Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80
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4332–4338 Nucleic Acids Research, 1998, Vol. 26, No. 19 1998 Oxford University Press Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80 A. Errami1,2, N. J. Finnie3, B. Morolli1,2, S. P. Jackson3, P. H. M. Lohman1 and M. Z. Zdzienicka1,2,* 1MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, LUMC, Leiden, The Netherlands, 2J. A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, Leiden, The Netherlands and 3Wellcome/Cancer Research Campaign Institute and Department of Zoology, Cambridge University, Cambridge CB2 1QR, UK Received July 13, 1998; Revised and Accepted August 21, 1998 ABSTRACT instability, genetic loss or cell death (1). In eukaryotes, two major DSB repair pathways have been identified that appear to be Downloaded from http://nar.oxfordjournals.org/ by guest on September 23, 2015 Ku, a heterodimer of ∼70 and ∼80 kDa subunits, is a conserved from yeast to humans: homologous recombination, nuclear protein that binds to double-stranded DNA which requires the presence of homologous sequences, and ends and is a component of the DNA-dependent non-homologous DNA end-joining, by which the two ends of a protein kinase (DNA-PK). Cell lines defective in Ku80 DSB are joined by a process that is independent of DNA sequence belong to group XRCC5 of ionizing radiation-sensitive homology (2). mutants. Five new independent Chinese hamster cell In order to examine the mammalian cellular response to mutants, XR-V10B, XR-V11B, XR-V12B, XR-V13B and ionizing radiation, X-ray-sensitive mutants have been isolated. At XR-V16B, that belong to this group were isolated. To least 11 groups have been established and the genes defective in shed light on the nature of the defect in Ku80, the these groups have been designated XRCC1–XRCC11 (3). Analysis molecular and biochemical characteristics of these of these mutants has led to the recognition that the repair of DSBs mutants were examined. All mutants, except XR-V12B, by non-homologous end-joining involves the DNA-dependent express Ku80 mRNA, but no Ku80 protein could clearly protein kinase (DNA-PK). It has been shown that DNA-PK plays be detected by immunoblot analysis in any of them. a key role not only in DSB repair, but also in lymphoid V(D)J DNA sequence analysis of the Ku80 cDNA from these recombination (4–6). The DNA-PK enzyme complex is composed mutants showed a deletion of 252 bp in XR-V10B; a 6 bp of the Ku heterodimer, formed by Ku80 (Ku86) and Ku70, and deletion that results in a new amino acid residue at a catalytic subunit (DNA-PKcs), which are encoded by the position 107 and the loss of two amino acid residues XRCC5/Ku80, XRCC6/Ku70 and XRCC7/SCID/Prkdc genes, at positions 108 and 109 in XR-V11B; a missense respectively. Ku binds to double-stranded DNA ends, nicks, gaps mutation resulting in a substitution of Cys for Tyr at and DNA hairpins, and activates DNA-PKcs (7–10). It has been position 114 in XR-V13B; and two missense mutations shown that, at least in vitro, DNA-PK can autophosphorylate (11) in XR-V16B, resulting in a substitution of Met for Val at and can phosphorylate a variety of transcription factors, such as position 331 and Arg for Gly at position 354. All these Sp1, c-Jun (12), c-Myc and p53 (13–15). mutations cause a similar, 5–7-fold, increase in X-ray It has been reported that the Prkdc gene is mutated in rodent cell sensitivity in comparison to wild-type cells, and a mutants of group XRCC7 (16–19). A variety of cell lines defective complete lack of DNA-end binding and DNA-PK in XRCC5/Ku80 have been isolated in several laboratories (3,6,20) activities. This indicates that all these mutations lead and mutational changes in Ku80 have been identified (21–23). to loss of the Ku80 function due to instability of the Cell mutants defective in XRCC6/Ku70 have not been found so defective protein. far, but ES cells knocked out for Ku70 function show the anticipated characteristics (24,25). Ku80-deficient