interestingly, the analyses using genomic DNA demonstrated that the mutation occurs in only one allele (Fig. 3). The phenomenon that mRNA is transcribed from only one allele while the other allele is silent was also demonstrated in the ku86 gene locus of ku86-deficient Chinese hamster ovary (CHO) cell lines (18, 35). Referred to as "functional hemizygosity," it is often observed in rodent cells (36). Indeed, revertant exhibiting radio-resistant phenotype of SX9 is observed during long-term culture.

The identified T to C transition on 9572 in the dna-pkcs gene results in substitution of leucine (3191) to proline. Conformational change of the protein molecule may occur because the rotation of peptide bond of proline residue is severely restricted. Proline residue is virtually nonpermissive for conservative change during evolution.

The relationship between quantity and activity of DNA-PKcs products in cell lines SR-1, SX9, and SX10 was analyzed. The cell line SX10 exhibits a high sensitivity to ionizing radiation but a normal level of DNA-PK activity. Intriguingly, the quantity of DNA-PKcs molecules decreases to about one-half in SX10 as well as SX9 cells. However, it was shown that DNA-PK activity in SX10 cells is indistinguishable from that in normal cells (30). Semiquantitative RT-PCR of the dna-pkcs gene in SR-1, SX9 cell lines, and SX10 supports this observation (Fig. 2A). These investigations indicate that the reduction of DNA-PK activity in SX9 cells cannot be explained by the moderate reduction of its quantity. We conclude that the identified leucine to proline substitution in DNA-PKcs products is responsible for the aberration of SX9 cells.

The next question is how this mutation causes the radiosensitivity. To address this issue, we tested the V(D)J recombination in SX9 cells. The coding joint formation was severely impaired. The recombination frequency was reduced to less than 1/13. On the other hand, the frequency and the fidelity of signal joint formation are also reduced in SX9 cells. The frequency decreased to approximately 1/10 that of the parent cell. Moreover, ApaLI positive clones generated by precise joint formation were reduced to 12.2% of total recombined clones (Table I). These observations are somewhat different from those in other previously reported dna-pkcs-deficient cell lines such as scid, V3, and irs-20. In those cell lines, the coding joint formation is drastically impaired although the signal joint formation is nearly equal to normal control or only slightly reduced. Therefore, the drastic reduction of the fidelity of signal joint formation in SX9 is especially noteworthy. In that regard, SX9 cells resemble ku86-deficient mutant cells, xrs-6 rather than dna-pkcs-deficient cells. Both coding joint and signal joint formation are impaired in xrs-6 cells (16).

In detailed analyses, DNA sequence of 86 ApaLI negative signal joints demonstrated that almost all of the recombinations in the clones prepared from rag-introduced SX9 cells took place within the RSS (Fig. 4, A and B). Another interesting point is that the deletions in signal joint in SX9 cells occurred symmetrically as shown in Fig. 4, A and B. This finding suggests that the deletion took place when the two RSSs were assembled together and protected by some protein, because two intermediates, including RSS, with 23 or 12 spacers exist separately and are independently attacked by some exonuclease, deletion from the border of RSS by exonuclease activity may occur independently and the deletion in signal joint may not occur in a symmetric manner.

Recently, equine scid was reported as a mutant with a defect in signal joint formation as well as in coding joint formation (26). In addition, detailed analyses strongly suggested that the dna-pkcs gene is the responsible gene for equine scid, and the point mutation just upstream from the phosphatidylinositol 3-kinase domain making the nonsense codon resulting in the truncation of the product was suggested as a candidate for the responsible genetic aberration (25). Complete determination of the equine scid dna-pkcs gene sequence will shed light on the precise mechanism.

V(D)J recombination in SX9 and previously reported DNA-PKcs-deficient cell lines is shown (Table II). The mutation of SX9 and equine scid exist just upstream from the conserved phosphatidylinositol 3-kinase domain in DNA-PKcs but the mutation of mouse scid exists downstream from the phosphatidylinositol 3-kinase domain (Fig. 6). These findings suggest the crucial role of protein kinase activity in DNA-PKcs for signal joint formation.

