From the Kihara Institute for Biological Research and
Graduate School of Integrated Science, Yokohama City University,
Maioka-cho 641-12, Totsuka-ku, Yokohama 244-0813, the
§ Department of Radiation Oncology, Graduate School of
Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyou-ku, Tokyo
113-0033, the ¶ Department of Molecular and Cell Genetics, School
of Life Science, Faculty of Medicine, Tottori University, Yonago
683-8504, and the
Faculty of Biology-Oriented Science and
Technology, Kinki University, Uchita-cho, Wakayama 649-64, Japan
Received for publication, November 21, 2000
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ABSTRACT |
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The mouse carcinoma cell line SX10 is a
hypersensitive mutant to x-rays and bleomycin. An earlier
complementation test suggests that SX10 would belong to x-ray-cross
complementing group (XRCC) 4. However, in this study, a human XRCC4
expression vector failed to complement the SX10 phenotype. Consistent
with the previous report, SX10 showed the same level of
DNA-dependent protein kinase activity as the wild-type
SR-1. We isolated and analyzed hybrids between SX10 and human diploid
fibroblast cells and found that human chromosome 13 conferred the x-ray
resistance to the hybrids, suggesting that a candidate gene would be
located on this chromosome. Polymerase chain reaction analysis with
these hybrids and x-ray-resistant transformants obtained by introducing
human chromosomes into SX10 indicated that the mutant was likely to be
defective in DNA ligase IV. Sequence analysis of the DNA ligase
IV gene confirmed that a defect in SX10 was attributed to a
transition of G to A at nucleotide position 1413 of the gene, leading
to an amino acid substitution from Trp at residue 471 to a stop codon.
Revertant clones (Rev1-3) derived from SX10 showed a restored x-ray
resistance; Rev1 reverted to the original nucleotide G at position
1413, whereas Rev2 and Rev3 to C. Transfection of a mouse DNA
ligase IV cDNA vector into SX10 restored the resistance to
both x-rays and bleomycin. SX10 showed a reduced frequency of
chromosomal integration of transfected DNA, but the revertants restored
the frequency found in the wild-type cells. These results suggest a
possible involvement of DNA ligase IV in the integration event of
foreign DNA as well as a crucial role in DNA double-strand break repair.
DNA double-strand breaks
(DSBs)1 are caused by
ionizing radiation and DNA-damaging agents (1) or occur as
intermediates in certain cellular processes such as V(D)J recombination
(2). If unrepaired or repaired inadequately, DSBs cause cell death by
loss or inactivation of an essential gene, or various chromosomal rearrangements, which lead to genetic diseases and cancer (3). Therefore, very efficient mechanisms have evolved to repair such DNA
damage. In eukaryotes, there are two major pathways for DSB repair:
homologous recombination that involves the exchange of genetic
information between a damaged chromosome and its undamaged partner, and
nonhomologous end-joining (NHEJ) that requires little or no homology at
broken DNA ends (4). Although homologous recombination is a main
pathway in yeast cells, NHEJ predominantly acts in mammalian cells
(5).
Mammalian cell mutants hypersensitive to ionizing radiation or
radiomimetic agents have been isolated and greatly contributed to
understanding the mechanism of DNA repair. These mutants have been
shown to be deficient in DSBR and classified into at least nine x-ray
cross complementing (XRCC) groups (6). Among them, mutants in
XRCC4-7 groups exhibit an extremely high sensitivity to
ionizing radiation and show severe defects in DSBR and V(D)J recombination (7, 8). With the hamster mutant XR-1 cell line, the
XRCC4 gene has been cloned by rescue of its deficiency in
DSBR and V(D)J recombination (9). This gene encodes a protein that
interacts with DNA ligase IV and stimulates the enzyme activity (9-11). The XRCC5 and XRCC6 genes encode Ku86
and Ku70, respectively, which are subunits of Ku protein (12-14). The
Ku86-defective hamster cell line xrs-6 exhibits defects in both DSBR
and V(D)J recombination processes (12, 15). Ku protein binds to broken
DNA ends and recruits DNA-PKcs encoded by XRCC7 (16-20) to
form a large protein complex. This complex recruits DNA ligase IV bound
to XRCC4 and undergoes the end-joining reaction. Also, the complex
reveals an activated protein kinase activity, which phosphorylates
certain unidentified substrate(s) involved in the regulation of cell
cycle checkpoints and controls the processing of the cell cycle during the process of DSBR. The mouse SX9 (21) and hamster V3 (19, 20, 22)
mutant cell lines and severe-combined immune-deficient (scid) mice (20,
23, 24) all exhibit defects in DNA-PKcs and are defective in both DSBR
and V(D)J recombination. The NHEJ pathway is conserved in yeast cells.
