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Nucleic Acids Research Pages 3146-3153  

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation
Introduction
Materials And Methods
   Cell and culture conditions
   Chemicals
   Irradiation
   Survival curves
   Analysis of DSB rejoining kinetics by pulse-field gel electrophoresis
   Cell fusion and complementation analyses
   Microcell-mediated chromosome transfer
   Fluorescence in situ hybridization
   Immunodetection of the DNA-PKcs protein
   DNA-PK peptide kinase assays
   V(D)J recombination
Results
   Isolation of an X-ray-sensitive mutant, XR-C1, from CHO cells and its sensitivity to various DNA-damaging agents
   DNA double-strand rejoining
   Genetic complementation analyses
   Complementation of XR-C1 by human chromosome 8
   DNA-PKcs protein and DNA-PK activity in XR-C1 cells and in XR-C1 microcell hybrid clones with human chromosome 8
   V(D)J recombination
Discussion
   XR-C1 belongs to the XRCC7 complementation group
   DNA-PKcs is required for both V(D)J recombination joining reactions
   Molecular basis for the different group 7 mutant phenotypes
Acknowledgements
Note Added In Proof
References

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

Abdellatif Errami, Dong Ming He1, Anna A. Friedl2, Wilhelmina J. I. Overkamp, Bruno Morolli, Eric A. Hendrickson1, Friederike Eckardt-Schupp2, Mitsuo Oshimura3, Paul H. M. Lohman, Stephen P. Jackson4, Malgorzata Z. Zdzienicka*

Department of Radiation Genetics and Chemical Mutagenesis, MGC, Leiden University-Medical Center andJ. A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, Leiden, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands, 1Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA, 2GSF, Institute for Radiation Biology, Neuherberg, Germany, 3Tottori University, Yonago, Japan and 4Wellcome/CRC Institute, and Department of Zoology,University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

Received March 30, 1998; Revised and Accepted April 28, 1998

ABSTRACT

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7/SCID (Prkdc) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.

INTRODUCTION

In order to examine the cellular responses to ionizing radiation (IR), IR-sensitive rodent cell mutants have been isolated. Eleven complementation groups have been established and the genes defective in these groups have been designated XRCC1-11 (for a review see 1). Analysis of these mutants has lead to the identification of genes involved in DNA double-strand break (DSB) rejoining and V(D)J recombination (for reviews see 2,3). In particular, the DNA-dependent protein kinase (DNA-PK) complex, which is composed of the Ku heterodimer and a catalytic subunit (DNA-PKcs) has been shown to be involved in DSB repair (2,3). DNA-PKcs is the product of the scid (severe combined immune deficient) gene [also designated XRCC7 or Prkdc (1)], and homozygous scid mice are profoundly immune deficient due to impaired V(D)J recombination, which results in T- and B-cell maturational arrest (reviewed in 4). Furthermore, scid mice are IR sensitive (5) and impaired in DSB repair (6,7). Ku is a heterodimeric protein of 70 (Ku70:XRCC5) and 86 kDa (Ku86/Ku80:XRCC6) subunits which binds tightly to double-stranded DNA ends. Rodent cell lines which are mutated in the Ku86 gene are IR sensitive, impaired in DSB repair and defective for V(D)J recombination (reviewed in 2,3). In addition, inactivation of the Ku86 (8-10) or Ku70 (11,12) genes in either mice or murine embryonic stem cells produced the predicted deficits in IR sensitivity, DSB repair and V(D)J recombination. Thus, DNA-PK has been unequivocally identified as an important mammalian DNA repair complex, and mutations in any of the three DNA-PK subunits result in severe IR sensitivity and V(D)J recombination defects due to impaired DSB repair.

While the IR sensitivity and DSB repair activities of Ku70-, Ku86- and DNA-PKcs-defective cell lines are virtually identical, their V(D)J phenotypes are not. Thus, all Ku70-/- and Ku86-/- cell lines described to date are defective for both rejoining steps of V(D)J recombination (coding junction and signal junction formation), whereas the original DNA-PKcs mutant cell line (murine scid) is predominately defective for coding junction formation (reviewed in 13,14). The differing phenotypes for mutations of genes thought to act as a complex was unexpected and suggested either that the murine scid mutation was not completely penetrant or that DNA-PKcs and the Ku subunits performed functions independent of the DNA-PK complex. These hypotheses were not mutually exclusive and evidence has accumulated to support both views. Thus, there is abundant cellular and molecular data to suggest that the murine scid mutation is non-null (reviewed in 13). In addition, DNA-PKcs is active and able to bind DNA in the absence of Ku (15,16), and Ku can stimulate end joining in the absence of DNA-PKcs (17-19). This latter observation is also consistent with the presence of Ku homologues and the apparent lack of a DNA-PKcs homologue in yeast (reviewed in 2). Taken together, these data suggested that the Ku heterodimer was required for both V(D)J recombination joining reactions whereas DNA-PKcs might only be required for coding junction formation.