                              
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Table II
V(D)J recombination activity of radiosensitive mutants


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Fig. 6.   Mutants of DNA-PKcs. Expressed DNA-PKcs products from each cell are shown by an open box. Mutated amino acid number is shown above the rectangles. PI3K is phosphatidylinositol 3-kinase domain. The broken line indicates the region in which DNA sequence has not been determined.

Although it has not been proved experimentally, it seems likely that the DNA-PK activity in murine scid is partial. Therefore, in murine scid the association and/or activation of other participants (especially XRCC4-ligase IV complex) involved in V(D)J recombination, which is controlled by the kinase activity of DNA-PK, might be stronger than those in SX9. Furthermore, the activation energy for signal joint formation seems to be lower than that for coding joint, since coding joint formation is more complex and may require more proteins than signal joint formation (37). These may be the reasons why the signal joint formation is normal only in murine scid. In other words, the difference in DNA-PK activity causes the difference in activation of other participants involved in V(D)J recombination. The requirement of activity in the joining step of V(D)J recombination differs between signal and coding joint formation. This hypothesis should be tested.

Finally, to confirm the responsibility of DNA-PKcs for SX9 abnormal V(D)J recombination, full-length cDNA of dna-pkcs cDNA was introduced into SX9 cells and the V(D)J recombination activity was assessed. Restoration of frequency and fidelity in coding and signal joint formation was observed (Table I and Fig. 5). Although the restoration of frequency was not enough in coding joint and signal joint formation, it seems reasonable because the assay was performed under transient expression of rag-1, -2, assay vector, and dna-pkcs gene. If the probability of simultaneous introduction of these genes is considered, it seems natural that the restoration by the dna-pkcs gene was not complete. We obtained similar results when the full-length dna-pkcs gene constructed in other expression vectors were used (data not shown). Additionally, complete restoration of V(D)J recombination is generally difficult (16). Indeed, when the scid cell line was introduced by human chromosome 8, in which human dna-pkcs exists, radiosensitivity but not V(D)J recombination was restored completely (data not shown). We are currently trying to determine whether the radiosensitivity, DNA-PK activity, and activation of c-Abl are restored in the stable transformants.

To date, murine scid is the only DNA-PKcs-deficient mutant in which DNA sequence has been determined. A candidate for the responsible genetic aberration in dna-pkcs in equine scid was also suggested (25). Analyses of other DNA-PKcs-deficient mutants at the DNA sequence level are required to elucidate the biological mechanism.

SX9 is the second cell line in which the DNA sequence of the dna-pkcs gene was completely determined. This cell line should therefore contribute to a better understanding not only of V(D)J recombination but also of the repair mechanism including double strand break repair.

    ACKNOWLEDGEMENTS

We thank K. Yokoro for encouragement and B. F. Burke-Gaffney for editing the English during preparation of this manuscript. We also thank G. F. Whitmore for providing the V3 cell line; P. Jeggo for the xrs-6 cell line; H. Koyama for the SR-1 cell line; M. Gellert for pJH200 and pJH290; Y. Shinkai for BCMGSNeo-TAG1 and BCMGSNeo-TAG2; K. Maruyama for pME18S. We also thank E. Kubo, Y. Hoki, and T. Ohata for technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence data bases with the following accession numbers: AB007544 for Prkdc, AB010282 for ku70, and AB011543 for Prkdc in the expression vector.

Contributed equally to the results of this work.

Dagger Dagger To whom correspondence should be addressed: Dept. of Biology and Oncology, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba-shi, Chiba 263-8555, Japan. Tel.: 81-43-206-3219; Fax: 81-43-251-4593; E-mail: abemasum{at}uexs72.nirs.go.jp.

