Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80

Page created by Lauren Clark
 
CONTINUE READING
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,. Overkamp,W.J. I., Morolli,B.,
mutant and later examined for X-ray sensitivity, all mutants                       Hendrickson,E.A., Eckardt-Schupp,F., Oshimura,M., Lohman,P.H.M.,
showed a similar degree of the X-ray sensitivity (data not shown).                 Jackson,S.P. and Zdzienicka,M.Z. (1998) Nucleic Acids Res., 26, 3146–3153.
                                                                                20 Jeggo,P.A. and Kemp L.M. (1983) Mutat. Res., 112, 313–327.
Thus, the observed differences amongst newly isolated mutants                   21 Mizuta,R., Taccioli,G.E. and Alt,F.W. (1996) Int. Immunol., 8, 1467–1471.
most probably result from the presence of X-ray-resistant                       22 Errami,A., Smider,V., Rathmell,W.K., He,D.M., Hendrickson,E.A.,
revertants in the cell population. This is in agreement with the                   Zdzienicka,M.Z. and Chu,G. (1996) Mol. Cell. Biol., 16, 1519–1526.
4338 Nucleic Acids Research, 1998, Vol. 26, No. 19

23 Singleton,B.K., Priestley,A., Steingrimsdottir,H., Gell,D., Blunt,T.,      33 Stamato,T.D., Weinstein,R., Giaccia,A. and Mackenzie,L. (1983) Somat.
   Jackson,S.P., Lehmann,A.R. and Jeggo,P.A. (1997) Mol. Cell. Biol., 17,        Cell Genet., 9, 165–173.
   1264–1273.                                                                 34 Whitmore,G.F., Varghese,A.J. and Gulyas,S. (1989) Int. J. Radiat. Biol.,
24 Gu,Y, Jin,S, Weaver,D.T. and Alt,F.W. (1997) Proc. Natl Acad. Sci. USA,       56, 657–665.
   94, 8076–8081.                                                             35 Zdzienicka,M.Z., Tran,Q., van der Schans,G.P. and Simons,J.W.I.M.
25 Ouyang,H., Nussenzweig,A., Kurimasa,A., Soares,V.C., Li,X.,                   (1988) Mutat. Res., 336, 203–213.
   Cordon-Cardo,C., Li Wh, Cheong,N., Nussenzweig,M., Iliakis,G.,             36 Zdzienicka,M.Z., Jaspers,N.G., van der Schans,G.P., Natarajan,A.T. and
   Chen,D.J. and Li,G.C. (1997) J. Exp. Med., 186, 921–929.                      Simons,J.W. (1989) Cancer Res., 49, 1481–1485.
26 Nussenzweig,A., Chen,C., da Costa Soares,V., Sanchez,M., Sokol,K.,         37 Verhaegh,G.W., Jongmans,W., Morolli,B., Jaspers,N.G., van der Schans,G.P.,
   Nussenzweig,M.C. and Li,G.C. (1996) Nature, 382, 551–555.
                                                                                 Lohman P.H. and Zdzienicka,M.Z. (1995) Mutat. Res., 337, 119–129.
27 Zhu,Ch., Bogue,M.A., Lim,D.-A., Hasty,P. and Roth,D.B. (1996) Cell, 86,
                                                                              38 Zdzienicka,M.Z., Jongmans,W., Oshimura,M., Priestley,A., Whitmore,G.F.
   379–389.
                                                                                 and Jeggo,P.A. (1995) Radiation Res., 143, 238–244.
28 Smider,V., Rathmell,W.K., Lieber,M.R. and Chu,G. (1994) Science, 266,
   288–291.                                                                   39 Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem., 162, 156–159.
29 Taccioli,G.E., Gottlieb,T.M., Blunt,T., Priestly,A., Demengeot,J.,         40 White,R.J., Gotlieb,T.M., Downes,C.S. and Jackson,S.P. ( 1995)
   Mizuta,R., Lehmann,A.R., Alt,F.W., Jackson,S.P. and Jeggo,P.A. (1994)         Mol. Cell. Biol., 15, 1983–1992.
   Science, 265, 1442–1445.                                                   41 Finnie,N.J., Gotlieb,T.M., Blunt,T., Jeggo,P.A. and Jackson,S.P. (1995)
30 Wu,X. and Lieber,M.R. (1996) Mol. Cell. Biol., 16, 5186–5193.                 Proc. Natl Acad. Sci., USA, 92, 320–324.
31 Osipovich,O., Durum,S.K. and Muegge,K. (1997) J. Biol. Chem., 272,         42 Anderson,C.W. and Lees-Miller,S.P. (1992) Crit. Rev. Eukary. Gene Expr.,
   27259–27265.                                                                  2, 283–314.
32 Cary,R.B., Chen,F., Shen,Z. and Chen,D.J. (1998) Nucleic Acids Res., 15,   43 Zdzienicka,M.Z. and Simons,J. (1987) Mutat. Res., 178, 235–244.
   974–979.                                                                   44 Jeggo,P.A. and Holliday,R. (1986) Mol. Cell. Biol., 6, 2944–2949.

                                                                                                                                                              Downloaded from http://nar.oxfordjournals.org/ by guest on September 23, 2015
You can also read