To address the role of DNA-PKcs in V(D)J recombination more fully, additional mammalian cell mutants were isolated. Subsequently, two Chinese hamster cell mutants, V-3 (20) and irs-20 (21), a mouse mammary carcinoma cell line, SX-9 (22), a human glioma cell line, M059J, and an Arabian foal, equine scid, were shown to be defective in the Prkdc gene encoding DNA-PKcs (23-27). Subsequent experimentation demonstrated that the V(D)J recombination phenotype of V-3 and irs-20 was identical to murine scid cells, i.e. that coding junction formation was more severely affected than signal junction formation (23,26). The existence of three independent rodent mutant cell lines (murine scid, V-3 and irs-20) with identical phenotypes implied that DNA-PKcs protein was only required for coding junction formation. Confusingly, however, analysis of cells derived from the equine scid mutants revealed that, like XRCC5 and XRCC6 mutant cell lines, they were defective in both V(D)J joining reactions (28). To help clarify this issue, we have isolated and characterized a new X-ray-sensitive mutant cell line, XR-C1, derived from Chinese hamster ovary (CHO) cells which belongs to ionizing group 7 (XRCC7). Cellular and biochemical characteristics of XR-C1 showed that XR-C1 cells lacked detectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). However, unlike other rodent XRCC7 mutant cell lines, which are preferentially impaired in coding joint formation, XR-C1 was defective in both coding and signal joint formation. These data imply that DNA-PKcs, like Ku70 and Ku86, is required for both V(D)J joining reactions.

MATERIALS AND METHODS

Cell and culture conditions

The X-ray-sensitive hamster cell mutants XR-1 (29), XR-V15B (30), V-C8 (31) and V-3 (20) have been described. XR-1 cells were kindly supplied by Dr T.D.Stamato (Lankenau Medical Research Centre, Wynnewood, PA), and V-3 cells by Dr G.F.Whitmore (Ontario Cancer Institute, Toronto, Ontario). TOR (thioguanine resistant and ouabain resistant) derivatives of XR-C1, XR-1 and V-3 were obtained by the isolation of a spontaneous thioguanine-resistant clone followed by the isolation of an ouabain-resistant clone (31). scid cells (SCGR11) were obtained from Dr D.Weaver (Center for Blood Research, Boston, MA). Construction of monochromosomal hybrid clones of mouse A9 cells containing a single human chromosome 8 tagged with the pSV2neo plasmid DNA has been described (32). XR-C1, XR-1, XR-V15B, V-3 and V-C8 mutants and their respective parental cell lines CHO9, CHOK1, V79B, AA8 and V79, and scid cells (SGGR11), were cultured in plastic dishes (Greiner) in Ham's F-10 medium (without hypoxanthine and thymidine), supplemented with 10% fetal serum (Gibco), or 15% newborn calf serum and penicillin (100 U/ml) and streptomycin (0.1 mg/ml) for survival experiments. The monochromosomal hybrids were selected in medium supplemented with G418 at 400 µg/ml. Cells were maintained at 37°C in a 5% CO2 atmosphere, humidified to 95-100%. For each experiment, a frozen ampoule containing 106 cells was thawed, allowed to grow for 2-3 days, subcultured and grown for an additional 3 days. Subsequently, the cells were trypsinized and used for the experiments.

Chemicals

Cytochalasin-B, polyethylene glycol (PEG) 1450 mol. wt, human and mouse cot-1 DNA were purchased from Sigma (St Louis, MO, USA). Colcemid and Geneticin (G418) were from Gibco BRL (Gaithersburg, MD, USA), and phytohemaglutinin (PH-A) from Difco Laboratories (Detroit, MI, USA). Bleomycin (BLM) was from Londbeck (Amsterdam), and mitomycin C (MMC) Kyowa from Lampro B. V. (The Netherlands); ethyl methane sulphonate (EMS) from Eastman Co. (Rochester, NY, USA).

Irradiation

Cells were irradiated in medium at a dose rate of 3 Gy/min (200 kV, 4 mA, 0.78 mm Al). For irradiation with UV light of 254 nm, a Philips TUV germicidal lamp was used with a fluence rate of 0.19 W/m2, measured with an IL/770 germicidal radiometer.