Except for DNA-PKcs, four homologues corresponding to Ku70, Ku80,
XRCC4, and DNA ligase IV also exist in Saccharomyces
cerevisiae and function to repair DSBs in a similar manner
(7).
In 1986, Sato et al. (25) isolated the x-ray-sensitive
mutant SX10 by mutagenizing SR-1 cells of the mouse mammary carcinoma FM3A cell line with a potent mutagen
N-methyl-N'-nitro-N-nitrosoguanidine. SX10 possesses a greatly reduced rejoining capacity of DSBs compared with the wild-type cells (26) and have been suggested to fall into the
same complementation group as that of x-ray-hypersensitive mouse
lymphoma M10 cells (25). Although SX10 has been characterized in
several ways (25-27), a gene responsible for the defective phenotypes has not yet been identified. Here we show that SX10 is a DNA ligase IV-defective mutant and that the defect results from a nonsense mutation in the ligase gene. Also we show that SX10 has a lower activity to integrate transfected DNA into the genomic sites than wild-type cells, suggesting the involvement of NHEJ mechanism in the
integration event.
Cells and Culture Methods--
Mouse mammary carcinoma FM3A cell
lines, wild-type SR-1, mutant SX10, and its 6-thioguanine- and
ouabain-resistant derivative SX10TOR (25) were used; cells were grown
in suspension in ES medium (Nissui Seiyaku Co., Tokyo) (28)
supplemented with 4% bovine calf serum (HyClone). The mouse leukemia
cell lines L5178Y and its x-ray-hypersensitive mutant M10 (29) were
used; cells were grown in suspension in ES medium supplemented with
10% bovine calf serum. HeLa and normal human diploid fibroblast TIG-3
cells (obtained from the Tokyo Institute of Gerontology, Tokyo) were also used; cells were grown as monolayers in Dulbecco's modified Eagle's medium (Nissui Seiyaku Co.) supplemented with 10% calf serum
or 10% fetal bovine serum (HyClone), respectively, and were subcultured by dispersing with 0.1% trypsin solution. All cultures were incubated at 37 °C in an atmosphere of 5% CO2 in
air. Logarithmically growing cells were used for all experiments.
Survival and Growth Inhibition Assays--
Cells were exposed to
x-rays (MBR-1520, Hitachi Medico Tokyo) at a dose rate of 1.1 Gy/min
with a 0.5-mm Al/0.2-mm Cu filter at room temperature. Then the cells
were diluted appropriately with growth medium and plated at
100-104 cells/dish into 60-mm bacterial plastic dishes
containing 5 ml of 0.15% agarose-containing growth medium and cultured
for 14-18 days. For growth inhibition assays, x-ray-treated or
untreated cells were plated at 104 cells/well into 24-well
plates (NUNC), cultured for 3-5 days, and counted for cell number. To
determine sensitivity to bleomycin (Wako Pure Chem. Ind., Osaka), cells
were plated at 100-104 cells/dish into 60-mm dishes
containing 5 ml of 0.15% agarose-containing growth medium with
different drug concentrations and cultured for 14-18 days. Resulting
colonies were counted and plating efficiencies were calculated.
Cell Fusion and Hybrid Selection--
Cell fusion was carried
out as described previously (25). Human diploid fibroblast TIG-3 cells
were used as a partner of cell fusion. Briefly, SX10TOR and TIG-3 cells
were harvested and washed with serum-free ES medium; both cells (each
1 × 106) were mixed in a glass centrifuge tube and
centrifuged. To the cell pellet was gently added 0.3 ml of a 50%
PEG1500 solution (Roche Molecular Biochemicals). After 1 min at room
temperature, serum-free ES medium was gradually added and suspended.
The mixture was spun down at low centrifugation, and the cell pellet
was dispersed into growth medium, plated into 100-mm bacterial dishes,
and incubated. On the next day, hybrid selection started in growth
medium containing HAT/ouabain (75 µM hypoxanthine, 0.5 µM amethopterin, 20 µM thymidine, and 1 mM ouabain). Two weeks later, the resulting colonies were picked, transferred to normal medium, and subcultured.
