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Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80
Introduction
Materials And Methods
Cell culture
Chemicals
Irradiation
Survival curves
Cell fusion and complementation analysis
Northern blot analysis
Immunoblotting
DNA-PK kinase assay
RT-PCR analysis of hamster Ku80 mRNA from wild-type and mutant cells
DNA sequence analysis
Results
Isolation of the X-ray-sensitive Chinese hamster V79 cell mutants
Genetic complementation analysis
Cross-sensitivity to other DNA-damaging agents
Expression of Ku80 in the XR-V mutants
DNA end-binding and DNA-PK activity in XR-V mutants
Sequence analysis of Ku80 cDNA in XR-V mutants
Discussion
Acknowledgements
References
Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80
ABSTRACT
INTRODUCTION
DNA double strand breaks (DSBs) are generated following exposure to ionizing radiation or to radiomimetic chemicals. DSBs are also introduced as intermediates during V(D)J recombination in differentiating lymphocytes, transposition events and meiotic recombination. DSBs, if not repaired, can lead to genomic instability, genetic loss or cell death (1). In eukaryotes, two major DSB repair pathways have been identified that appear to be conserved from yeast to humans: homologous recombination, which requires the presence of homologous sequences, and non-homologous DNA end-joining, by which the two ends of a DSB are joined by a process that is independent of DNA sequence homology (2).
In order to examine the mammalian cellular response to ionizing radiation, X-ray-sensitive mutants have been isolated. At least 11 groups have been established and the genes defective in these groups have been designated XRCC1-XRCC11 (3). Analysis of these mutants has led to the recognition that the repair of DSBs by non-homologous end-joining involves the DNA-dependent protein kinase (DNA-PK). It has been shown that DNA-PK plays a key role not only in DSB repair, but also in lymphoid V(D)J recombination (4-6). The DNA-PK enzyme complex is composed of the Ku heterodimer, formed by Ku80 (Ku86) and Ku70, and a catalytic subunit (DNA-PKcs), which are encoded by the XRCC5/Ku80, XRCC6/Ku70 and XRCC7/SCID/Prkdc genes, respectively. Ku binds to double-stranded DNA ends, nicks, gaps and DNA hairpins, and activates DNA-PKcs (7-10). It has been shown that, at least in vitro, DNA-PK can autophosphorylate (11) and can phosphorylate a variety of transcription factors, such as Sp1, c-Jun (12), c-Myc and p53 (13-15).
It has been reported that the Prkdc gene is mutated in rodent cell mutants of group XRCC7 (16-19). A variety of cell lines defective in XRCC5/Ku80 have been isolated in several laboratories (3,6,20) and mutational changes in Ku80 have been identified (21-23). Cell mutants defective in XRCC6/Ku70 have not been found so far, but ES cells knocked out for Ku70 function show the anticipated characteristics (24,25). Ku80-deficient mice have been generated and they also exhibit radiosensitivity and a profound deficiency in V(D)J rearrangements, confirming the role of Ku80 in processing of DSBs in vivo (26,27). The mutations in the XRCC5/Ku80 hamster cell mutants reported to date comprise of large deletions or lack of expression of Ku80 protein and they show a similar mutant phenotype. All these mutants are X-ray-sensitive, are impaired in DSB repair, lack Ku-mediated DNA binding and DNA-PK activities and have abnormal V(D)J recombination (4,5,28,29).
To gain more insights into the functional domains of Ku80, the assembly and structure of Ku80 and its interactions with DNA in vitro were examined in yeast cells, with apparently contradictory results. In one study the C-terminus of Ku80 (amino acid residues 449-732) was shown to be essential for subunit interaction and DNA end-binding (30), whereas other studies reported that the central part of Ku80 plays an important role in these functions (31,32). The results of these studies, in general, are in agreement with the presence of deletions in the central part of the Ku80 gene in the mutant cell lines XR-V9B, XR-V15B (22). To shed light on the function of Ku80 in vivo, we have analyzed the biochemical and molecular characteristics of newly isolated X-ray-sensitive hamster cell XRCC5 mutants, XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B. Notably, four of them have mutations in Ku80, which are localized between amino acid residues 107 and 355. In the XR-V12B mutant, no Ku80 mRNA was detected. Single base substitutions (XR-V13B, XR-V16B) as well as large deletions (XR-V10B) caused a high degree of X-ray sensitivity, and a complete lack of Ku-mediated DNA-end binding and DNA-PK activities, suggesting that all these changes of the Ku80 structure prevent the formation of a stable Ku heterodimer.