Survival curves

Cultures in exponential growth were trypsinized and 300-3 000 cells were plated, in duplicate, on 10 cm dishes and left to attach for 4 h. The cells were then treated with MMC, BLM or EMS for 24 or 1 h, respectively or were UV- or X-irradiated. After the treatment with chemicals, medium was removed, the cells were rinsed twice with PBS, normal medium was added, and then the cells were incubated for 8-10 days. The dishes were then rinsed with NaCl (0.9%), air dried and stained with methylene blue (0.25%) and visible colonies counted. Each survival curve represents the mean of at least three independent experiments.

Analysis of DSB rejoining kinetics by pulse-field gel electrophoresis

Subconfluent cells were trypsinized and agarose plugs were made as described (33), except that the plugs were held in medium. To establish the damage-response calibration curve part of the plugs were irradiated with 0, 10, 20, 30, 40 and 50 Gy (60Co-[gamma]-rays) and the cells were lysed directly afterwards. For repair analysis, the remaining plugs were X-irradiated with 0 and 40 Gy, transferred to fresh, pre-warmed growth medium and incubated in an incubator at 37°C before lysis. The plugs were cast into a 0.8% agarose gel which were run in a CHEF electrophoresis system (Biorad) for 30 h at 1.3 V/cm with a 75 min pulse time. Ethidium-bromide-staining and measurement of fluorescence intensity have been described (33). On each gel, repair samples and calibration samples from the same experiment were loaded. For each lane, the total fluorescence signal and the fraction (Fr) representing molecules released from the well were determined using a home-made evaluation program. The Fr values determined for the calibration samples were plotted against dose to give a gel- and cell-population-specific DNA damage response curve which served for converting the Fr values obtained in repair samples into dose-equivalent values. For determination of the rejoining kinetics, the dose equivalents were plotted against repair incubation time.

Cell fusion and complementation analyses

The TOR hybridization/selective system (31) was used with fusion of one doubly-marked line to one unmarked line by polyethylene glycol (PEG). Populations of hybrids (>100 clones) were collected from each cross and then used to determine survival to ionizing radiation. The modal chromosome number was determined by fluoresence in situ hybridization with mouse cot-1 DNA to metaphase chromosome of scid/hamster cell hybrids.

Microcell-mediated chromosome transfer

Microcells with single human chromosomes were obtained as described (32). The microcell suspension was added to a monolayer of recipient cells (~1.5 × 106 mutant cells per 6 cm dish) and allowed to attach at room temperature for 20 min. The cells were fused by treatment with 2 ml of 47% PEG (in serum-free medium containing 10% dimethyl sulfoxide) for 1 min followed by washing in serum-free medium containing 10% dimethyl sulfoxide. After 24 h, the cells were trypsinized and split into three 10 cm dishes with selective medium containing 400 µg/ml G418. The resulting microcell hybrids were isolated and cloned 10-14 days after selection.

Fluorescence in situ hybridization

Metaphase chromosomes were generated following 2 h treatment with colcemid (0.1 µg/ml). The cells were harvested by trypsinization and treated with 75 mM KCl for 30 min before being fixed in a 3:1 mixture of methanol:glacial acetic acid. In situ hybridization to metaphase spreads of the cell lines with biotin-labelled human or mouse cot-1 DNA was performed as described (34), using RNase A and pepsin-treated chromosomes. The biotin-labelled DNA was detected with FITC-conjugated antibodies and the rodent chromosomes were counter stained with propidium iodide.

Immunodetection of the DNA-PKcs protein

Whole cell extracts were prepared as described (35). Briefly, a frozen cell pellet (~5 × 107 cells) was thawed on ice and resuspended in an equal volume of freshly made extraction buffer [50 mM NaF, 20 mM HEPES (pH 7.6), 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (0.5 µg/ml), trypsin inhibitor (1.0 µg/ml), aprotinin (0.5 µg/ml), bestatin (40 µg/ml)] before being sequentially snap frozen on dry-ice and thawed at 30°C three times. After microcentrifugation (14 000 r.p.m.) for 10 min at 4°C, supernatants were aliquoted and stored at -70°C. Protein concentrations were determined using the Bradford protein assay with BSA as the standard. Protein (100 µg) was resolved by SDS-PAGE, transferred to nitrocellulose filters and probed with polyclonal antisera FLA, which recognizes the full-length DNA-PKcs protein, and with monoclonal 42-27, which recognizes the DNA-PKcs kinase domain (36). The filters were then probed with horseradish-peroxidase-conjugated goat antibody to rabbit or mouse IgG (TAGO). Antibody binding was detected by enhanced chemiluminescence (Amersham).