1 The abbreviations used are: DNA-PK, DNA-dependent protein kinase; XRCC, x-ray cross-complementing groups; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; SCID, severe combined immune deficiency; RT-PCR, reverse transcriptase polymerase chain reaction; RAG, recombination activating gene; RSS, recombination signal sequence; LA-PCR, long and accuracy-PCR; CHO, Chinese hamster ovary.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Caldecott, K., and Jeggo, P. (1991) Mutat. Res. 255, 111-121[Medline] [Order article via Infotrieve]
  2. Tambini, C. E., George, A. M., Rommens, J. M., Tsui, L. C., Scherer, S. W., and Thacker, J. (1997) Genomics 41, 84-92[CrossRef][Medline] [Order article via Infotrieve]
  3. Grawunder, U., Wilm, M., Wu, X., Kulesza, P., Wilson, T. E., Mann, M., and Lieber, M. R. (1997) Nature 388, 492-495[CrossRef][Medline] [Order article via Infotrieve]
  4. Li, Z., Otevrel, T., Gao, Y., Cheng, H. L., Seed, B., Stamato, T. D., Taccioli, G. E., and Alt, F. W. (1995) Cell 83, 1079-1089[Medline] [Order article via Infotrieve]
  5. Weaver, D. T., and Alt, F. W. (1997) Nature 388, 428-429[CrossRef][Medline] [Order article via Infotrieve]
  6. Wilson, T. E., Grawunder, U., and Lieber, M. R. (1997) Nature 388, 495-498[CrossRef][Medline] [Order article via Infotrieve]
  7. Critchlow, S. E., Bowater, R. P., and Jackson, S. P. (1997) Curr. Biol. 7, 588-598[Medline] [Order article via Infotrieve]
  8. Teo, S. H., and Jackson, S. P. (1997) EMBO J. 16, 4788-4795[Abstract/Free Full Text]
  9. Anderson, C. W. (1993) Trends Biochem. Sci. 18, 433-437[CrossRef][Medline] [Order article via Infotrieve]
  10. Kharbanda, S., Pandey, P., Jin, S., Inoue, S., Bharti, A., Yuan, Z. M., Weichselbaum, R., Weaver, D., and Kufe, D. (1997) Nature 386, 732-735[CrossRef][Medline] [Order article via Infotrieve]
  11. Araki, R., Fujimori, A., Hamatani, K., Mita, K., Saito, T., Mori, M., Fukumura, R., Morimyo, M., Muto, M., Itoh, M., Tatsumi, K., and Abe, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2438-2443[Abstract/Free Full Text]
  12. Blunt, T., Gell, D., Fox, M., Taccioli, G. E., Lehmann, A. R., Jackson, S. P., and Jeggo, P. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10285-10290[Abstract/Free Full Text]
  13. Danska, J. S., Holland, D. P., Mariathasan, S., Williams, K. M., and Guidos, C. J. (1996) Mol. Cell. Biol. 16, 5507-5517[Abstract]
  14. Hamatani, K., Matsuda, Y., Araki, R., Itoh, M., and Abe, M. (1996) Immunogenetics 45, 1-5[CrossRef][Medline] [Order article via Infotrieve]
  15. Itoh, M., Hamatani, K., Komatsu, K., Araki, R., Takayama, K., and Abe, M. (1993) Radiat. Res. 134, 364-368[Medline] [Order article via Infotrieve]
  16. Taccioli, G. E., Gottlieb, T. M., Blunt, T., Priestley, A., Demengeot, J., Mizuta, R., Lehmann, A. R., Alt, F. W., Jackson, S. P., and Jeggo, P. A. (1994) Science 265, 1442-1445[Medline] [Order article via Infotrieve]
  17. Mizuta, R., Taccioli, G. E., and Alt, F. W. (1996) Int. Immunol. 8, 1467-1471[Abstract]