mice have INTRODUCTION been generated and they also exhibit radiosensitivity and a profound deficiency in V(D)J rearrangements, confirming the DNA double strand breaks (DSBs) are generated following role of Ku80 in processing of DSBs in vivo (26,27). The exposure to ionizing radiation or to radiomimetic chemicals. DSBs mutations in the XRCC5/Ku80 hamster cell mutants reported to are also introduced as intermediates during V(D)J recombination in date comprise of large deletions or lack of expression of Ku80 differentiating lymphocytes, transposition events and meiotic protein and they show a similar mutant phenotype. All these recombination. DSBs, if not repaired, can lead to genomic mutants are X-ray-sensitive, are impaired in DSB repair, lack *To whom correspondence should be addressed at: MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, Sylvius Laboratory, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Tel: +31 71 5276175; Fax: +31 71 5221615; Email: zdzienicka@rullf2.leidenuniv.nl
4333 Nucleic Acids Nucleic Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.119 4333 Ku-mediated DNA binding and DNA-PK activities and have Survival curves abnormal V(D)J recombination (4,5,28,29). To gain more insights into the functional domains of Ku80, the Cultures in exponential growth were trypsinized and 300–3000 cells assembly and structure of Ku80 and its interactions with DNA in were plated, in duplicate, on 10 cm dishes and left to attach for 4 h. vitro were examined in yeast cells, with apparently contradictory Cells were then irradiated or treated with MMC or EMS, for 24 results. In one study the C-terminus of Ku80 (amino acid residues or 4 h, respectively. After treatment with chemicals, medium was 449–732) was shown to be essential for subunit interaction and removed, cells were rinsed twice with phosphate buffered saline DNA end-binding (30), whereas other studies reported that the (PBS), then normal medium was added and cells were incubated central part of Ku80 plays an important role in these functions for 8–10 days. After incubation, dishes were rinsed with NaCl (31,32). The results of these studies, in general, are in agreement (0.9%), air dried and stained with methylene blue (0.25%), then with the presence of deletions in the central part of the Ku80 gene visible colonies were counted. Each survival curve represents the in the mutant cell lines XR-V9B, XR-V15B (22). To shed light mean of at least three independent experiments. on the function of Ku80 in vivo, we have analyzed the biochemical and molecular characteristics of newly isolated Cell fusion and complementation analysis X-ray-sensitive hamster cell XRCC5 mutants, XR-V10B, XR- The TOR hybridization/selective system described earlier (38) V11B, XR-V12B, XR-V13B and XR-V16B. Notably, four of was used with fusion of one double-marked line (TOR→ them have mutations in Ku80, which are localized between amino thioguanine-resistant and ouabain-resistant) to one unmarked line by acid residues 107 and 355. In the XR-V12B mutant, no Ku80 PEG. Populations of hybrids (>100 clones) were collected from mRNA was detected. Single base substitutions (XR-V13B, each cross, then used for X-ray survival and the determination of XR-V16B) as well as large deletions (XR-V10B) caused a high the modal chromosome number. degree of X-ray sensitivity, and a complete lack of Ku-mediated Downloaded from http://nar.oxfordjournals.org/ by guest on September 23, 2015 DNA-end binding and DNA-PK activities, suggesting that all Northern blot analysis these changes of the Ku80 structure prevent the formation of a stable Ku heterodimer. 