DNA-PK peptide kinase assays

DNA-PK pull-down assays were performed as described (37). Briefly, 200 µg of whole cell extract was incubated with 40 µl of preswollen dsDNA-cellulose (Sigma) in Z[prime]0.05 buffer [25 mM HEPES/KOH (pH 7.6), 12.5 mM MgCl2, 20% glycerol, 0.1% Nonidet P-40, 1 mM DTT and 50 mM KCl] for 2 h at 4°C. The DNA-cellulose was then washed twice in 1 ml of Z[prime]0.05 buffer and resuspended in 60 µl Z[prime]0.05 buffer. Samples were divided into three aliquots, 0.5 µl [[gamma]-32P]ATP (300 Ci/mmol) was added and kinase assays were conducted in the presence of 4 nmol of peptide (0.2 mM) for 10 min at 30°C in a total volume of 20 µl. Reactions were then stopped by adding an equal volume of 30% acetic acid and analyzed by spotting the samples onto phosphocellulose paper, followed by washing and liquid scintillation counting as described (38). The sequences of wild-type and mutant p53 peptides were EPPLSQEAFADLLKK and EPPLSEQAFADLLKK, respectively. All assays were performed several times with at least two different extract preparations.

V(D)J recombination

Extrachromosomal V(D)J recombination assays were carried out as described (39), using pJH200 and pJH290 (40,41) as substrate plasmids to monitor signal and coding joint formation, respectively. Briefly, 10 µg of pJH200 or pJH290 plasmid was transfected with 3.5 µg RAG-1 and 4.2 µg RAG-2 (42) into 106 exponentially growing cells by coprecipitation with calcium phosphate. Cells were then incubated for 48 h at 37°C with 0.15 mM sodium butyrate (43) prior to recovery of plasmid DNA by the Hirt protocol. DNA was digested with DpnI to eliminate unreplicated plasmids, transfected into electrocomponent DH-10B cells (Gibco) and then plated on ampicillin (100 µg/ml) or ampicillin and chloramphenicol (16.5 µg/ml) plates. Unrearranged plasmids conferred resistance to ampicillin (Ampr), whereas rearranged plasmids conferred resistance to both ampicillin and chloramphenicol (Ampr/Camr). The ratio of double-drug-resistant colonies to the number of ampicillin-resistant colonies represents the frequency of V(D)J recombination for a cell line. The formation of a signal junction in pJH200 creates a new Apa LI restriction site and coding junction formation in pJH290 results in the loss of a ClaI restriction site. Thus, restriction enzyme analysis was performed to confirm the authenticity of the double-drug-resistant bacterial colonies as having arisen from legitimate V(D)J recombination events.

RESULTS

Isolation of an X-ray-sensitive mutant, XR-C1, from CHO cells and its sensitivity to various DNA-damaging agents

XR-C1 was isolated from an ENU-mutagenized population of CHO9 cells by replica plating (44). XR-C1 was ~3.3-fold more sensitive to X-irradiation than the parental CHO9 cell line (Fig. 1) and XR-C1 maintained its X-ray sensitivity for >3 months of continuous culture (data not shown). The sensitivity of XR-C1 was also compared with wild-type cells with regard to killing by UV-irradiation, bleomycin, and mono- (EMS) and bi- (MMC) functional alkylating agents. XR-C1 was slightly hypersensitive to bleomycin and EMS and showed a normal response to MMC (Table 1). The doubling times of CHO9 and XR-C1 were very similar, 17.5 and 18 h, respectively, as were the cloning efficiencies, ~95% for CHO9 cells and ~80% for XR-C1 cells (data not shown). Thus, the XR-C1 cell line appeared to be relatively healthy and was selectively sensitive to DNA damage caused by X-irradiation.


Figure 1. X-ray survival curves of CHO9 ([cir]) and XR-C1 (s). Error bars represent 1 SEM.

Table 1. Cross-sensitivities of XR-C1 to various DNA damaging agents
Cell line D10
  X-ray
(Gy)		 UV
(J/m2)		 BLM
(g/ml)		 EMS
(mM)		 MMC
(ng/ml)
CHO9 5.0 15.4 10.6 30.5 98.0
XR-C1 1.5 12.7 7.1 24.6 99.0
The numbers represent the mean of D10 value, i.e. the dose required to kill 90% of the cells. All values are the average of at least three independent survival experiments performed in duplicate.

