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J Biol Chem, Vol. 274, Issue 36, 25571-25575, September 3, 1999
From the Laboratory of Radiation and Life Science, School
of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
The product of the ATM gene, which is
mutated in ataxia telangiectasia, is a nuclear phosphoprotein, and it
involves the activation of the p53 pathway after ionizing radiation.
Here we show that the ATM protein is constitutively associated with
double strand DNA and that the interaction increases when the DNA is
exposed to ionizing radiation. The ATM protein also had affinity to
restriction endonuclease PvuII-digested DNA, but not to
UV-irradiated DNA nor X-irradiated single-stranded DNA. The
immunoprecipitation experiment detected very weak association between
ATM and DNA-PK proteins, and immunodepletion of DNA-PK showed little or
no effect on the interaction of the ATM protein with damaged DNA,
indicating that an interaction with DNA-PK might not be required for
the recruitment of the ATM protein to damaged DNA. Furthermore, the association was also confirmed in xrs-5 and xrs-6e cells, which are
Chinese hamster ovary mutant cell lines defective in Ku80 function.
These results indicate that the ATM protein is recruited to the site of
DNA damage and it recognizes double strand breaks by itself or through
an association with other DNA-binding protein other than DNA-PK and
Ku80 proteins.
Ataxia telangiectasia
(AT)1 is a human autosomal
recessive disorder characterized by a variety of clinical symptoms. At
the cellular level, AT cells exhibit hypersensitivity to ionizing radiation, radio-resistant DNA synthesis, and a high frequency of
chromosome aberrations (reviewed in Refs. 1-3). Recently, the gene
defective in AT cells has been cloned and designated ATM
(4). Sequence analysis revealed that the ATM protein shows homology to
a family related to phosphatidylinositol 3-kinase, those include
phosphatidylinositol 3-kinase p110 subunit, FRAP, RAFT1, and DNA-PK
proteins in mammalian cells (2, 5-7). Because p53 accumulation is
diminished or severely delayed in AT cells irradiated with ionizing
radiation, ATM protein has been suggested to be involved in p53
activation (8-11). p53 is a nuclear protein whose phosphorylation is
required for its stability and activity (reviewed in Refs. 12-15). Our
study and others (16, 17) have reported that ionizing radiation causes
p53 protein accumulation, and recently phosphorylation of p53 at serine
15 has been shown to control p53 accumulation through inhibition of its
interaction with MDM2 (18-20). Furthermore, recent in vitro
experiments have indicated that ATM phosphorylates p53 protein at
serine 15 (21, 22). Although there has been a report indicating the
phosphorylation of serine 15 in AT cells (23), it is highly possible
that the activity of p53 protein is mediated by ATM kinase activity.
Very little is known about the mechanism by which ATM protein is
activated by DNA damage. Previous studies have shown that ATM protein
levels do not change during cell cycle progression or after exposure to
ionizing radiation (24-26), and therefore, it has been hypothesized
that ATM protein may not be an inducible effector, but may act as a
transducer or, more likely, a sensor for DNA damage. Previous studies
have indicated that ATM protein harbors a putative leucine zipper motif
(6), whose structure is commonly detected in DNA binding proteins that
form homo- or heterodimers. Furthermore, the association of ATM protein
with meiotic chromosomes has been reported, indicating that the ATM protein may sense double strand break related structures occurring in
synapsing meiotic chromosomes (27). Thus, it is possible that ATM
protein also interacts with DNA double strand breaks induced by
ionizing radiation. Because DNA-PK, a protein kinase that shares amino
acid homology with ATM, has been shown to be recruited through its
association with Ku70/80 proteins bound to the DNA ends of double
strand breaks, and DNA-PK has a putative leucine zipper motif (5, 28),
it can be hypothesized that ATM protein interacts with damaged DNA by
itself or through an association with DNA-PK or Ku-like proteins.
In the present study, we examined whether ATM protein interacts with
damaged DNA in vitro. Our results show that the ATM protein constitutively associates with double strand DNA and that its interaction is potentiated when the DNA is exposed to ionizing radiation. The ATM protein also interacted with
PvuII-treated DNA but not with DNA exposed to UVC light.