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, MATERIALS AND METHODS transferred to a nitrocellulose filter in 20 SSC, and baked at 80C for 2 h. The probe was obtained by PCR amplification and Cell culture was a 1 kb fragment encoding N-terminus region hamster Ku80. The Chinese hamster wild-type cell line, V79B, was used for the As a control for equal loading, the same blot was also incubated isolation of the XR-V10B, XR-V11B, XR-V12B, XR-V13B and with a GAPDH probe. The probes were labelled by random XR-V16B X-ray-sensitive mutants. XR-1, V-3, XR-V9B, V-E5 priming and hybridization was carried out under standard and V-C8 and their parental cell lines, CHOK1, AA8, V79B and conditions. V79, respectively, were described previously (33–37). All cells were cultured in Ham’s F10 medium (without hypoxanthine and Immunoblotting thymidine), supplemented with 15% new-born calf serum or in Whole cell extracts were prepared as described previously (40). Dulbecco’s modified Eagle’s medium supplemented with 10% Briefly, 40 106 cells were harvested and washed twice with fetal calf serum and 1% L-glutamine. Culture media also included PBS. Cell pellets were resuspended in equal volume of extraction penicillin (100 U/ml), and streptomycin (0.1 mg/ml). Cells were buffer [50 mM NaF, 20 mM HEPES (pH 7.8), 450 mM NaCl, maintained at 37C in a 5% CO2 atmosphere, humidified to 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM 95–100%. phenylmethylsulfonyl fluoride, leupeptine (0.5 µg/ml), protease inhibitor (0.5 µg/ml), trypsin inhibitor (1.0 µg/ml), aprotinin Chemicals (0.5 µg/ml), bestatin (40 µg/ml)], snap frozen on dry-ice and thawed at 30C three times. After centrifugation for 10 min at Cytochalasin-B, Polyethylene glycol (PEG; 1450 mol wt), 4C, supernatants were aliquoted and stored at –70C. Protein human and mouse cot-1 DNA were purchased from Sigma concentrations were determined using the Bradford protein assay (St Louis, MO); colcemid and geneticin (G418) were from Gibco using BSA as the standard. 100 µg of proteins were resolved by BRL and phytohemaglutinin (PH-A) from Difco Laboratories; SDS–PAGE, transferred to nitrocellulose filters, and probed with ethylnitrosourea (ENU) from Pfaltz and Bauer (Stanford, CA); polyclonal antibodies specific for Ku70 and Ku80 followed by ethyl methanesulfonate (EMS) from Eastman Co. (Rochester, horseradish peroxidase-conjugated goat antibody to rabbit IgG NY); mitomycin C (MMC) from Lampro B.V; bleomycin (BLM) (TAGO, Burlingame, CA). Antibody binding was detected by from Londbeck (Amsterdam, The Netherlands). enhanced chemiluminescence (Amersham, Arlington Heights, IL). DNA-PK kinase assay Irradiation DNA-PK pull-down assays were performed as described previously For X-ray irradiation the cells were irradiated in tissue culture (41). Briefly, 200 µg of whole cell extract was incubated with medium at a dose rate of 3 Gy/min (200 kV, 4 mA , 0.78 mm Al). 40 µl of pre-swollen dsDNA–cellulose (Sigma) in Z′ buffer For irradiation with UV light of 254 nm, a Philips TUV (25 mM HEPES/KOH pH 7.9, 50 mM MgCl2, 20% glycerol, germicidal lamp was used with a fluence rate of 0.19 W/m2, 0.1% Nonidet P-40, 1 mM dithiothreitol) for 2 h at 4C. The DNA measured with the IL/770 germicidal radiometer. cellulose was then washed twice in 1 ml Z′ buffer and
4334 Nucleic Acids Research, 1998, Vol. 26, No. 19 Figure 1. X-ray survivals of (A) wild-type V79B (f), XR-V10B (▲), XR-V11B (■), XR-V12B (z), XR-V13B (▼) and XR-V16B (●); (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). Downloaded from http://nar.oxfordjournals.org/ by guest on September 23, 2015 resuspended in 60 µl of Z′ buffer. Samples were divided into three samples were resolved in a 6% polyacrylamide gel at 40 W and aliquots, 0.5 µl [γ-32P]ATP (300 Ci/mmol) was added, and kinase gels were exposed to X-ray film. Sequence analysis was also assays were conducted in the presence of 4 nmol of peptide performed after cloning the PCR products in a TA cloning vector (0.2 mM) for 10 min at 30_C, in a total volume of 20 µl. Reactions (Promega, Madison, WI). were then stopped by adding equal volume of 30% acetic acid and analyzed by spotting onto phosphocellulose paper, washing and RESULTS subjecting to liquid scintillation counting as described previously (42). The sequences of wild-type (wt) and mutant p53 peptides Isolation of the X-ray-sensitive Chinese hamster V79 cell are