DNA double-strand rejoining

Many X-ray-sensitive cell lines are also impaired in DSB repair (reviewed in 2,3). To determine if this paradigm could be extended to XR-C1 cells, the kinetics of DSB rejoining following X-irradiation (40 Gy) was determined in XR-C1 and CHO9 cells by pulse-field gel electrophoresis. After 4 h, ~45% of the DNA lesions were still unrepaired in XR-C1 cells, in comparison with only 30% in CHO9 cells (Fig. 2). This defect in DNA DSB repair could be observed at all time of the assay (Fig. 2) and was highly reminiscent of the impaired DSB repair profiles from other IR-sensitive cell lines (7,30,45). Thus, XR-C1 cells were impaired in the process of DSB repair.


Figure 2. Measurement of DSB repair rejoining by pulse field electrophoresis in CHO9 ([cir]) and XR-C1 (s) cells. Error bars represent 1 SEM.


Figure 3. X-ray survival curves of (A) XR-V15B (z), XR-1 ([diams]), hybrid cell lines, XR-V15B/XR-C1TOR, XR-1/XR-C1TOR and V79B (shaded lines); (B) AA8 (o), V-3TOR (l) and hybrid clones XR-C1/V-3TOR (shaded lines); (C) CHO9 ([cir]), scid ([squf]) and hybrid clones scid/XR-C1TOR (shaded lines). Error bars represent 1 SEM.

Genetic complementation analyses

To determine whether the XR-C1 mutant represented a complementation group distinct from other IR-sensitive, DSB-repair-deficient rodent cell mutants, genetic complementation analysis by cell fusion was performed. Hybrids were generated between XR-C1 and XR-1, XR-V15B, V-3 and V-C8 cells (representing IR groups 4, 5, 7 and 11, respectively), and these hybrids were analyzed for their IR sensitivity. Hybrids between XR-C1 cells and XR-V15B or XR-1 (Fig. 3A) or V-C8 cells (data not shown) showed an IR sensitivity indistinguishable from wild-type cells. In striking contrast, hybrids between XR-C1 and V-3 cells (Fig. 3B) or scid cells (Fig. 3C) showed only partial complementation of radiation resistance. A similar degree of complementation has been reported in hybrids between V-3 and scid, mutants known to be defective in the Prkdc gene (46). The reason for this partial complementation in hybrids formed between mutants of IR group 7 remains unknown. Thus, from these experiments we concluded that XR-C1 cells do not belong to complementation groups 4, 5 or 11, but rather that they belonged to complementation group 7.

Complementation of XR-C1 by human chromosome 8

The gene encoding DNA-PKcs (Prkdc), which complements V-3 and scid cells, is located on human chromosome 8, and both cell lines can be complemented by this chromosome (46-50). Therefore, to confirm the results of the complementation analysis, a single human chromosome 8 was transferred to XR-C1 cells by microcell-mediated chromosome transfer (see Materials and Methods) and resulting microcell hybrids were examined for their IR sensitivity. XR-C1 cells were almost fully complemented by human chromosome 8 (Fig. 4), and all three hybrid clones examined contained a cytologically normal human chromosome 8, as determined by in situ hybridization using human cot-1 DNA as a probe (Fig. 5). These data were consistent with the assignment of XR-C1 cells to complementation group 7.


Figure 4. Survival curves of CHO9 ([cir]), XR-C1 (s) and minicell XR-C1/#8 hybrids ([Delta]) containing a cytogenetically normal human chromosome 8. Three independent hybrid clones were examined. Error bars represent 1 SEM.


Figure 5. Fluorescence in situ hybridization with human cot-1 DNA to hybrid metaphase chromosome of monochromosomal hybrid clones of XR-C1 containing a cytologically normal human chromosome 8.

DNA-PKcs protein and DNA-PK activity in XR-C1 cells and in XR-C1 microcell hybrid clones with human chromosome 8

DNA-PKcs protein in the XR-C1 mutant was evaluated by western blot analyses using polyclonal and monoclonal antibodies against DNA-PKcs. DNA-PKcs protein was not detected in extracts from XR-C1 cells, whereas the presence of this protein could be detected in the parental CHO9 cells, HeLa cells and in two independent microcell hybrids of XR-C1 containing a single human chromosome 8 (Fig. 6). DNA-PKcs protein expression in serial dilutions of the parental CHO9 and XR-C1 cell extracts indicated that the level of DNA-PKcs in XR-C1 is at least 30-fold lower than that in CHO9 (data not shown).