Although a very weak interaction between ATM and DNA-PK was observed,
immunodepletion of DNA-PK protein did not affect the interaction of ATM
protein with damaged DNA. Furthermore, ATM associated with damaged DNA in Chinese hamster cells deficient in Ku80. These results suggested that association of the ATM protein with damaged DNA is independent of
DNA-PK or Ku80 proteins.
Cell Cultures and Irradiation--
Normal human embryo cells
(HE49) were cultured in minimum Eagle's medium supplemented with 10%
fetal bovine serum (Trace Bioscience PTY Ltd., Australia) as described
previously (30). 1 × 106 cells were seeded in T75
flasks (75 cm2) and subcultured every 4-5 days to maintain
exponential cell growth. AT5BI and AT2KY cells were obtained from
Japanese Cancer Research Resources Bank. The xrs-5 cells and xrs-6e
cells were obtained from Dr. Tom K. Hei, Columbia University, New York
and from Dr. William F. Morgan, University of California San Fransisco, respectively. Exponentially growing cells were irradiated with x-rays
from a x-ray generator at 150 kVp and 5 mA with a 0.1-mm copper filter.
The dose rate was 0.44 Gy/min. Ultraviolet light was exposed by
UV-light box (Ultraviolet Products, Upland, CA) equipped with two
germicidal lamps. DNA-cellulose was irradiated with x-rays in RIPA
buffer or exposed to UV under semidried condition.
Western Blot Analysis--
Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Nonidet
P-40, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. The cell lysate
was cleared by centrifugation at 15,000 rpm for 10 min at 4 °C, and
the supernatant was used as total cellular protein (31). Protein
concentration was determined by BCA protein assay (Pierce). For
extraction of the nuclear protein, cells were lysed in Lysis buffer (10 mM HEPES, pH 8.0, 50 mM NaCl, 0.5 M
sucrose, 0.1 mM EDTA, 0.5% Triton X-100, 1 mM
dithiothreitol, 5 mM MgCl2) as described
previously (31). The lysate was centrifuged at 5000 rpm for 1 min and
the supernatant was collected as cytoplasmic protein. The resultant
pellet was washed repeatedly with Lysis buffer, suspended in Lysis
buffer containing 0.5 M NaCl and 5 mM
spermidine, and incubated for 30 min at 4 °C. The lysate was cleared
by centrifugation at 12,000 rpm for 30 min at 4 °C, and the
supernatant was used as nuclear protein. Protein concentration was
determined with a protein assay kit (Bio-Rad, Tokyo, Japan). Sixteen to
thirty-two µg of total or nuclear proteins were electrophoresed on
SDS-polyacrylamide gels as described (31). The proteins were electrophoretically transferred to polyvinyl difluoride membrane in
transfer buffer (100 mM Tris, 192 mM glycine),
and the membrane was incubated with blocking solution (10% skim
milk) overnight. The membrane was incubated with anti-ATM mouse
monoclonal antibody (clone 1A1, Gene Tex, San Antonio, TX), anti-DNA-PK
mouse monoclonal antibody mixture (MC-365, Kamiya Biomedical Co.,
Seattle, WA), and anti-p53 monoclonal antibody (clone BP53-12, Lab
Vision Corp., Fremont, CA) for 2 h. The membrane was then
incubated with biotinylated secondary antibody followed by
streptavidin-alkalinephophatase. The band was visualized after addition
of nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as a substrate.
DNA Binding Assay--
Total cell lysates (500 µg) were
incubated with native or denatured DNA-cellulose (Amersham Pharmacia
Biotech, Tokyo) equilibrated in RIPA buffer (50 mM
Tris-HCl, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, 1% sodium
deoxycholate, 0.1% SDS). Native double-stranded DNA-cellulose was
irradiated with 20 Gy of x-rays, 5000 J/m2 UV light, or
1000 units of PvuII for 4 h. The cell lysates were also
incubated with native DNA-cellulose (75 µg of DNA) equilibrated in
RIPA buffer containing between 150 and 550 mM NaCl. For
DNA-PK- or ATM-depleted samples the cell lysate was incubated with
monoclonal antibodies against DNA-PK or ATM for 6 to 12 h, and
with protein A/G-agarose (Oncogene Science Inc.) for 4 h, then
subjected to DNA binding assay. The reaction mixture was inverted
gently for 6-12 h at 4 °C. DNA-cellulose was recovered by
centrifugation, washed four times with sufficient amounts of RIPA
buffer, and DNA binding proteins were dissolved in Laemmli's sample
buffer (31).
