From the Department of Molecular Medicine/Institute
of Biotechnology, The University of Texas Health Science Center at
San Antonio, San Antonio, Texas 78245, the
Department of
Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, the ** Department of Biochemistry and Molecular
Biophysics, Howard Hughes Medical Institute, Columbia University,
New York, New York 10032, the
Department
of Molecular Pathology, MD Anderson Cancer Center, Houston, Texas
77030, and the §§ Life Sciences Division,
Los Alamos National Laboratory, Los Alamos, New Mexico 87545
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ABSTRACT |
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Cells from individuals with the recessive
cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to
ionizing radiation (I-R). ATM (mutated in A-T) is a protein kinase
whose activity is stimulated by I-R. c-Abl, a nonreceptor tyrosine
kinase, interacts with ATM and is activated by ATM following I-R. Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair. Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM
and c-Abl. ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell
extracts. Consistent with the physical interaction, c-Abl
phosphorylates Rad51 in vitro and in vivo. In
assays using purified components, phosphorylation of Rad51 by c-Abl
enhances complex formation between Rad51 and Rad52, which cooperates
with Rad51 in recombination and repair. After I-R, an increase in
association between Rad51 and Rad52 occurs in wild-type cells but not
in cells with mutations that compromise ATM or c-Abl. Our data suggest signaling mediated through ATM, and c-Abl is required for the correct
post-translational modification of Rad51, which is critical for the
assembly of Rad51 repair protein complex following I-R.
Ataxia telangiectasia
(A-T)1 is an autosomal
recessive genetic disease characterized by diverse clinical symptoms
that include neuronal degeneration, immune deficiency, gonadal
abnormalities, cancer predisposition, premature aging, and
oculocutaneous telangiectasias (1, 2). Cells from individuals with A-T
are hypersensitive to ionizing radiation (I-R) and chemicals that cause
DNA double-strand breaks (DSB). Upon I-R, A-T cells exhibit defects in
multiple cell cycle checkpoint functions, including an aberrant p53
response and DNA DSB repair (1, 3). The DSB repair defect in A-T cells
is subtle, but nevertheless contributes significantly to the
radiosensitivity and chromosomal instability phenotype of these cells
(4-6). At a time when rejoining of I-R-induced DSB is completed in
normal mitotic cells, A-T cells still contain a significant number of
DSBs, which may be converted into chromosomal breaks (7, 8).
Additionally, the DSB repair process in A-T cells is error-prone (9,
10). A-T cells also possess an elevated level of spontaneous
intrachromosomal recombination (11). In meiosis of A-T cells, the
synaptonemal complexes fail to form at the pachytene stage, thus
aborting gametogenesis (12-14). Little is known about the molecular
basis for the meiotic and DNA DSB repair defects in A-T cells.
In eukaryotes, DNA DSB can be repaired by either homologous
recombination or nonhomologous end-joining pathways (15). Homologous recombination is achieved through multiple enzyme-catalyzed steps that
include processing and resection of the DSB, searching for and pairing
of DNA homologs, strand invasion, DNA synthesis, and finally,
resolution of recombination intermediates (16, 17). Rad51, a eukaryotic
homolog of the bacterial recombinase RecA, is required for
recombinational repair of DSB. In in vitro recombination assays using purified components, Rad51 catalyzes strand exchange in an
ATP-dependent reaction (16, 17). Interactions between yeast
Rad51 and other recombination factors including RPA, Rad52, Rad54,
Rad55, and Rad57 have been described (18, 19). Human Rad51 also binds
Rad52 (20, 21). Recent biochemical studies have demonstrated that the
strand exchange activity of Rad51 is stimulated by Rad52, Rad54, and
the Rad55·Rad57 complex, indicating that the protein-protein
interactions are functionally significant (22-26). The results of
studies with human Rad51 and Rad52 suggest that the mechanisms of DSB
repair by homologous recombination are conserved in higher
eukaryotes (27).