EPPLSQEAFADLLKK and EPPLSEQAFADLLKK, re- mutants spectively. All assays were performed multiple times with at least The X-ray-sensitive mutants, XR-V10B, XR-V11B, XR-V12B, two different extract preparations. XR-V13B and XR-V16B, were isolated from two mutagenized population of Chinese hamster V79B cells on the basis of their RT–PCR analysis of hamster Ku80 mRNA from wild-type hypersensitivity to X-rays, by the replica plating method (43). and mutant cells Mutant cell lines XR-V10B, XR-V11B and XR-V13B were isolated from EMS-mutagenized V79B cells (70 mM EMS killed Total RNA was isolated by the guanidium isothiocyanate method ∼99% of treated cells), whereas XR-V12B and XR-V16B were as described previously (39). First-strand Ku80 cDNA synthesis isolated from ENU-mutagenized V79B cells (4 mM ENU killed was performed as follows: total RNA (1 µg) was added to a ∼90% of the treated cells). They are 5–7-fold more sensitive to reverse transcription solution consisting of 10 mM Tris–HCl X-rays, in comparison to wild-type parental V79B cells, as (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM dNTPs, 2.5 µM judged by D10 (a dose required to reduce survival to 10%) oligo(dT)15 primer, 20 U RNasin and 50 U MuLV (Murine (Fig. 1A). The generation time and the cloning efficiency of these Leukaemia Virus) reverse transcriptase (Geneamp RNA PCR mutants are similar to that of wild-type V79B cells. All the mutants Kit). The reaction mixture (final volume, 20 µl) was incubated for maintained their X-ray sensitivity for >3 months of continuous 30 min at 42_C, heated to 95_C for 5 min and then chilled on ice. culture. The newly generated RNA:cDNA hybrids were amplified by the polymerase chain reaction (PCR) with specific Ku80 primers Genetic complementation analysis resulting in a 2.3 kb product. Primer sequences were described previously (22). PCR was performed with 30 cycles consisting of Genetic complementation between XR-V10B, XR-V11B, XR- denaturation for 30 s at 94_C, primer annealing at 55_C for 30 s V12B, XR-V13B and XR-V16B was determined after cell fusion and extension at 72_C for 2 min (DNA thermal cycles, by measuring the colony forming ability of X-irradiated hybrid Perkin-Elmer Cetus, Norwalk, CT). clones. Hybrids derived from crosses between these mutants showed no complementation of X-ray sensitivity, indicating that DNA sequence analysis all the mutants belong to the same complementation group (data not shown). To examine whether these mutants represent a new PCR primers were biotinylated and, following PCR, the amplified complementation group, hybrid clones between XR-V10B and 2.3 kb cDNA fragment was gel-purified (Qiaex, Qiagen, the mutants representing different complementation groups, Chatsworth, CA), and magnetic Dynabeads M-280 (Dynal AS, XR-1 (XRCC4), XR-V9B (XRCC5), V-3 (XRCC7), V-E5 Oslo, Norway) were used to prepare single-stranded, immobilized (XRCC8) and V-C8 (XRCC11), were analyzed. Only hybrids with templates. DNA sequence was obtained with a T7 sequence kit XR-V9B remained X-ray-sensitive (Fig. 1B), indicating that (Pharmacia Biotech, Uppsala, Sweden), using [α-32P]dATP. The XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B
4335 Nucleic Acids Nucleic Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.119 4335 belong to complementation group XRCC5, like XR-V15B and the xrs series mutants. 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. Cross-sensitivities of XR-V cells to various DNA damaging agents 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. Cell line D10 X-ray UV BLM EMS MMC (Gy) (J/m2) (µg/ml) (mM) (ng/ml) DNA end-binding and kinase activities in these mutants is in V79B 6.8 16 18.8 63 127 agreement with the results of western blot analysis of Ku80, which showed the absence of the Ku80 protein in all these XR-V10B 1.3 12 0.9 38 122 XRCC5 mutants. Downloaded from http://nar.oxfordjournals.org/ by guest on September 23, 2015 XR-V11B 1.4 12 1.2 47 101 XR-V12B 1.4 14 0.5 61 155 Sequence analysis of Ku80 cDNA in XR-V mutants