Chromosome-mediated Gene Transfer (CMGT)--
This method was
carried out as described previously (30). Briefly, metaphase chromosome
preparations from human HeLa cells were passed through a needle and
precipitated with calcium phosphate, and the precipitate was added to
SX10 cells. On the next day, the cells were harvested, plated, and
grown in 0.15% agarose-containing growth medium with 500 ng/ml
bleomycin. The drug-resistant colonies were isolated, subcultured in
growth medium without bleomycin, and assayed for sensitivity to x-rays.
The presence of human Alu sequences was screened by PCR
using Alu-PCR probe 451 as described (31). Also, the human
DNA ligase IV sequence was examined by PCR by using the
following primers h21F (5'-GATGTATTGATGGTTAATAATAAAAAGCTAGGG-3') and
hR1 (5'-AAAGCTAGCTTTAAATCAAATACTGGTTTTCTTC-3'). The PCR was performed
in 20 µl of a reaction mixture composed of 1× PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 200 µM each of four
deoxynucleoside triphosphates, 0.2 µl of the above primers, 40 ng of
template genomic DNA, and 0.12 µl of Taq polymerase (5 units/µl, Life Technologies, Inc.). The reaction conditions were: 94 °C for 3 min, 94 °C for 40 s, 56 °C for 30 s, and
72 °C for 1 min for 35 cycles and 72 °C for 10 min.
Identification of a Mutation--
Total cellular DNA and RNA
were prepared as described previously (32). To examine a mutation in
the mouse DNA ligase IV gene, the following primers
were used for both genomic PCR and RT-PCR: mF1
(5'-CAAACCGGAGGATTTGTTGTCGCT-3'), mF2
(5'-CCCATTTATTCACAATGCGTTCGGGA-3'), mR1
(5'-CATCATCACTGTCTGCTGTTGTCTGACA-3'), mR2
(5'-TAGGTCAGCCTTTGGATAACCATCTAA-3'), mR3
(5'-AGATGGCCTGTCACCAGGAGGTGGTG-3'), and mR4
(5'-GGGTTGTAGGCCATCATCTCACCG-3'). The PCR was carried out as
described above. The reaction conditions were: 94 °C for 3 min,
94 °C for 40 s, 56 °C for 30 s, and 72 °C for 1 min
for 35 cycles and 72 °C for 10 min. Amplified fragments were
cloned into pGEM-T Easy Vector (Promega) and directly sequenced using a
DNA sequencer model 4000 (Li-COR) with SequiTherm Long-Read cycle
sequencing kits LC (Epicenter Technologies) as described (32).
Otherwise, the fragments were digested with the restriction enzyme
DdeI (New England BioLabs), electrophoresed on a 8%
polyacrylamide gel, stained with 0.5 µg/ml ethidium bromide, and photographed.
Southern Blot Analysis--
Southern blotting was performed as
described previously (33). Briefly, 10 µg of genomic DNA purified
from cells were digested with DdeI, electrophoresed in a
1.0% agarose gel, and transferred to a nylon membrane
(Hybond-N+, Amersham Pharmacia Biotech). Then the membrane
was probed with a 32P-labeled 487-bp fragment of the mouse
DNA ligase IV sequence (see Fig. 3A). The probe
had been prepared by amplifying with mF2 and mR2 primers and SR-1 cell
DNA as a template, and by digesting the 1099-bp product with
DdeI.
Construction of an XRCC4 Expression Vector--
Based on the
reported sequence of human XRCC4 cDNA (9), we amplified
the fragment of the entire open reading frame and inserted it into a
pcDNA3 vector (Invitrogen). The resulting vector, designated
phXRCC4, was purified by 2 cycles of CsCl density centrifugation and
used for transfection.
Transfection by Electroporation--
To isolate stable
transfectants with the above phXRCC4 plasmid and the mouse DNA ligase
IV expression vector pCLig4 (a gift from Frederic W. Alt), transfection
was carried out with the plasmids linearized by digestion with
restriction enzymes BglII and PvuI (Amersham
Pharmacia Biotech), respectively, which cut the vector backbone
once. SX10 was transfected by electroporation as described previously
and incubated in growth medium for 24 h (33). The cells were then
harvested, replated, and cultured in 0.15% agarose-containing growth
medium with 0.8 mg/ml active Geneticin (G418, Life Technologies, Inc.)
for about 2 weeks. The resulting colonies were picked with micropipets,
grown to mass culture in the presence of the drug, and used for further analysis.