MATERIALS AND METHODS
Cell culture
The Chinese hamster wild-type cell line, V79B, was used for the isolation of the XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B X-ray-sensitive mutants. XR-1, V-3, XR-V9B, V-E5 and V-C8 and their parental cell lines, CHOK1, AA8, V79B and V79, respectively, were described previously (33-37). All cells were cultured in Ham's F10 medium (without hypoxanthine and thymidine), supplemented with 15% new-born calf serum or in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1% l-glutamine. Culture media also included penicillin (100 U/ml), and streptomycin (0.1 mg/ml). Cells were maintained at 37°C in a 5% CO2 atmosphere, humidified to 95-100%.
Chemicals
Cytochalasin-B, Polyethylene glycol (PEG; 1450 mol wt), human and mouse cot-1 DNA were purchased from Sigma(St Louis, MO); colcemid and geneticin (G418) were from Gibco BRL and phytohemaglutinin (PH-A) from Difco Laboratories; ethylnitrosourea (ENU) from Pfaltz and Bauer (Stanford, CA); ethyl methanesulfonate (EMS) from Eastman Co. (Rochester, NY); mitomycin C (MMC) from Lampro B.V; bleomycin (BLM) from Londbeck (Amsterdam, The Netherlands).
Irradiation
For X-ray irradiation the cells were irradiated in tissue culture 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 the IL/770 germicidal radiometer.
Survival curves
Cultures in exponential growth were trypsinized and 300-3000 cells were plated, in duplicate, on 10 cm dishes and left to attach for 4 h. Cells were then irradiated or treated with MMC or EMS, for 24 or 4 h, respectively. After treatment with chemicals, medium was removed, cells were rinsed twice with phosphate buffered saline (PBS), then normal medium was added and cells were incubated for 8-10 days. After incubation, dishes were rinsed with NaCl (0.9%), air dried and stained with methylene blue (0.25%), then visible colonies were counted. Each survival curve represents the mean of at least three independent experiments.
Cell fusion and complementation analysis
The TOR hybridization/selective system described earlier (38) was used with fusion of one double-marked line (TOR-> thioguanine-resistant and ouabain-resistant) to one unmarked line by PEG. Populations of hybrids (>100 clones) were collected from each cross, then used for X-ray survival and the determination of the modal chromosome number.
Northern blot analysis
Total RNA was isolated by the guanidium isothiocyanate method as described previously (39). An aliquot of 20 µg of total RNA was electrophoresed on a 0.8% agarose-formaldehyde gel, transferred to a nitrocellulose filter in 20× SSC, and baked at 80°C for 2 h. The probe was obtained by PCR amplification and was a 1 kb fragment encoding N-terminus region hamster Ku80. As a control for equal loading, the same blot was also incubated with a GAPDH probe. The probes were labelled by random priming and hybridization was carried out under standard conditions.