XR-V13B 1.1 10 1.1 28 96 To determine the nature of the Ku80 mutations in the XR-V10B, XR-V16B 1.7 12 1.5 31 150 XR-V11B, XR-V13B and XR-V16B cell lines, PCR amplified Ku80 cDNAs from these mutants were sequenced and compared The numbers represent the D10 value, i.e. the dose required to kill 90% of the cells. to the wild-type Chinese hamster V79B Ku80 sequence described earlier (22) (DDBJ/EMBL/GenBank accesion no. L48606). In Expression of Ku80 in the XR-V mutants this Chinese hamster Ku80 sequence, a difference at bp 168 (A is present in place of G) was detected. The mutations identified Expression of the Ku80 transcript and protein in XR-V10B, in the new hamster mutants are summarized in Table 2. In XR-V13B, XR-V14B and XR-V16B and wild-type V79B cells XR-V10B, a deletion of 252 bp from nucleotides +799 to +1050, was examined by northern and western blot analyses. All which does not shift the reading frame, was found. This deletion mutants, except XR-V12B, expressed the Ku80 mRNA at levels corresponds to a deletion of 84 amino acid residues from codon and size similar to that observed in wild-type V79B parental cells 267 to 350. In XR-V11B a deletion of 6 bp, from nucleotides +320 (Fig. 2). This indicates that the XR-V10B, XR-V13B, XR-V14B to +325 was detected. This 6 bp deletion from codon 107 to 109 and XR-V16B mutants do not harbour major truncations in the results in an appearance of a new amino acid residue, Asn, instead Ku80 transcript nor mutations that render the gene inoperative. of Ile, Leu and Asp. A missense mutation was found in the Ku80 Since no detectable amplification product has been observed in cDNA of XR-V13B cells, which results in a substitution of Cys XR-V12B by RT–PCR, this mutant most probably does not for Tyr at position 114. In XR-V16B cells, two missense express Ku80. The presence of the Ku80 protein in these mutants mutations resulting in a substition of Met for Val at position 331 was evaluated by western blot analysis using the polyclonal and Arg for Gly at position 354 were found. Since we were not antibody Ku80-4. The Ku80 protein could not clearly be detected able to amplify the Ku80 cDNA in the XR-V12B mutant, no in the extracts of all five mutant cell lines, whereas in the wild-type mutations could be identified in this mutant. Previously, it has V79B cells, Ku80 protein was evidently detected (Fig. 4). Notably, been reported that the mutant cell lines xrs-1, xrs-5 and xrs-7 also the Ku70 protein was also not present in any of these mutant cell express low levels of Ku80 transcripts, which leads to hypersensi- lines as shown by western blot analysis using antiserum Ku70-5 tivity to X-rays (23). (data not shown). These results support the model that wild-type Ku80 is required for the stabilization of Ku70. DISCUSSION DNA end-binding and DNA-PK activity in XR-V mutants In this report we described the genetic, biochemical and molecular characteristics of five newly isolated X-ray-sensitive Previously, it has been shown that the mutants of group XRCC5 Chinese hamster V79 mutants: XR-V10B, XR-V11B, XR-V12B, lack dsDNA end-binding activity, which leads to defective XR-V13B and XR-V16B. Cross-sensitivity to various DNA- DNA-PK activity (10,41). The XR-V10B, XR-V12B, XR- damaging agents suggested that these mutants are predominantly V13B, XR-V14B and XR-V16B mutants also lack DNA sensitive to free radical-producing agents, which generate DNA end-binding properties of Ku, as measured by UV crosslinking DSBs. Genetic complementation analysis with other DSB repair experiments (data not shown). In addition, through use of the mutants and lack of DNA-end binding and DNA-PK activities in pull-down DNA-PK peptide kinase assay as described by Finnie all the examined mutants clearly indicated that they belong to et al. (41), it was found that all the XR-V mutant cells lack group 5, which is defective in the XRCC5/Ku80 gene. Further, detectable DNA-PK kinase activity (Fig. 5). The observed lack of biochemical and molecular analyses confirmed that mutations in