To examine the ability of SR-1 and SX10 cells to integrate foreign DNA
into their chromosomes, quantitative transfection assays were carried
out with plasmid pSV2neo linearized with EcoRI before use
(33). Briefly, 2 µg of the plasmid DNA was transfected into 4 × 106 cells by electroporation, and the cells were diluted
appropriately in growth medium and cultured for 24 h. Then, the
cells were collected, diluted, and replated at 1-5 × 105 and 102 cells/60-mm dish into 0.15%
agarose-containing growth medium with and without active G418 (0.9 mg/ml), respectively. They were cultured for 14-18 days at 37 °C.
The number of resulting colonies was scored, and the integration
frequency was calculated by dividing the number of G418r
colonies with surviving cells.
SX10 Is Likely to Differ from XRCC1-9 in the Complementation Group--
Sato et al. (25) reported that SX10 would belong to
the same complementation group as that of the M10 mouse leukemia line, which is hypersensitive to x-rays (26). Because M10 had been suggested
to belong to the XRCC4 group (34), we constructed a human
XRCC4 expression vector, phXRCC4, transfected it into wild-type SX10
and M10, and obtained G418-resistant clones. We assayed their
sensitivity to x-rays using two representative clones, SX10/hXRCC4 and
M10/hXRCC4. Fig. 1 clearly shows that
SX10/hXRCC4 was as sensitive to x-ray irradiation as SX10 but that
M10/hXRCC4 restored the resistance of L5178Y cells, the parental cells
of M10, indicating that the XRCC4 vector was able to complement M10 but
not SX10. We therefore conclude that SX10 is not part of the XRCC4 group. Although we measured DNA-PK activity in whole
cell extracts by pull-down assays (35), no difference was found between SR-1 and SX10 cells (data not shown), indicating that SX10 would fall
into a complementation group distinct from those of
XRCC5-7. We measured the level of sister chromatid exchange
in SR-1 and SX10 and observed no significant difference between the two
cell types (data not shown). Because the elevated sister chromatid exchange level is characteristic for a Human Chromosome 13 Complements the X-ray Sensitivity of
SX10--
To determine a human chromosome that could complement the
defect in SX10, we established three hybrid lines between SX10 and normal human diploid fibroblast TIG-3 cells by selection in growth medium containing HAT/ouabain after cell fusion as described under "Experimental Procedures." We first isolated one of the hybrid clones, CC3, which exhibited a high resistance to x-rays, and subcloned
CC3-2J and CC3-2P after serial passages. We examined their sensitivity
to x-rays by growth inhibition assays. As shown in Fig.
2A, CC3-2J was almost as
resistant as wild-type SR-1 whereas CC3-2P showed a striking
sensitivity like SX10. Moreover, subclone CC3-2J-13D derived from the
CC3-2J line lost the resistance to x-rays, indicating that a human
chromosome conferring the x-ray resistance segregated from the CC3-2J
cells. Then, with these hybrid lines, PCR analysis was performed using
primer sets (MapPaires, Research Genetics) specific for each of human
chromosomes. As a result, we found that only a 205-bp fragment
amplified by using a primer set (D13S325) specific for human chromosome
13 was correlated with the x-ray resistance of the hybrid lines (Fig.
2B). These results imply that the restored resistance was
concordant with human chromosome 13, thus suggesting a candidate gene
residing in chromosome 13.
SX10 Appears to Be Defective in DNA Ligase IV--
So far, the
genes responsible for DNA repair residing in human chromosome 13 are
DNA ligase IV (41), BRCA2 (42), and
XPG (43). Because SX10 is highly sensitive to x-rays but
insensitive to UV (25), we reasoned that the DNA ligase gene
could be a responsible gene. Therefore, we established three
x-ray-resistant transformants (Tor1-3) by the CMGT with human
chromosomes prepared from HeLa cells, as described under
"Experimental Procedures." The transformants all showed restored
resistance to x-ray treatment, although Tor2 was less resistant than
Tor1 and Tor3, as determined by a growth inhibition assay (Fig.