  18. Singleton, B. K., Priestley, A., Steingrimsdottir, H., Gell, D., Blunt, T., Jackson, S. P., Lehmann, A. R., and Jeggo, P. A. (1997) Mol. Cell. Biol. 17, 1264-1273[Abstract]
  19. Fulop, G. M., and Phillips, R. A. (1990) Nature 347, 479-482[CrossRef][Medline] [Order article via Infotrieve]
  20. Taccioli, G. E., Rathbun, G., Oltz, E., Stamato, T., Jeggo, P. A., and Alt, F. W. (1993) Science 260, 207-210[Medline] [Order article via Infotrieve]
  21. Zhu, C., Bogue, M. A., Lim, D. S., Hasty, P., and Roth, D. B. (1996) Cell 86, 379-389[Medline] [Order article via Infotrieve]
  22. Lieber, M. R., Hesse, J. E., Lewis, S., Bosma, G. C., Rosenberg, N., Mizuuchi, K., Bosma, M. J., and Gellert, M. (1988) Cell 55, 7-16[Medline] [Order article via Infotrieve]
  23. Lin, J. Y., Muhlmann-Diaz, M. C., Stackhouse, M. A., Robinson, J. F., Taccioli, G. E., Chen, D. J., and Bedford, J. S. (1997) Radiat. Res. 147, 166-171[Medline] [Order article via Infotrieve]
  24. Taccioli, G. E., Cheng, H. L., Varghese, A. J., Whitmore, G., and Alt, F. W. (1994) J. Biol. Chem. 269, 7439-7442[Abstract/Free Full Text]
  25. Shin, E. K., Perryman, L. E., and Meek, K. (1997) J. Immunol. 158, 3565-3569[Abstract]
  26. Wiler, R., Leber, R., Moore, B. B., VanDyk, L. F., Perryman, L. E., and Meek, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11485-11489[Abstract]
  27. Sato, K., Ito, A., Shiomi, T., Hama-Inaba, H., Ishikawa, H., Yoshizumi, T., and Nakazawa, T. (1986) Jpn. J. Cancer Res. 77, 456-461[Medline] [Order article via Infotrieve]
  28. Sato, K., Chen, D. J., Eguchi-Kasai, K., Itsukaichi, H., Odaka, T., and Strniste, G. F. (1995) J. Radiat. Res. (Tokyo) 36, 38-45[Medline] [Order article via Infotrieve]
  29. Hesse, J. E., Lieber, M. R., Gellert, M., and Mizuuchi, K. (1987) Cell 49, 775-783[Medline] [Order article via Infotrieve]
  30. Peterson, S. R., Stackhouse, M., Waltman, M. J., Chen, F., Sato, K., and Chen, D. J. (1997) J. Biol. Chem. 272, 10227-10231[Abstract/Free Full Text]
  31. Fujimori, A., Araki, R., Fukumura, R., Saito, T., Mori, M., Mita, K., Tatsumi, K., and Abe, M. (1997) Genomics 45, 194-199[CrossRef][Medline] [Order article via Infotrieve]
  32. Eastman, Q. M., Leu, T. M., and Schatz, D. G. (1996) Nature 380, 85-88[CrossRef][Medline] [Order article via Infotrieve]
  33. McBlane, J. F., van Gent, D. C., Ramsden, D. A., Romeo, C., Cuomo, C. A., Gellert, M., and Oettinger, M. A. (1995) Cell 83, 387-395[Medline] [Order article via Infotrieve]
  34. Tonegawa, S. (1983) Nature 302, 575-581[Medline] [Order article via Infotrieve]
  35. Errami, A., Smider, V., Rathmell, W. K., He, D. M., Hendrickson, E. A., Zdzienicka, M. Z., and Chu, G. (1996) Mol. Cell. Biol. 16, 1519-1526[Abstract]
  36. Gupta, R. S., Chan, D. Y., and Siminovitch, L. (1978) Cell 14, 1007-1013[Medline] [Order article via Infotrieve]
  37. Ramsden, D. A., Paull, T. T., and Gellert, M. (1997) Nature 388, 488-491[CrossRef][Medline] [Order article via Infotrieve]


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