Immunoprecipitation and Immunodepletion--
Total cell extract
was incubated with monoclonal antibodies against ATM or DNA-PK proteins
for 12 and 6 h at 4 °C. Then Protein A/G-agarose was added and
incubated for a further 12 and 6 h at 4 °C. The immunocomplexes
were recovered by centrifugation at 15,000 rpm for 10 min at 4 °C.
The supernatant was used as immunodepleted cell lysate. The resultant
pellet was washed four times with RIPA buffer, dissolved in sample
buffer, and electrophoresed on 5% SDS-polyacrylamide gel. The proteins
were detected by Western blotting analysis as described above.
DNA Coprecipitation--
Total cell extract containing closed
circular pREP4 and PvuII-digested was incubated with
monoclonal antibodies against ATM protein for 6 h. Then protein
A/G-agarose was added and incubated for further 6 h at 4 °C.
The immunocomplexes were recovered by centrifugation at 15,000 rpm for
10 min at 4 °C. The resultant pellet was washed four times with RIPA
buffer and resuspend in TE buffer (10 mM Tris-HCl, pH 7.2, 1 mM EDTA) containing 0.1 mg/ml proteinase K. The samples
were incubated for 1 h at 50 °C, extracted with
phenol/chloroform/isoamyl alcohol, and precipitated with ethanol. The
plasmid DNA was resuspended in TE buffer and electrophoresed on 1%
agarose gel.
Detection of ATM Protein in Normal Human Cells--
The
anti-human ATM mouse monoclonal antibody used in this study was raised
against human ATM amino acid sequence between 2577 and 3056. As shown
in Fig. 1, the antibody recognized a
single protein in normal human cells whose molecular mass was estimated to have approximately 370 kDa. The expression of the ATM protein was examined in two AT fibroblasts, AT2KY and AT5BI. These two cell
lines have deletions in the ATM gene and these mutations are
expected to cause ATM protein truncation. We found that no ATM protein
was expressed in these two cell lines, while the immunoblotting using
anti-DNA-PK antibody confirmed the equal amount of DNA-PK protein
expressed in all three cells. The results indicate that the antibody
detects ATM protein in normal human cells and that the mutations
in two AT cell lines destabilize ATM protein.
Expression of ATM Protein in Normal Human Cells--
The level of
ATM protein in normal human cells irradiated with 4 Gy of x-rays was
examined by Western blotting (Fig. 2).
The ATM protein was found in the nuclear proteins, and its amount did
not change for up to 12 h after X-irradiation (Fig. 2,
middle). The abundance of ATM protein in the cytoplasm also
did not change (Fig. 2, top), indicating that ionizing
radiation did not affect ATM protein stability or nuclear
localization. In contrast, bi-phasic accumulation of the p53 protein
was detected (Fig. 2, bottom). p53 protein levels increased
1 h after irradiation, transiently decreased to a control
level, and increased again at 8 h after irradiation. We also
confirmed that transient decreases in p53 protein between 4 and 6 h are due to increased expression of MDM2 protein induced by p53 (data
not shown).
DNA Binding Ability of ATM Protein--
In order to examine
whether ATM protein recognizes damaged DNA, total cell lysate prepared
from unirradiated normal human cells or cells incubated for 2 h
after X-irradiation with 6 Gy was mixed with double strand
DNA-cellulose, and the proteins associated with DNA-cellulose were
subjected to Western blot analysis (Fig. 3). ATM protein in lysate from
unirradiated cells was found in the proteins bound to native double
strand DNA (Fig. 3A), and the amount of ATM protein did not
change when cells were irradiated with 6 Gy of x-rays and incubated for
2 h, which caused the efficient accumulation of p53 protein (Fig.
2). In contrast, the association of the ATM protein to double strand
DNA was significantly increased by more than 3-fold when DNA-cellulose
irradiated with 20 Gy of x-rays in vitro was used (Fig.
3A). In order to confirm whether the association was
specific to DNA, cell lysate first incubated with unirradiated
DNA-cellulose was mixed with irradiated DNA-cellulose (Fig.