2C). To examine the existence of the human DNA ligase
IV gene in the transformants, genomic PCR using h21F and h21R
primers specific for the gene sequence was carried out. Like human
diploid fibroblast TIG-3 cells, a 1645-bp fragment was amplified in the
three transformants, whereas no fragment was amplified in either mouse
SR-1 or SX10 cells (Fig. 2D). These results strongly suggest
that SX10 is complemented by DNA ligase IV located in human chromosome 13.
A Point Mutation Is Present at the DNA Ligase IV Gene in
SX10--
To verify directly the defect in SX10, we determined the
nucleotide sequences of the DNA ligase IV gene in SR-1 and SX10. Because the mouse DNA ligase IV gene consists of only one
exon (44), we were able to amplify a genomic sequence, including the
entire open reading frame, using several pairs of primers prepared
based on the mouse DNA ligase IV sequence and genomic DNA as
a template (Fig. 3). We identified a
point mutation from G to A at nucleotide position 1413 of the ligase
gene in SX10 cells, resulting in an amino acid substitution from Trp at
residue 471 to a stop codon (TGA). No other change was detected in the sequence in the mutant cells.
During the CMGT experiments mentioned above, we isolated three
additional clones that were found on PCR amplification with either
human Alu-PCR primers or the h21F and h21R primers for the
human DNA ligase IV gene. We examined whether these clones, designated as Rev1-3, were sensitive to x-rays. Fig.
4 shows that they all were resistant to
the radiation (data not shown for Rev3 cells). So we determined their
sequences of the DNA ligase IV gene. As shown in Fig.
3A (upper panel), the mutated nucleotide A in
SX10 was changed to the original G in Rev1, and to C in both Rev2 and
Rev3. Therefore, we conclude that these cells resulted from reversion
of the mutated gene in SX10.
SX10 Is Hemizygous for the DNA Ligase IV Gene--
We asked
whether the mutation found in SX10 cells was homozygous or hemizygous.
Because the transition of G to A at position 1413 results in the
appearance of the recognition site (CTGAG) for DdeI, this
mutation will generate 245- and 84-bp fragments from the 329-bp
fragment observed in SR-1 (see Fig. 3, lower panel). We
amplified a region (including the mutation site) of DNA ligase IV cDNA from SR-1, SX10, and the two revertants and a pair of mF2 and mR3, and analyzed them on a polyacrylamide gel (Fig.
5A). Without DdeI
digestion, cDNAs from all the cell lines gave a 559-bp fragment.
When digested with DdeI, the wild-type cDNA was split into 329- and 209-bp fragments, whereas the SX10 cDNA was into 245-, 209-, and 84-bp fragments (Fig. 5A). The two
revertants Rev1 and Rev2 revealed the pattern of the wild-type cells,
reflecting a nucleotide change at the mutated site (Fig. 3, upper
panel). The 21-bp fragment was too small to be visible on the gel
for any cell line. These results suggest that SX10 has only a single mutated allele of DNA ligase IV. To further confirm this, we analyzed genomic DNA digested with DdeI by Southern blotting. Fig.
5B shows that, different from SR-1, Rev1, and Rev2, only
SX10 displayed a smaller 245-bp band that came from the mutated
sequence. These results demonstrate that SX10 is hemizygous for the
DNA ligase IV gene.
Transfection of the Mouse DNA Ligase IV Expression Vector Is Able
to Complement the Defect in SX10--
To prove complementation with
exogenous DNA ligase IV, we transfected the PvuI-linearized,
mouse DNA ligase IV expression vector pCLig4 into SX10 cells and
isolated several G418r clones. Two representative clones,
designated as Tec1 and Tec2, were assayed for their radiosensitivity.
It is evident that both transfectants restored a normal level of
resistance to treatment with either x-rays (Fig.
6A) or bleomycin (Fig.
6B). We assayed the growth rates of SR-1, SX10, revertants,
and transfectants by culturing at a density of 104
cells/well per ml in 24-well plates and counting the cell number every
day. SX10 grew with a longer doubling time of 23 h compared with
that of 14 h found in SR-1. Rev1 and Rev2 grew with a doubling time of 19 and 20 h, respectively; Tec1 and Tec2 cells revealed the doubling times of 21 and 20 h, respectively. These results indicate that the lower growth rate in SX10 was slightly recovered in
the revertants and transfectants.