Figure 6. The ~460 kDa DNA-PKcs protein is not expressed in the X-ray-sensitive mutant XR-C1. Hybrid clones of XR-C1 with human chromosome 8, all show expressed DNA-PKcs. HeLa cells were used as a control for a DNA-PKcs-positive cell line. After subjecting samples (100 µg of total protein in each lane; except for HeLa cells, where 25 µg of HeLa extract was loaded) to SDS-PAGE, western-blot analysis was performed using either 42-27 monoclonal antibody directed against the kinase domain or the FLA antibody directed against the whole DNA-PKcs protein (data not shown). To serve as a control for equivalent loading of the proteins, blots were re-probed with an antibody raised against [beta]-actin.


Figure 7. DNA-PK peptide phosphorylation pull-down assays in: (A) the X-ray-sensitive hamster cell mutant XR-C1 and wild-type CHO9 cells; (B) hybrid clones of XR-C1 with a single human chromosome 8. 200 µg of whole cell extracts were analyzed by DNA-PK pull-down peptide assay with wild-type p53 peptide (solid columns) that is recognized effectively by DNA-PK, or with a mutated peptide (hatched columns), which is not an effective substrate for DNA-PK. All experiments were performed at least three times and error bars represent standard deviations between results of these experiments.

DNA-PKcs protein is required for the kinase activity of the DNA-PK complex (for reviews see 2,3). Thus, extracts from the parental CHO9 cells, the XR-C1 mutant cells and XR-C1 microcell hybrids containing a single human chromosome were assayed for DNA-PK activity. CHO9 cell extracts readily phosphorylated a good DNA-PK substrate (a p53-derived peptide; see Materials and Methods) and they were much less effective in phosphorylating a mutated version of this substrate (Fig. 7A). XR-C1 extracts were incapable of phosphorylating either peptide substrate (Fig. 7A). In striking contrast, the addition of a single human chromosome 8 into XR-C1 microcell hybrid clones restored peptide-specific kinase activity to the extracts (Fig. 7B). Together, these results unequivocally demonstrate that XR-C1 cells are defective in the stable expression of DNA-PKcs protein and that the deficiency in DNA-PK activity can be complemented by a chromosome containing the human Prkdc gene.

V(D)J recombination

Of the four DNA-PKcs mutant cell lines described to date, the three rodent cell lines (murine scid, V-3 and irs-20) are preferentially defective in V(D)J mediated coding junction formation (23,26,51-53), whereas only the equine scid cell line is defective in both coding and signal joining events (24,28). Thus, it was of interest to determine the V(D)J recombination potential of the XR-C1 mutant. XR-C1 and wild-type CHO9 cells were co-transfected with RAG-1 and RAG-2 expression vectors (42) and either a coding junction testor plasmid (pJH290) or a signal junction testor plasmid (pJH200) (39). Valid recombination events were scored by drug selection (chloramphenicol resistance) and then confirmed by restriction analysis. In four independent experiments CHO9 cells efficiently carried out both coding and signal junction formation, though the pJH290 (coding junction) plasmid was more effectively rearranged than the pJH200 (signal junction) plasmid (Table 2). This rearrangment bias using these extrachromosomal vectors has been previously observed (54,55). XR-C1 cells were profoundly defective in the rejoining of both signal and coding junctions (Table 2). XR-C1 cells carried out coding junction formation 63-fold lower and signal junction formation 74-fold lower than the frequency observed in CHO9 cells (Table 2). In addition, XR-C1 cells were >94-fold reduced for inversional (which requires both signal and coding junction formation) V(D)J rearrangement (data not shown). Finally, XR-C1 microcell hybrids containing a single human chromosome 8 were fully complemented for both coding and signal junction formation (data not shown). Thus, the XR-C1 mutant, unlike other rodent cell IR group 7 mutants, was impaired in both V(D)J-mediated joining events.

DISCUSSION

We have isolated and characterized a new IR-sensitive Chinese hamster cell mutant, XR-C1, derived from CHO9 cells that is defective in DSB repair. Genetic complementation analysis indicated that XR-C1 belonged to IR group 7, which is defective in the gene encoding DNA-PKcs (Prkdc). It seems likely that the mutation in XR-C1 is more severe than that in other rodent mutants of this group, as (i) it is the most IR-sensitive mutant (>3-fold, whereas scid or V-3 are only ~2-fold more IR sensitive than their parental cell lines (5,20), (ii) immunoblot analysis failed to detect a DNA-PKcs cross-reactive protein in XR-C1 cells (Fig. 6) and (iii) both V(D)J coding and signal joint formation are severely defective in XR-C1 cells (Table 2).