Immunoblotting
Whole cell extracts were prepared as described previously (40). Briefly, 40 × 106 cells were harvested and washed twice with PBS. Cell pellets were resuspended in equal volume of extraction buffer [50 mM NaF, 20 mM HEPES (pH 7.8), 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, leupeptine (0.5 µg/ml), protease inhibitor (0.5 µg/ml), trypsin inhibitor (1.0 µg/ml), aprotinin (0.5 µg/ml), bestatin (40 µg/ml)], snap frozen on dry-ice and thawed at 30°C three times. After centrifugation for 10 min at 4°C, supernatants were aliquoted and stored at -70°C. Protein concentrations were determined using the Bradford protein assay using BSA as the standard. 100 µg of proteins were resolved by SDS-PAGE, transferred to nitrocellulose filters, and probed with polyclonal antibodies specific for Ku70 and Ku80 followed by horseradish peroxidase-conjugated goat antibody to rabbit IgG (TAGO, Burlingame, CA). Antibody binding was detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
DNA-PK kinase assay
DNA-PK pull-down assays were performed as described previously (41). Briefly, 200 µg of whole cell extract was incubated with 40 µl of pre-swollen dsDNA-cellulose (Sigma) in Z[prime] buffer (25 mM HEPES/KOH pH 7.9, 50 mM MgCl2, 20% glycerol, 0.1% Nonidet P-40, 1 mM dithiothreitol) for 2 h at 4°C. The DNA cellulose was then washed twice in 1 ml Z[prime] buffer and resuspended in 60 µl of Z[prime] 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 equal volume of 30% acetic acid and analyzed by spotting onto phosphocellulose paper, washing and subjecting to liquid scintillation counting as described previously (42). The sequences of wild-type (wt) and mutant p53 peptides are EPPLSQEAFADLLKK and EPPLSEQAFADLLKK, respectively. All assays were performed multiple times with at least two different extract preparations.
RT-PCR analysis of hamster Ku80 mRNA from wild-type and mutant cells
Total RNA was isolated by the guanidium isothiocyanate method as described previously (39). First-strand Ku80 cDNA synthesis was performed as follows: total RNA (1 µg) was added to a reverse transcription solution consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM dNTPs, 2.5 µM oligo(dT)15 primer, 20 U RNasin and 50 U MuLV (Murine Leukaemia Virus) reverse transcriptase (Geneamp RNA PCR Kit). The reaction mixture (final volume, 20 µl) was incubated for 30 min at 42°C, heated to 95°C for 5 min and then chilled on ice. The newly generated RNA:cDNA hybrids were amplified by the polymerase chain reaction (PCR) with specific Ku80 primers resulting in a 2.3 kb product. Primer sequences were described previously (22). PCR was performed with 30 cycles consisting of denaturation for 30 s at 94°C, primer annealing at 55°C for 30 s and extension at 72°C for 2 min (DNA thermal cycles, Perkin-Elmer Cetus, Norwalk, CT).
DNA sequence analysis
PCR primers were biotinylated and, following PCR, the amplified 2.3 kb cDNA fragment was gel-purified (Qiaex, Qiagen, Chatsworth, CA), and magnetic Dynabeads M-280 (Dynal AS, Oslo, Norway) were used to prepare single-stranded, immobilized templates. DNA sequence was obtained with a T7 sequence kit (Pharmacia Biotech, Uppsala, Sweden), using [[alpha]-32P]dATP. The samples were resolved in a 6% polyacrylamide gel at 40 W and gels were exposed to X-ray film. Sequence analysis was also performed after cloning the PCR products in a TA cloning vector (Promega, Madison, WI).
RESULTS
Isolation of the X-ray-sensitive Chinese hamster V79 cell mutants
The X-ray-sensitive mutants, XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B, were isolated from two mutagenized population of Chinese hamster V79B cells on the basis of their hypersensitivity to X-rays, by the replica plating method (43). Mutant cell lines XR-V10B, XR-V11B and XR-V13B were isolated from EMS-mutagenized V79B cells (70 mM EMS killed ~99% of treated cells), whereas XR-V12B and XR-V16B were isolated from ENU-mutagenized V79B cells (4 mM ENU killed ~90% of the treated cells). They are 5-7-fold more sensitive to X-rays, in comparison to wild-type parental V79B cells, as judged by D10 (a dose required to reduce survival to 10%) (Fig.