3B). We found that the ATM protein was again recovered as the protein associated with irradiated DNA. Similarly, when cell lysate
mixed with unirradiated DNA was then incubated with unirradiated DNA,
ATM protein was recovered as DNA-associated protein, although there was
little or no ATM protein recovered if the cell lysate was first
incubated with irradiated DNA-cellulose. The results indicate that ATM
protein specifically associates with double strand DNA and that ATM
protein interacts more efficiently with damaged DNA.
In order to clarify whether the enhanced interaction with
irradiated DNA is dependent on DNA double strand breaks, cell lysate was mixed with double strand DNA-cellulose treated with either 5 kJ/m2 UVC or 1000 units of PvuII for 4 h or
with single strand DNA exposed to 20 Gy of x-rays (Fig.
4A). We found that increased association of ATM protein was observed only if the double strand DNA
was treated with PvuII, indicating that the ATM protein
preferentially recognizes double strand breaks. In Fig. 4B,
the association of ATM protein with x-ray-irradiated double strand DNA
was examined in a buffer containing different concentrations of NaCl.
ATM association was not affected at NaCl concentrations between 150 and
450 mM, but it was significantly reduced at 550 mM NaCl. The interaction of ATM protein with damaged DNA
was further examined by DNA coprecipitation assay (Fig.
5). Closed circular and
PvuII-digested plasmid DNA was mixed with total cell
extract, and then immunoprecipitates using anti-ATM antibody were
subjected to agarose gel electrophoresis. As shown in Fig.
5B, the immunoprecipitation coprecipitated linear PvuII-digested DNA, confirming the association of ATM
protein with damaged double strand DNA.
Role of DNA-PK and Ku80 on DNA Double Strand Break Recognition by
the ATM Protein--
The putative leucine zipper motif at codons
1217-1238 suggests that the ATM protein may form homodimers or
heterodimers with other proteins. DNA-PK also has a leucine zipper
motif, and it recognizes DNA double strand breaks by interaction with
Ku proteins. Therefore, we examined whether ATM and DNA-PK form
heterodimers to recognize damaged DNA. We found that immunoprecipitates
using anti-ATM antibody contained small amounts of DNA-PK protein,
while the antibody immunoprecipitated significant amounts of ATM
protein (Fig. 6). Similarly, anti DNA-PK
antibody immunoprecipitated large amounts of DNA-PK protein, whereas
only small amounts of ATM protein was recovered. These results indicate
that only a small fraction of ATM protein interacts with DNA-PK protein
in vitro. In order to clarify whether DNA-PK protein is
required for the association of ATM protein with damaged DNA,
immunodepleted cell lysate was used for DNA binding assays. As shown in
Fig. 7, ATM depletion significantly
reduced the amount of ATM protein but not DNA-PK protein in cell lysate
(Fig. 7A and 7B). Similarly, depletion of DNA-PK did not affect the
amount of ATM protein. As shown in Fig. 7C, ATM protein
depletion significantly decreased the amount of ATM protein interacting
with DNA, whereas DNA-PK depletion did not affect its association.
Thus, it can be concluded that the association of ATM protein with
damaged DNA does not required DNA-PK protein. Furthermore, cell lysates
obtained from xrs-5 and xrs-6 cells were used in order to examine
whether Ku proteins are involved in an association of the ATM protein
with damaged DNA (Fig. 8). In xrs-5
cells, there were no mutations in the Ku80 gene; however, very low
levels of Ku80 transcription were found (29). The Ku80 gene in xrs-6e
cells carries an insertion that results in a frameshift and the
resultant Ku80 protein was predicted to be a truncated protein (29).
Immunoblotting analysis proved there was no detectable Ku80 protein in
either cells (data not shown). As shown in Fig. 8, the antibody used in
this study recognizes ATM protein in CHO, xrs-5 and xrs-6e cells,
although the amount of ATM protein was slightly decreased in xrs-5 and
xrs-6e cells. The cell lysates obtained from CHO, xrs-5, and
xrs-6 cells were mixed with double strand DNA-cellulose irradiated with
20 Gy of x-rays. Although the amounts of ATM protein associated with
damaged DNA were less in xrs-5 and xrs-6e cells than in CHO cells, the ATM protein in all of the cells could interact with damaged DNA.