SX10 Exhibits Reduced Chromosomal Integration of Transfected
DNA--
Transfection of foreign DNA into mammalian cells results in
the integration of the DNA into random chromosomal sites (45). To
assess whether the defect in DNA ligase IV affected the integration event, we carried out quantitative transfection assays with
EcoRI-linearized pSV2neo and compared integration
frequencies in SR-1, SX10, and two revertants. In Fig.
7, the basal level of the integration frequency in wild-type SR-1 was 5.6 × 10 In this study, we have presented evidence showing that the mouse
SX10 cell line that is hypersensitive to x-rays has a mutated DNA
ligase IV gene.
The mutation identified is a transition of G to A at nucleotide
position 1413 of the DNA ligase IV gene, resulting in the generation of a stop codon (Fig. 3A). The mutation site in
SX10 cells is located at the C-terminal portion of the catalytic
domain. The mouse and human DNA ligase IV genes have no
intron, consisting of one open reading frame (44, 46). DNA ligase IV is
composed of a catalytic domain in the N-terminal region and two domains containing BRCT motifs in the C-terminal region; it binds to the XRCC4
protein in the region between the two motifs (47). We have not directly
examined the catalytic activity of the mutated gene product.
Nevertheless, the product must have no activity, because it is a
truncated form that has lost both parts of the catalytic domain and the
BRCT domains along with the XRCC4-binding site. Recently, the
radiosensitive 180BR cell line derived from a leukemia patient has been
shown to possess a mutation at position 833 of the DNA ligase
IV cDNA, leading to an Arg to His substitution (48). This
mutation decreases the ability of the ligase IV to form an
enzyme-adenylate complex.
SX10 was found to be hemizygous for the DNA ligase IV gene.
We could find only one allele in both revertants Rev1 and Rev2 (Fig. 5,
A and B), which were obtained in the CMGT
experiments. We had examined the reversion of SX10 by selecting over
107 cells in bleomycin-containing plates but failed to
obtain any revertants. Introduction of foreign DNA or chromosomes into
cells seems to induce a mutation or rearrangements in the host
genome.2 We have not examined
whether the parental SR-1 cells are hemizygous for the DNA ligase
IV gene. However, it may be possible that the hemizygosity has
been established prior to mutant selection following mutagenesis
with
N-methyl-N'-nitro-N-nitrosoguanidine,
because the karyotype of SX10 considerably differs from that of SR-1
(data not shown).
Hybrids between SX10 and XRCC4-deficient M10 were unable to complement
each other for their radiosensitivity, suggesting that the two mutant
lines would belong to the same complementation group (25). However, in
this study, we found that this was not the case. The expression vector
of the human XRCC4 failed to rescue the SX10 defect (Fig. 1). The
reason for this difference is not known, but the complementing
chromosome in the hybrid cells might have segregated during the early
passage after the hybrid selection, or it is also possible that, in the
hybrid cells, the mutated XRCC4 protein would interfere with the normal
XRCC4 that specifically binds to DNA ligase IV and acts in the NHEJ process.
Transfection of SX10 with the mouse DNA ligase IV-expressing vector
pCLig4 (44) restored the x-ray and bleomycin resistance seen in
wild-type cells (Fig. 6). This result clearly demonstrates the
deficiency of DNA ligase IV in SX10 cells. The extent of the recovery
in the transfectants was lesser than that found in revertant Rev1 (data
not shown). In addition, Rev2 slightly resumed the resistance compared
with Rev1 (data not shown). This would be attributed to a reversion of
the mutated nucleotide A to C in Rev2 in place of G, a wild-type
nucleotide observed in Rev1. This incomplete recovery of the
sensitivity to x-rays in the revertants and transfectants may suggest
that SX10 has a genetic or epigenetic defect(s) other than the nonsense
mutation in the DNA ligase IV gene.