Figure 1. X-ray survivals of (A) wild-type V79B ([open circle]), XR-V10B ([solid triangle]), XR-V11B ([solid square]), XR-V12B ([solid diamond]), XR-V13B ([solid cone]) and XR-V16B ([solid circle]); (B) hybrid XR-1TOR/XR-V10B, V-3TOR/XR-V10B, V-E5/XR-V10BTOR and V-C8/XR-V10BTOR cell lines are resistant to X-rays (upper shaded lines); hybrid XR-V10B/XR-V9BTOR, XR-V11B/XR-V9BTOR, XR-V12B/XR-V9BTOR, XR-V13B/XR-V9BTOR, XR-V16B/XR-V9BTOR cell lines remained X-ray sensitive (lower shaded lines).
Genetic complementation analysis
Genetic complementation between XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B was determined after cell fusion by measuring the colony forming ability of X-irradiated hybrid clones. Hybrids derived from crosses between these mutants showed no complementation of X-ray sensitivity, indicating that all the mutants belong to the same complementation group (data not shown). To examine whether these mutants represent a new complementation group, hybrid clones between XR-V10B and the mutants representing different complementation groups, XR-1 (XRCC4), XR-V9B (XRCC5), V-3 (XRCC7), V-E5 (XRCC8) and V-C8 (XRCC11), were analyzed. Only hybrids with XR-V9B remained X-ray-sensitive (Fig.
Cross-sensitivity to other DNA-damaging agents
The sensitivities of XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B and wild-type V79B cells were compared with regard to killing by UV, BLM and mono- or bi-functional alkylating agents: EMS and MMC, respectively. As shown in Table 1, all mutants are clearly sensitive to BLM (15-20-fold) as compared to wild-type V79B cells, ~2-fold more sensitive to EMS, and only slightly or more sensitive to UV or MMC.
Table 1.
| Cell line | D10 X-ray (Gy) |
UV (J/m2) |
BLM (µg/ml) |
EMS (mM) |
MMC (ng/ml) |
| V79B | 6.8 | 16 | 18.8 | 63 | 127 |
| XR-V10B | 1.3 | 12 | 0.9 | 38 | 122 |
| XR-V11B | 1.4 | 12 | 1.2 | 47 | 101 |
| XR-V12B | 1.4 | 14 | 0.5 | 61 | 155 |
| XR-V13B | 1.1 | 10 | 1.1 | 28 | 96 |
| XR-V16B | 1.7 | 12 | 1.5 | 31 | 150 |
Expression of Ku80 in the XR-V mutants
Expression of the Ku80 transcript and protein in XR-V10B, XR-V13B, XR-V14B and XR-V16B and wild-type V79B cells was examined by northern and western blot analyses. All mutants, except XR-V12B, expressed the Ku80 mRNA at levels and size similar to that observed in wild-type V79B parental cells (Fig.
Figure 2. Northern blot analysis. An aliquot of 20 µg of total RNA was loaded in each lane. As a control the same blot was also incubated with a GAPDH probe.
DNA end-binding and DNA-PK activity in XR-V mutants
Previously, it has been shown that the mutants of group XRCC5 lack dsDNA end-binding activity, which leads to defective DNA-PK activity (10,41). The XR-V10B, XR-V12B, XR-V13B, XR-V14B and XR-V16B mutants also lack DNA end-binding properties of Ku, as measured by UV crosslinking experiments (data not shown). In addition, through use of the pull-down DNA-PK peptide kinase assay as described by Finnie et al. (41), it was found that all the XR-V mutant cells lack detectable DNA-PK kinase activity (Fig.
Sequence analysis of Ku80 cDNA in XR-V mutants
To determine the nature of the Ku80 mutations in the XR-V10B, XR-V11B, XR-V13B and XR-V16B cell lines, PCR amplified Ku80 cDNAs from these mutants were sequenced and compared to the wild-type Chinese hamster V79B Ku80 sequence described earlier (22) (DDBJ/EMBL/GenBank accesion no. L48606). In this Chinese hamster Ku80 sequence, a difference at bp 168 (A is present in place of G) was detected. The mutations identified in the new hamster mutants are summarized in Table 2. In XR-V10B, a deletion of 252 bp from nucleotides +799 to +1050, which does not shift the reading frame, was found. This deletion corresponds to a deletion of 84 amino acid residues from codon 267 to 350. In XR-V11B a deletion of 6 bp, from nucleotides +320 to +325 was detected. This 6 bp deletion from codon 107 to 109 results in an appearance of a new amino acid residue, Asn, instead of Ile, Leu and Asp. A missense mutation was found in the Ku80 cDNA of XR-V13B cells, which results in a substitution of Cys for Tyr at position 114. In XR-V16B cells, two missense mutations resulting in a substition of Met for Val at position 331 and Arg for Gly at position 354 were found. Since we were not able to amplify the Ku80 cDNA in the XR-V12B mutant, no mutations could be identified in this mutant. Previously, it has been reported that the mutant cell lines xrs-1, xrs-5 and xrs-7 also express low levels of Ku80 transcripts, which leads to hypersensitivity to X-rays (23).