The present study shows that approximately 350-kDa protein was
detected as ATM protein in normal human embryo cells using anti-ATM
monoclonal antibody used in this study (Fig. 1). We did not detect ATM
protein in two AT cell lines, AT2KY and AT5BI. AT2KY cells have a
deletion in the ATM gene at nucleotide position 7883, which is
predicted to form a truncated ATM protein (32). AT5BI cells have a
deletion of 6 base pairs in the ATM gene, which may cause deletion of
two amino acids at codon 2427 (32). Because the antibody used in this
study recognizes amino acids between 2577 and 3056, it is possible that
the ATM protein in these AT cell lines will be destabilized, as
suggested previously (25, 26).
The time course experiments showed that the amount of ATM protein in
both nuclear and cytoplasm did not change up to 12 h after
X-irradiation, while p53 accumulated significantly during this period.
Previous studies have also shown that ionizing radiation and UV
irradiation do not affect the amount of ATM protein (24-26). Therefore, it can be hypothesized that ATM protein is not an inducible mediator or a transducer in signal transduction which activates p53
protein, but it acts as a sensor for DNA damage. Recently, it has been
reported that ATM protein associates with chromatin in meiotic prophase
(27). Furthermore, in ATM The amino acid sequence of the ATM protein has a putative leucine
zipper motif at codons 1217-1238 (6, 35), suggesting that ATM protein
may form homo- or heterodimers with other nuclear protein(s). Recently,
the biological significance of the leucine zipper motif was examined by
Morgan et al. (36). They found that overexpression of the
ATM protein fragment containing this motif in RKO cells abrogated the
S-phase check point and enhanced radiosensitivity. This dominant
negative effect indicates that this motif is required for ATM function.
Therefore, we examined whether nuclear protein association
enhanced association of ATM protein with damaged DNA. Since
DNA-PK has been reported to have a putative leucine zipper motif at
codons 1503-1538 (5), we determined whether ATM protein associated
with damaged DNA through interaction with DNA-PK. As shown in Fig. 6,
the association between ATM and DNA-PK was very weak. Furthermore,
depletion of DNA-PK did not affect the association of ATM with damaged
DNA (Fig. 7). The association of ATM protein with damaged DNA was also
detected in Chinese hamster cells deficient in Ku80 protein (Fig. 8).
Therefore, both DNA-PK and Ku80 proteins are probably not involved in
the recognition of damaged DNA by ATM protein. So far, DNA-PK and ATM
have implicated to function independently (37); however, recent reports
have indicated that c-Abl protein interacts with both DNA-PK and ATM
proteins (38-40), and therefore, there is a possibility that these two
independent pathways may communicate or cooperate to transduce signals.
Further studies will be needed to clarify this point.
In conclusion, we have shown that ATM protein associates with DNA
in normal human cells, and the association is potentiated by DNA double
strand breaks induced by ionizing radiation. The recruited ATM protein
may be activated by an unknown mechanism and induce a signal to
stabilize p53, thereby regulating cell cycle, DNA replication,
apoptosis, and DNA repair.
We thank T. K. Hei, W. F. Morgan,
and P. A. Jeggo for materials that made this study feasible.
*
This work was supported by a grant for Scientific Research
from the Japanese Ministry of Education, Science, Sports and Culture, and by a grant for the Regional Links Research Program at Nagasaki for
the Research Development Corporation of 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.
The abbreviations used are:
AT, ataxia
telangiectasia;
Gy, gray;
RIPA, radioimmune precipitation buffer;
CHO, Chinese hamster ovary.
Recruitment of ATM Protein to Double Strand DNA Irradiated with
Ionizing Radiation*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
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Fig. 1.
Detection of ATM and DNA-PK proteins in human
cells. Total cell extracts were obtained from normal human cells
(HE49) and from two ataxia telangiectasia cells, AT2KY and AT5BI.
Expression of ATM (A) and DNA-PK (B) proteins
were determined by Western blot analysis. The proteins were
electrophoresed on SDS-polyacrylamide gels, and ATM and DNA-PK proteins
were detected using mouse monoclonal antibodies against human ATM
protein (clone 1A1) and human DNA-PK protein (the mixture of clones
18-2, 25-4, and 42-26), respectively. Equivalent protein loading was
confirmed by staining the gels with Coomassie Brilliant Blue R.