The growth rate of SX10 was not completely recovered in either
revertants or transfectants. The doubling time of SX10 was 23 h,
whereas that of the parental SR-1 was 14 h. Rev1, Rev2, Tec1, and
Tec2 grew with reduced doubling times of 19-21 h, thus showing a
20-40% recovery of growth rates compared with SX10 cells. This
partial recovery might also result from genetic or epigenetic defect(s)
other than the defect in DNA ligase IV. Recently, DNA ligase
IV-deficient mice have been reported to be embryonic lethal due to
massive neuronal cell death at the developmental stage (44, 49, 50);
however, fibroblast cells cultured from the mutant embryos are viable
and grow but reveal a reduced growth rate and a lowered saturation
density (44). Furthermore, we have established DNA ligase IV-negative
mutants from chicken B-cell lymphoma DT40 cells by gene targeting; the
mutants exhibit a small but significant reduction of growth
rate.3 Taken together, these
observations may suggest that DNA ligase IV could partially be involved
in DNA replication or repair-coupled DNA replication. Although in
mammalian cells DNA ligase I is thought to function in DNA replication,
it is not ruled out that other DNA ligases, including ligase IV, may
also contribute to DNA replication (51). However, human pre-B cell
mutants deficient in DNA ligase IV do not exhibit growth retardation
(46); this may imply that the mutants have a level of DNA ligase I
activity enough to fully support the replication.
Finally, the present study shows that the chromosomal integration of
transfected, linear DNA into SX10 cells was greatly reduced as compared
with wild-type SR-1 cells (Fig. 7) and this defect was restored to
normal levels in the two revertants. DNA ligase IV has been implicated
in DSB repair through NHEJ (51). This end-joining mechanism has long
been postulated to be involved in the chromosomal integration of
transfected DNA; that is, the integration event could occur by joining
of both ends of the input DNA and broken chromosomal DNA (45, 52),
although there is no direct evidence to support this view. Our finding
indicates that DNA ligase IV is involved in an integration process,
and, in other words, suggests that the integration event occurs through an NHEJ process in support of the above view (45, 52). Recently, we
have obtained the data supporting the contribution of NHEJ to the
integration process by an extensive structural analysis of integration
junctions.4 However, this
observation is inconsistent with the data by Merrihew et al.
(53), which do not agree with the end-joining mechanism for the
chromosomal integration. We are now obtaining further evidence for the
involvement of an end-joining mechanism in random integration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ray-sensitive Chinese hamster ovary mutant EM-9 classified into XRCC1 (36), our
result suggests that SX10 does not fall into the XRCC1
group. Like the XRCC4-7 mutant lines reported, SX10
revealed much greater sensitivity to ionizing irradiation than
XRCC2 (37), XRCC3 (38), XRCC8 (39),
and XRCC9 (40); therefore, we infer that SX10 would belong
to a complementation group different from these groups.
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Fig. 1.
X-ray survival of transfectants generated
with the human XRCC4 cDNA vector. X-ray
treatment and survival assays were performed as described under
"Experimental Procedures." The data are expressed as the percentage
of surviving colonies in x-ray-treated cells relative to that in
untreated cells. Open circles, wild-type SR-1; closed
circles, SX10; open triangles, L5178Y; closed
triangles, M10; open squares, SX10/hXRCC4; and
closed squares, M10/hXRCC4.
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Fig. 2.
Analysis of hybrids and transfectants.
A, x-ray sensitivity of hybrid cells between SX10TOR and
human diploid fibroblast TIG-3 cells. Growth inhibition assays were
performed as described under "Experimental Procedures." The data
are expressed as the percentage of cell number in x-ray-treated cells
relative to that in untreated cells. Open circles, wild-type
SR-1; closed circles, SX10; open triangles,
CC3-2J; closed triangles, CC3-2P; and open
squares, CC3-2J-13D. B, pattern of PCR products
amplified using a primer set (D13S325) specific for human chromosome 13 as described under "Experimental Procedures." The arrow
represents a 205-bp band amplified. C, x-ray sensitivity of
transformants generated by introducing human chromosomes into SX10
cells. Growth inhibition assays were performed as in A. Open circles, wild-type SR-1; closed circles,
SX10; open triangles, Tor1; closed triangles,
Tor2; and open squares, Tor3. D, detection of the
human DNA ligase IV gene by PCR in the transformants. PCR
was performed using a pair of primers h21F and h21R specific for the
human DNA ligase IV sequence as in B. TIG-3, human diploid fibroblast cells. For other cell lines,
refer to the legend in C.
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Fig. 3.