Table 2. V(D)J recombination is defective in the XR-C1 mutant
Cell line Experiment Ampra Camr + Amprb %Recc
Coding Junction (pJH290)  
CHO9 1 2400 270 11.25
  2 2700 710 26.30
  3 210 400 2760 1.31
  4 28 130 1070 3.80
     Average 10.676 ± 11.24
XR-C1 1 2100 0 0.00
  2 5200 30 0.58
  3 162 270 130 0.08
  4 53 400 10 0.02
     Average 0.17 ± 0.27
Signal Junction (pJH200)  
CHO9 1 2500 60 2.40
  2 128 260 710 0.55
  3 128 200 160 0.13
  4 16 870 280 1.66
     Average 1.19 ± 1.04
XR-C1 1 NDd ND ND
  2 113 000 30 0.03
  3 149 070 30 0.02
  4 5830 0 0.00
     Average 0.02 ± 0.01
aAmpr, the total number of ampicillin-resistant bacterial colonies.
bCar + Amr, the total number of doubly chloramphenicol- and ampicillin-resistant bacterial colonies.
c% Rec, the recombination frequency (Camr + Ampr/Ampr × 100).
dND, not done.

XR-C1 belongs to the XRCC7 complementation group

The eleven complementation groups that have been defined for IR-sensitive mammalian cell lines can be divided into two classes: those that are proficient for DSB repair and those that are deficient (reviewed in 1). The latter class includes the groups XRCC4, XRCC5, XRCC6, XRCC7 and XRCC11. The relatively IR-specific sensitivity of XR-C1 cells (Fig. 1 and Table 1) and the significant deficiency in DSB repair following IR-exposure (Fig. 2) suggested that XR-C1 represented either a new addition to one of these five complementation groups or that it defined a novel group. Full complementation of the IR sensitivity in hybrids of XR-C1 fused with XR-1 or XR-V15B mutants (Fig. 3A) or V-C8 cells (data not shown) indicated that XR-C1 was not defective in the XRCC4, XRCC5 or XRCC11 genes. Since cell lines defective in XRCC6 (Ku70) (11,12) are just now becoming generally available, we could not test these cell lines for complementation with XR-C1. However, additional biochemical experiments suggested that DNA end-binding activity, provided in part by XRCC6, was unaltered in XR-C1 cells (data not shown). Also, normal levels of Ku70 protein in these cells was detected by western blot analysis using polyclonal antibodies against Ku70 (data not shown). Thus, it is unlikely that a mutation in XRCC6 is responsible for the phenotypes of the XR-C1 mutant. In contrast with full complementation found in hybrids of XR-C1 with XR-1, XR-V15B or V-C8, only partial complementation was observed when XR-C1 cells were fused with scid or V-3 (XRCC7) cell lines (Fig. 3B and C). In addition, the IR sensitivity of XR-C1 was complemented by a single human chromosome 8 that contains the XRCC7:Prkdc gene (Fig. 4) and immunoblot analysis confirmed that DNA-PKcs protein was not expressed in XR-C1 cells (Fig. 6). Taken together, these multiple, independent experimental techniques confirmed that the XR-C1 cell mutant is a member of the XRCC7 complementation group and that the phenotypes of XR-C1 are due to the defective expression of DNA-PKcs.

DNA-PKcs is required for both V(D)J recombination joining reactions

Murine scid cells are defective in coding joint formation during V(D)J recombination (51-53). A preferential inability to form coding joints was observed in the other rodent cell mutants of XRCC7 group, V-3 and irs-20 (23,26), implying that DNA-PKcs protein was only required for coding junction formation. However, the observation that equine scid cells were defective for both coding and signal junction joining (28) suggested a role for DNA-PKcs in both V(D)J joining reactions. Subsequently, another murine XRCC7 group cell line, SX-9, was described (22,27). Very recently, it was demonstrated that this cell line is defective for both steps of V(D)J joining (56). We have now extended this analysis to XR-C1 cells and shown that they are also clearly defective for both coding and signal junction formation (Table 2). Thus, the existence of three independent cell lines, equine scid, SX-9 and XR-C1 from three different species (horse, mouse and hamster, respectively) provides compelling evidence that DNA-PKcs is necessary for V(D)J signal junction formation as well as for its documented role in V(D)J coding junction formation.