Figure 3. Hamster Ku80 cDNA and the location of all the mutations identified so far in hamster mutants. Hatched boxes correspond to regions deleted in the Ku80 cDNA of mutants XR-V9B, XR-V10B and XR-V15B. Arrows indicate the mutated amino acid residues of the corresponding hamster mutants. Black boxes correspond to the putative leucine zipper motive and a proline-rich domain.
DISCUSSION
In this report we described the genetic, biochemical and molecular characteristics of five newly isolated X-ray-sensitive Chinese hamster V79 mutants: XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B. Cross-sensitivity to various DNA-damaging agents suggested that these mutants are predominantly sensitive to free radical-producing agents, which generate DNA DSBs. Genetic complementation analysis with other DSB repair mutants and lack of DNA-end binding and DNA-PK activities in all the examined mutants clearly indicated that they belong to group 5, which is defective in the XRCC5/Ku80 gene. Further, biochemical and molecular analyses confirmed that mutations in the gene for Ku80 are responsible for the phenotype of the XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B mutants (Table 2). Although much is known about the biochemical properties of Ku, its precise cellular function still remains unknown. Therefore, extended studies with the mutants defective in the Ku80 gene are of great importance for unraveling specific architectural and catalytic roles of Ku80.
Table 2.
| Cell line | Codon | Nucleotide position |
Mutation | Amino acid residues |
| XR-V10B | 267-350 | 799-1050 | 252 bp deletion | 84 deleted |
| XR-V11B | 107-109 | 320-325 | 6 bp deletiona | (Ile, Leu, Asp)->Asn |
| XR-V13B | 114 | 341 | TGC->TAC | Cys->Tyr |
| XR-V16B | 331 | 991 | ATG->GTG | Met->Val |
| 354 | 1060 | AGA->GGA | Arg->Gly |
Surprisingly, we found that the independently isolated XR-V10B mutant expresses a Ku80 mRNA that contains a 252 bp in-frame deletion, causing loss of a 84 amino acid residue protein region. The deletion which has been identified previously in XR-V9B (22). The XR-V10B hamster cell mutant is derived from an ENU-mutagenized V79B population, whereas XR-V9B is derived from an EMS-mutagenized V79B population. Therefore, most likely, the same deletion in these mutants results from different genomic mutations that affect RNA splicing. In XR-V12B, no mutation was identified due to the absence or very low levels of Ku80 mRNA. In order to unravel the defect in XR-V12B, the entire Ku80 gene and the regulatory sequences need to be sequenced.