Sequence analysis of the DNA ligase IV
gene. The sequence of DNA ligase IV gene in
wild-type SR-1, SX10, and revertants was amplified by PCR with their
genomic DNA and various pairs of primers shown (mF1-2 and mR1-4), and
the products were cloned and sequenced as described under
"Experimental Procedures." The lower panel depicts the
region at the DNA ligase IV locus where the mutation site
was examined by PCR amplification and sequencing. The arrows
represent the primers used for both PCR and RT-PCR (for Fig. 5).
D represents the restriction enzyme site of
DdeI.
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Fig. 4.
X-ray survival of revertants. The
survival assays were performed as described in the legend to Fig. 1.
Open circles, wild-type SR-1; closed circles,
SX10; open triangles, Rev1; and closed triangles,
Rev2.
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Fig. 5.
Hemizygosity of SX10 for the DNA ligase
IV gene. A, mutation detected by RT-PCR analysis.
The sequence of the DNA ligase IV gene was amplified by
RT-PCR with total RNA from wild-type SR-1, SX10, and two revertants and
the primers mF2 and mR3 (see Fig. 3, lower panel). The
transition of G to A at nucleotide position 1413 results in the
occurrence of sequence CTCAG, a recognition site of DdeI, so
that digestion of the amplified 559-bp fragment with DdeI
generates 329- and 209-bp fragments in SR-1, Rev1, and Rev2, but 245-, 209-, and 84-bp fragments in SX10. B, Southern blot
analysis. Genomic DNA from SR-1, SX10, and the two revertants were
digested with DdeI and analyzed by Southern blotting using a
probe shown in Fig. 3A (lower panel).
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Fig. 6.
Survival of transfectants generated with the
mouse DNA ligase IV expression vector after treatment with x-rays
(A) or bleomycin (B). Survival
assays were performed as in the legend to Fig. 1. The data are
expressed as the percentage of surviving colonies in x-ray- or
bleomycin-treated cells relative to those in untreated cells.
Open circles, wild-type SR-1; closed circles,
SX10; open triangles, Tec1; closed triangles,
Tec2; and open squares, SX10/pcDNA3 (a transfectant
generated with control vector pcDNA3).
4 per
surviving cell on average (three determinations). The frequency in SX10
was reduced to 5% of the frequency found in SR-1. The two revertants
Rev1 and Rev2 considerably resumed the integration activity, although
Rev1 exhibited slightly lower frequencies whereas Rev2 had higher
frequencies. This difference between the wild-type and SX10 cells could
come from a reduced uptake of the DNA into the nucleus following
transfection. To test this possibility, we carried out transient assays
by transfecting expression plasmids pSV2Luc or pSV2CAT linearized at
the backbone and determining the expression of luciferase or CAT
activities, respectively, 24 h after transfection; no significant
differences were found in the expression levels per cell between SR-1
and SX10 (data not shown). This rules out a possible reduction of DNA
uptake into the nucleus in the mutant.
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Fig. 7.
Integration frequency of transfected DNA into
random chromosomal sites. Quantitative transfection assays were
performed as described under "Experimental Procedures." Integration
frequencies were calculated by the number of G418r colonies
divided by surviving cells. The data are expressed as the ratio of the
frequencies observed in SX10 and two revertants to that in wild-type
SR-1. The data show the mean ± S.D. of three
determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Y Aratani and N. Adachi for valuable discussions and A. Onozuka and C. Nishigaki for technical assistance. We also thank Dr. W. Alt for a gift of the mouse DNA ligase IV expression vector.
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FOOTNOTES |
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* This work was supported by a grant-in-aid from the Ministry of Health and Welfare, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Tel.: 81-45-820-1907; Fax: 81-45-820-1901; E-mail: koyama@yokohama-cu.ac.jp.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M010530200
2 D. Ayusawa, and H. Koyama, unpublished data.
3 N. Adachi, T. Ishino, Y. Ishii, S. Takeda, and H. Koyama, manuscript in preparation.
4 T. Hosoiri, Y. Aratani, and H. Koyama, manuscript in preparation..
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ABBREVIATIONS |
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The abbreviations used are: DSB, double-strand break; DSBR, double-strand break repair; XRCC, x-ray repair cross-complementing; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; CMGT, chromosome-mediated gene transfer; RT-PCR, reverse transcriptase polymerase chain reaction; NHEJ, nonhomologous end-joining; bp, base pair(s); HAT, hypoxanthine/amethopterin/thymidine.
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