Molecular basis for the different group 7 mutant phenotypes

DNA-PKcs is a protein of 465 kDa that is encoded by an ~13 kb mRNA (57) which consists of 86 exons dispersed over >250 kb of genomic DNA (58). The daunting size of this gene has slowed molecular analyses of the group XRCC7 mutant cell lines. Nonetheless, a nonsense mutation resulting in an 83 amino acid truncation of DNA-PKcs was identified in the murine scid cDNA (59-61). Correspondingly, an ~457 kDa truncated protein was observed by immunoblot analysis suggesting that this cell line is not null for DNA-PKcs expression (60). A 5 nt deletion in the equine scid cDNA was identified that results in a frame shift and should generate an ~350 kDa truncated protein (28). This protein could not be detected by immunoblot analysis, suggesting that the truncation resulted in an unstable protein. It should be noted, however, that the first 570 bp of the equine scid cDNA were not sequenced and thus, while unlikely, it cannot unequivocally be ruled out that a second mutation in the extreme N-terminus of this gene exists (28). Regardless, it seems likely that little or no DNA-PKcs protein is expressed in equine scid cells. Intriguingly, a missense mutation has been identified at amino acid 3191 in the SX-9 cDNA (56), which is close to the location (amino acid 3155) of the equine scid mutation. SX-9 cells express diminished, but detectable, levels of DNA-PKcs protein (27). Unfortunately, the missense mutation in SX-9 cells was only observed on one chromosome suggesting, though not proving, that the other (wild-type) chromosomal allele was not expressed (56) and thus complicating the interpretation of this mutation with respect to the cells' phenotype(s). No DNA sequence data is yet available on the hamster mutants, V-3 and irs-20, nor on our XR-C1 mutant, due, principally, to the lack of a corresponding hamster DNA-PKcs cDNA. We are currently trying to determine both the hamster wild-type and the XR-C1 DNA-PKcs sequences.

The molecular data available on XRCC7 mutants and the existence of two distinct V(D)J phenotypes in XRCC7 cell lines can be most simply explained if three of these cell lines (equine scid, SX-9 and XR-C1) correspond to null mutations in the Prkdc gene while the other three cell lines (murine scid, V-3 and irs-20) represent incompletely penetrant mutations. This hypothesis is consistent with the limited amount of molecular data available, but would clearly be greatly strengthen by the identification of the inactivating mutations in the XR-C1, V-3 and irs-20 cell lines. An alternative hypothesis, where the equine scid, SX-9 and XR-C1 cell lines represent incompletely penetrant mutations and the murine scid, V-3 and irs-20 cell lines correspond to null mutations seems less likely, since we have shown by complementation analysis that XR-C1, at least, does not bear a dominant mutation (Fig. 3A). XR-C1/CHO9 hybrids also had a level of radiation resistance comparable with the wild-type cells (data not shown), confirming the recessive character of XR-C1. Thus, it is hard to imagine how a recessive mutation could yield a more severe phenotype than a null mutation. The construction of a fuctionally null/knockout mouse strain will clearly be helpful in addressing this issue.

In summary, we have described a hamster IR-sensitive cell line, XR-C1, that belongs to the XRCC7 complementation group and whose phenotype supports the notion that DNA-PKcs is required for both V(D)J coding and signal joint formation. We believe that further studies on XR-C1 will help to delineate the specific architectural and catalytic functions of DNA-PKcs in V(D)J recombination and DSB repair.

ACKNOWLEDGEMENTS

We would like to thank Laura Wetselaar and Marjolein Sonneveld for their skilful technical assistance, Drs Dik van Gent and Roland Kanaar for discussion, and we wish to acknowledge the help of Dr G.W.C.T.Verhaegh with pilot experiments on complementation analysis of XR-C1 cells. We also would like to thank Graeme C.M.Smith, Nicholas D.Lakin and David Gell for advice on performing DNA-PK assays. This work was supported by European Union Grants F14PCT90010, BMH1-CT93 1510, a grant from the Cancer Research Campaign and a grant AI35763 (E.A.H.) from the National Institutes of Health. E.A.H. is a Leukemia Scholar of America.

NOTE ADDED IN PROOF

A mutation in DNA-PKcs that causes substitution of lysine for glutamic acid in the fourth residue from the C-terminus (4124) has recently been identified in irs-20 by Priestley et al. (62).

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*To whom correspondence should be addressed at: Department of Radiation Genetics and Chemical Mutagenesis, Leiden University-Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Tel: +31 71 5276175; Fax: +31 71 5221615; Email: zdzienicka@rullf2.leidenuniv.nl

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