4336 Nucleic Acids Research, 1998, Vol. 26, No. 19 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. the gene for Ku80 are responsible for the phenotype of the XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B Downloaded from http://nar.oxfordjournals.org/ by guest on September 23, 2015 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. Types and location of mutations in the Ku80 cDNA of hamster cell mutants group 5 (XRCC5/Ku80) Cell line Codon Nucleotide Mutation Amino acid position residues XR-V10B 267–350 799–1050 252 bp deletion 84 deleted Figure 4. The Ku80 protein is not expressed in the X-ray-sensitive mutants. XR-V11B 107–109 320–325 6 bp deletiona (Ile, Leu, V79B cells were used as a positive cell line. After subjecting samples (100 µg Asp)→Asn 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 XR-V13B 114 341 TGC→TAC Cys→Tyr as a control for equivalent loading of the proteins, blots were re-probed with an antibody raised against β-actin. XR-V16B 331 991 ATG→GTG Met→Val 354 1060 AGA→GGA Arg→Gly a6bp were deleted (underlined) from the three codons: ATC107 TTG108 GAC109→AAC107 mutation in the N-terminal region of the Ku80 cDNA that substitutes a Cys for a Tyr at position 114. Cys contains a Surprisingly, we found that the independently isolated XR- sulfhydryl group (-SH), which can form disulfide bonds within or V10B mutant expresses a Ku80 mRNA that contains a 252 bp between proteins and can mediate hydrogen bond interactions. in-frame deletion, causing loss of a 84 amino acid residue protein Thus, this mutation could affect the conformation of Ku80 or its region. The deletion which has been identified previously in interactions with other proteins, and most likely is the inactivating XR-V9B (22). The XR-V10B hamster cell mutant is derived mutation in XR-V13B. Previous studies in which effects of Ku80 from an ENU-mutagenized V79B population, whereas XR-V9B deletions were examined in xrs-6 cells, have indicated that the is derived from an EMS-mutagenized V79B population. Therefore, N-terminal part of Ku80 is dispensable for DNA end-binding and most likely, the same deletion in these mutants results from dimerization with Ku70 (30–32). Therefore, it is most likely that different genomic mutations that affect RNA splicing. In the mutations in the N-terminal part of Ku80 in XR-V13B and XR-V12B, no mutation was identified due to the absence or very XR-V11B affect the tertiary structure of the protein, which is low levels of Ku80 mRNA. In order to unravel the defect in required for DNA end-binding and/or other functions in vivo. XR-V12B, the entire Ku80 gene and the regulatory sequences Whatever these structural changes are, they clearly result in need to be sequenced. unstable Ku80 protein, since western blot analysis showed the The mutations in the XRCC5 gene reported to date comprise of absence of Ku80 protein in these cell mutants. Thus, the same large deletions of the protein or lead to lack of Ku80 expression phenotype of XR-V13B, XR-V11B and other group 5 mutants is (22,23). Here, in addition to such mutations, we describe a novel most probably due to the degradation of the Ku80 protein. A type of Ku80 mutation that leads to a severe X-ray-sensitive summary of all the identified mutations in the Ku80 gene is phenotype. The XR-V13B mutant carries a single missense depicted in Figure 3.
4337 Nucleic Acids Nucleic Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.119 4337 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 Figure 5. DNA-PK peptide phosphorylation pull-down assays in the X-ray- Radiopathology and Radiation Protection and European Union sensitive hamster cell mutants XR-V10B, XR-V11B, XR-V12B, XR-V13B, grant F14PCT90010, The Netherlands, and in the S.P.J. laboratory XR-V16B and wild-type V79B cells. 200 µg of whole cell extracts were was funded by the Cancer Research Campaign. Downloaded from http://nar.oxfordjournals.org/ by guest on September 23, 2015 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 REFERENCES between results of these experiments. 