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Fig. 2.
Expression of ATM and p53 proteins in normal
human cells irradiated with 4 Gy of x-rays. Exponentially growing
HE49 cells were irradiated with 4 Gy of x-rays. Both cytoplasmic
(top) and nuclear (middle and bottom)
proteins were prepared at each time point indicated. Proteins were
subjected to SDS-PAGE, and ATM and p53 proteins were detected using
anti-ATM monoclonal antibody (clone 1A1) and anti-p53 monoclonal
antibody (clone Bp53-12), respectively. Equivalent protein loading was
confirmed by staining the gels with Coomassie Brilliant Blue R.
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Fig. 3.
Association of ATM protein with double strand
DNA. DNA binding assay was performed using native double-stranded
DNA-cellulose as described under "Experimental Procedures."
A and C, total cell extracts were obtained from
control cells (lanes 1 and 3) or cells irradiated
with 4 Gy of x-rays and incubated for 2 h (lanes 2 and
4). Cell extracts were incubated with unirradiated
(lanes 1 and 2) or 20 Gy-irradiated DNA-cellulose
(lanes 3 and 4) for 12 h at 4 °C, washed
four times with sufficient amounts of RIPA buffer (150 mM
NaCl), and the proteins bound to DNA-cellulose were analyzed by Western
blot analysis. B, cell extracts were incubated with
unirradiated (lanes 1 and 3) or with 20 Gy-irradiated DNA-cellulose (lanes 2 and 4) for
12 h at 4 °C. The samples were centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatant was collected and incubated with
unirradiated (lanes 2 and 3) or 20 Gy-irradiated
DNA-cellulose (lane 1) for a further 12 h at 4 °C.
The binding proteins were analyzed by Western blot analysis.
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Fig. 4.
Association of ATM protein with DNA.
A and C, total cell extracts were obtained from
control cells. Cell extracts were incubated for 6 h with
unirradiated double strand DNA-cellulose (lane 1) or with
double strand DNA-cellulose treated with 5000 J/m2 UVC
light (lane 2) or with 1000 units of PvuII for
4 h (lane 3). Cell extracts were also incubated with
single strand DNA-cellulose irradiated with 20 Gy of x-rays for 6 h (lane 4). B, total cell extracts were incubated
for 6 h with double strand DNA-cellulose suspended in RIPA buffer
containing various concentrations of NaCl between 150 and 550 mM (lanes 1-5). After incubation, DNA-cellulose
was washed extensively with sufficient amounts of RIPA buffer, and the
proteins bound to DNA-cellulose were extracted by Laemmli's sample
buffer. Then the samples were subjected to SDS-PAGE and ATM protein was
detected by Western blot analysis.
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Fig. 5.
DNA coprecipitation. Control cells were
lysed in RIPA buffer (150 mM NaCl), and 400 µl of the
extract (1 mg/ml) containing 5 µg of genomic DNA extracted from
normal human (HE49) cells, 5 µg of closed circular pREP4 plasmid
(Invitrogen, NV Leek, The Netherlands), and 5 µg of
PvuII-digested pREP4 was incubated with monoclonal antibody
against ATM protein for 6 h at 4 °C. Then 40 µl of protein
A/G-agarose (Oncogene Research Products, Cambridge, MA) was added, and
incubated for further 6 h at 4 °C. The immunocomplexes were
recovered by centrifugation at 15,000 rpm for 10 min at 4 °C. The
resultant pellet was washed 4 times with RIPA buffer, and resuspend in
TEN buffer (10 mM Tris-HCl, pH 7.2, 1 mM EDTA,
150 mM NaCl) containing 0.1 mg/ml proteinase K. The samples
were incubated for 1 h at 50 °C, extracted twice with
phenol/chloroform/isoamyl alcohol, and precipitated with ethanol. The
plasmid DNA was resuspended in TE buffer (10 mM Tris-HCl,
pH 8.0, 1 mM EDTA), and electrophoresed on 1% agarose gel.
A, the immunoprecipitates were subjected to SDS-PAGE and ATM
protein was detected by Western blot analysis. Total cell extract
(lane 1) was incubated in the absence (lane 2) or
in the presence (lane 3) of ATM antibody. B,
total cell extract containing both closed circular and
PvuII-digested plasmids (lane 1) was
immunoprecipitated in the absence (lane 2) or in the
presence (lane 3) of ATM antibody.