The mutations in the XRCC5 gene reported to date comprise of large deletions of the protein or lead to lack of Ku80 expression (22,23). Here, in addition to such mutations, we describe a novel type of Ku80 mutation that leads to a severe X-ray-sensitive phenotype. The XR-V13B mutant carries a single missense mutation in the N-terminal region of the Ku80 cDNA that substitutes a Cys for a Tyr at position 114. Cys contains a sulfhydryl group (-SH), which can form disulfide bonds within or between proteins and can mediate hydrogen bond interactions. Thus, this mutation could affect the conformation of Ku80 or its interactions with other proteins, and most likely is the inactivating mutation in XR-V13B. Previous studies in which effects of Ku80 deletions were examined in xrs-6 cells, have indicated that the N-terminal part of Ku80 is dispensable for DNA end-binding and dimerization with Ku70 (30-32). Therefore, it is most likely that the mutations in the N-terminal part of Ku80 in XR-V13B and XR-V11B affect the tertiary structure of the protein, which is required for DNA end-binding and/or other functions in vivo. Whatever these structural changes are, they clearly result in unstable Ku80 protein, since western blot analysis showed the absence of Ku80 protein in these cell mutants. Thus, the same phenotype of XR-V13B, XR-V11B and other group 5 mutants is most probably due to the degradation of the Ku80 protein. A summary of all the identified mutations in the Ku80 gene is depicted in Figure
Figure 4. The Ku80 protein is not expressed in the X-ray-sensitive mutants. V79B cells were used as a positive cell line. After subjecting samples (100 µg of total protein in each lane was loaded) to SDS-PAGE, western blot analysis was performed using 80-4 polyclonal antibody directed against Ku80. To serve as a control for equivalent loading of the proteins, blots were re-probed with an antibody raised against [beta]-actin. Figure 5. DNA-PK peptide phosphorylation pull-down assays in the X-ray-sensitive hamster cell mutants XR-V10B, XR-V11B, XR-V12B, XR-V13B, XR-V16B and wild-type V79B cells. 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, 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. Recent studies of Ku80 deletion mutants in a yeast two-hybrid assay have shown that amino acid residues 449-732 are important for Ku70-Ku80 subunit interactions. In addition, using an in vitro translation system, it has been found that amino acid residues 334-732 are essential for DNA-end binding activity (30). Osipovich et al. (31) defined a minimal region from amino acid residue 449 to 477 that is necessary for Ku80 to heterodimerize with Ku70. In agreement with these results, Cary et al. (32) found that the central part of Ku80 (amino acid residues 241-555) is important for DNA end-binding and heterodimerization. These findings are supported by the identification of deletions in the central part of Ku80 in XR-V9B, XR-V10B and XR-V15B cells (22) and point mutations in the XR-V16B mutant. The mutations in these cases are, however, outside the minimal region defined by Osipovich et al. (31) as necessary for Ku70 interaction. Most probably these mutations result in an unstable protein that is targeted for degradation. Our results with the XR-V11B and XR-V13B mutant cell lines clearly also indicate that mutations in the N-terminal part of Ku80, outside of the core region identified by Cary et al. (32) and Osipovich et al. (31), are important for Ku stability. It is worthy of mention that no mutant cell lines have been identified that harbour mutations in the very C-terminal portion of the Ku80 protein. This might suggest that mutations in this region do not lead to the degradation of Ku80 protein and/or this region of Ku80 does not affect X-ray sensitivity. In initial studies, the XR-V mutants showed different degrees of sensitivity towards X-ray (e.g. XR-V9B seemed to be less sensitive than XR-V15B), suggesting that this might reflect mutations in different functional domains of Ku80. However, when subclones from a single colony were isolated from each mutant and later examined for X-ray sensitivity, all mutants showed a similar degree of the X-ray sensitivity (data not shown). Thus, the observed differences amongst newly isolated mutants most probably result from the presence of X-ray-resistant revertants in the cell population. This is in agreement with the observation that several mutants of group 5 revert with a high frequency due to the demethylation of the second intact allele (22,23,28,44). In summary, an variety of different Ku80 mutations lead to a similar phenotype due to them all resulting in the generation of an unstable Ku80 protein. This instability seems to be relatively independent of the introduced mutations: similar effects are observed when large deletions, frameshift mutations or certain point mutations are induced in Ku80. Therefore, in vivo studies with rodent cell mutants has not afforded precise information about functional domains of Ku80, although they revealed that an intact Ku80 is necessary to provide Ku function in mammalian cells.
ACKNOWLEDGEMENTS
This work in part was supported by grant 9.0.6 to M.Z.Z. from the J. A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection and European Union grant F14PCT90010, The Netherlands, and in the S.P.J. laboratory was funded by the Cancer Research Campaign.
REFERENCES
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