1 Frankenberg-Schwager,M. and Frankenberg,D. (1990) Int. J. Radiat. Biol., 58, 569–575. Recent studies of Ku80 deletion mutants in a yeast two-hybrid 2 Roth,D. and Wilson,J. (1988) In Kucherlapati,R. and Smith,G.R. (eds), Genetic Recombination. Amerian Society for Microbiology, Washington, DC, assay have shown that amino acid residues 449–732 are important pp. 621–653. for Ku70–Ku80 subunit interactions. In addition, using an in vitro 3 Zdzienicka,M.Z. (1996) In Lindahl,T. (ed.), Cancer Surveys. Genomic translation system, it has been found that amino acid residues Instability and Cancer. Cold Spring Harbor Laboratory Press, NY. Vol. 28, 334–732 are essential for DNA-end binding activity (30). pp. 281–293. 4 Pergola,F., Zdzienicka,M.Z. and Lieber,M.R. (1993) Mol. Cell. Biol., 13, Osipovich et al. (31) defined a minimal region from amino acid 3464–3471. residue 449 to 477 that is necessary for Ku80 to heterodimerize 5 Taccioli,G.E., Rathbun,G., Oltz,E., Stamato,T., Jeggo,P.A. and Alt,F.W. with Ku70. In agreement with these results, Cary et al. (32) found (1993) Science, 260, 207–210. that the central part of Ku80 (amino acid residues 241–555) is 6 Lee,S.E., Pulaski,C.R., He,D.M., Benjamin,D.M., Voss,M.J., Um,J.Y. and important for DNA end-binding and heterodimerization. These Hendrickson,E.A. (1995) Mutat. Res., 336, 279–291. 7 Mimori,T. and Hardin,J.A. (1986) J. Biol. Chem., 261, 10375–10379. findings are supported by the identification of deletions in the 8 Mimori,T., Ohosone,Y., Hama,N., Suwa,A., Akizuki,M., Homma,M., central part of Ku80 in XR-V9B, XR-V10B and XR-V15B cells Griffith,A.J. and Hardin,J.A. (1990) Proc. Natl Acad. Sci. USA, 87, (22) and point mutations in the XR-V16B mutant. The mutations 1777–1781. in these cases are, however, outside the minimal region defined 9 Carter,T., Vancurova,I., Sun,I., Lou,W. and DeLeon,S. (1990) Mol. Cell. Biol., 10, 6460–6471. by Osipovich et al. (31) as necessary for Ku70 interaction. Most 10 Gottlieb,T.M. and Jackson,S.P. (1993) Cell, 72, 131–142. probably these mutations result in an unstable protein that is 11 Chan,D.W. and Lees-Miller,S.P. (1996) J. Biol. Chem., 271, 8936–8941. targeted for degradation. Our results with the XR-V11B and 12 Bannister,A.J., Brown,H.J., Sutherland,J.A. and Kouzarides,T. (1994) XR-V13B mutant cell lines clearly also indicate that mutations in Nucleic Acids Res., 22, 5173–5176. the N-terminal part of Ku80, outside of the core region identified 13 Chen,Y.R., Lees-Miller,S.P., Tegtmeyer,P. and Anderson C.W. (1991) J.Virol., 65, 5131–5140. by Cary et al. (32) and Osipovich et al. (31), are important for Ku 14 Jackson,S.P., MacDonald,J.J., Lees-Miller,S.P. and Tjian,R. (1990) Cell, stability. It is worthy of mention that no mutant cell lines have 63, 155–165. been identified that harbour mutations in the very C-terminal 15 Lees-Miller,S.P., Chen,Y.R. and Anderson,C.W. (1990) Mol. Cell. Biol., portion of the Ku80 protein. This might suggest that mutations in 10, 6472–6481. 16 Blunt,T., Gell,D., Fox,M., Taccioli,G., Lehmann,A.R., Jackson,S.P. and this region do not lead to the degradation of Ku80 protein and/or Jeggo,P.A. (1996) Proc. Natl Acad. Sci. USA, 93, 10285–10290. this region of Ku80 does not affect X-ray sensitivity. 17 Danska,J.S., Holland,D.P., Mariathasan,S., Williams,K.M. and Guidos,C.J. In initial studies, the XR-V mutants showed different degrees (1996) Mol. Cell. Biol., 16, 5507–5517. of sensitivity towards X-ray (e.g. XR-V9B seemed to be less 18 Priestley,A., Beamish,H.J., Gell,D., Amatucci,A.G., Muhlmann-Diaz,M.C., sensitive than XR-V15B), suggesting that this might reflect Singleton,B.K., Smith,G.C., Blunt,T., Schalkwyk,L.C., Bedford,J.S., Jackson,S.P., Jeggo,P.A. and Taccioli,G.E. (1998) Nucleic Acids Res., 26, mutations in different functional domains of Ku80. However, 1965–1973. when subclones from a single colony were isolated from each 19 Errami,A., He,D.M., Friedl,A.A,. 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