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Fig. 6.
Association of ATM protein with DNA-PK
protein. Total cell extracts were obtained from control cells.
Cell extracts were incubated with anti-DNA-PK antibody (A,
lane 2; B, lane 2) for 12 h or
with anti-ATM antibody (A, lane 3; B,
lane 3) overnight, washed four times with RIPA buffer, and
incubated with protein A/G-agarose for a further 12 h at 4 °C.
The immunoprecipitates were washed extensively with sufficient amounts
of RIPA buffer, and the proteins were extracted in Laemmli's sample
buffer. The samples were subjected to SDS-PAGE and DNA-PK
(A) and ATM (B) proteins were detected by Western
blot analysis. NS, nonspecific band.
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Fig. 7.
Effect of immunodepletion of ATM and DNA-PK
proteins on ATM association with damaged DNA. Total cell extracts
obtained from control cells were incubated with antibodies against ATM
(ATM-depleted) or DNA-PK (DNA-PK-depleted) for 12 and 6 h, respectively. The samples were then mixed with protein
A/G-agarose, incubated for 6-12 h at 4 °C, and centrifuged at
15,000 rpm for 10 min at 4 °C. The supernatant was screened for ATM
and DNA-PK protein amounts (A and B,
respectively), or it was subjected to a DNA binding assays
(C). The expression levels of ATM (A and
C) and DNA-PK (B) proteins were analyzed by
Western blot analysis.
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Fig. 8.
Association of ATM protein with double strand
DNA in Chinese hamster cells. Total cell extracts were obtained
from CHO (A and B, lanes 1 and
4), xrs-5 (A and B, lanes 2 and 5), and xrs-6e (A and B,
lanes 3 and 6) cells. Sixteen µg of samples
were subjected to SDS-PAGE, and the proteins were analyzed by Western
blot analysis (A and B, lanes 1-3).
DNA binding assay was performed using native double-stranded
DNA-cellulose irradiated with 20 Gy of x-rays. Cells were lysed in RIPA
buffer containing 350 mM NaCl, and total cell extracts
obtained from control cells were incubated with 20 Gy-irradiated
DNA-cellulose (A and B, lanes 4-6)
for 12 h at 4 °C, washed four times with sufficient amounts of
RIPA buffer (350 mM NaCl), and the proteins bound to
DNA-cellulose were analyzed by Western blot analysis. The proteins were
detected using monoclonal antibodies against ATM (A) and
DNA-PK (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ knockout mice, it has been
shown that meiosis is arrested at prophase, which results in an
abnormal chromosomal synapsis and chromosome fragmentation (33).
Because DNA double strand breaks are thought to occur in meiotic
prophase, it has been hypothesized that ATM recognizes DNA strand
breaks (27, 33). Therefore, we examined whether ATM protein associates
with damaged DNA. As shown in Figs. 3 and 4, ATM protein was found to
interact with double strand DNA, and its association markedly increased
when DNA was exposed to ionizing radiation, indicating that ATM
constitutively interacts with DNA, and it monitors DNA damage. We found
that irradiation of cells did not alter the amount of ATM protein
associated with damaged DNA. Thus, it seems likely that increased
association with damaged DNA may not require posttranslational
modification, although ATM protein is a phosphoprotein (34). The
results presented in Fig. 4 demonstrate that the association of ATM
protein with DNA was also increased using DNA-damaged by
PvuII endonuclease treatment, but not by DNA exposed to UV
light. X-irradiated denatured DNA was not a good substrate for ATM
association. PvuII treatment produces blunt-ended double
strand breaks, and UV light causes pyrimidine dimers and (6-4)
photoproducts predominantly. Thus, it is implicated that ATM protein is
recruited to the site of DNA double strand breaks and it functions as a
sensor for DNA damage.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom all correspondence should be addressed: Laboratory of
Radiation and Life Science, School of Pharmaceutical Sciences, Nagasaki
University, 1-14 Bunkyo-machi, Nagasaki 852, Japan. Tel.:/Fax: 81-958-44-5504; E-mail: kzsuzuki@net.nagasaki-u.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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