ATM, the gene mutated in A-T, encodes a 370 kDa protein that
is predominantly nuclear (28-31). It contains a phosphatidylinositol kinase domain that is also present in several other proteins known to
function in cellular responses to DNA damage, cell cycle control, and
telomere maintenance (32, 33). Upon activation by DNA damage, the ATM
kinase phosphorylates p53 on serine 15, which results in stabilization
of p53 (34, 35). In addition to modifying p53, ATM is also required for
the activation of the nonreceptor tyrosine kinase c-Abl (36, 37). ATM
binds c-Abl constitutively and may phosphorylate c-Abl on serine 465, leading to enhanced kinase activity of c-Abl (37). Whether defective
c-Abl activation affects DSB repair and thus contributes to the
phenotypes of A-T cells remains to be addressed.
In this study, we tested the hypothesis that the aberrant DNA repair
phenotype of A-T cells is due at least in part to defective modulation
of protein components of the DNA DSB repair machinery by ATM
kinase-mediated signals. We provide evidence that ATM and c-Abl kinase
are required for correct post-translational modification and assembly
of Rad51 protein complexes.
Antibodies--
The monoclonal anti-ATM antibody 3E8 was made
using GST-ATM-LZP fusion protein as the immunogen. Polyclonal and
monoclonal antibodies against human Rad51 and Rad52 were produced using
HisRad51 and HisRad52 purified from bacteria. The anti-phosphotyrosine antibodies 4G10 and RC20, anti- Purification of Rad51, Rad52, and c-Abl--
Human Rad51 and
Rad52 were overexpressed in Escherichia coli. Consecutive
chromatographic steps involving Q-Sepharose, Ni-NTA-agarose (for
histidine-tagged Rad51 only), Affi-blue, hydroxyapatite, and Mono-Q
were performed to purify Rad51, and chromatography on Ni-NTA and Mono-S
columns was used to purify histidine-tagged Rad52. Myc-tagged c-Abl,
overexpressed in COS-7 cells by transient transfection, was purified by
affinity chromatography with the 9E10 antibody immobilized on agarose.
The Myc epitope peptide was used to compete off the c-Abl bound to the column.
Analysis of Protein Interactions--
Cells were lysed in EBC
buffer (50 mM Tris, pH 7.6, 120 mM NaCl, 0.5%
Nonidet P-40, 1 mM EDTA, 1 mM
To examine the effects of Rad51 phosphorylation by c-Abl on interaction
between Rad51 and Rad52, 1 µg of Rad51 was incubated with affinity
purified wild-type or kinase inactive (K290R) c-Abl in kinase buffer
(20 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM MnCl2, and 1 mM dithiothreitol,
100 µM ATP) at 30 °C for 20 min. After addition of
genistein to inactivate the c-Abl kinase, Rad51 in the kinase reactions
was subjected to immunoprecipitation with a rabbit polyclonal
anti-Rad51 antibody and the anti-phosphotyrosine antibody 4G10.
Secondary antibodies conjugated with magnetic beads were used to
capture the immune complex. 0.5 µg of Rad52 was then added to the
mixture containing free and antibody-bound Rad51 and incubated at
4 °C for 45 min. Rad51 and Rad52 levels in antibody-bound and
-unbound fractions were determined by immunoblotting with their
respective antibodies.
Mapping the Residues of Rad51 Phosphorylated by
c-Abl--
His6-Rad51 cDNA was cloned into pCMV vector
for expression in mammalian cells. Specific tyrosine residues of Rad51
were mutated to phenylalanine using Quick-changeTM
(Stratagene). 8 µg of pSV40-Myc-c-Abl or -K290R-c-Abl, together with
2 µg of pCMV-His6-Rad51, were used to transfect COS-7
cells in one 10-cm plate. Two days later, the transfected cells were lysed in 1% Triton X-100 in EBC buffer containing 1 mM
Na3VO4 and protease inhibitors.
His6-Rad51 in the cell lysates was absorbed to Ni-NTA
resin. Proteins bound to the Ni-NTA resin were eluted with 250 mM imidazole in EBC and were then subjected to
immunoprecipitation with rabbit anti-Rad51 antibody. Tyrosine
phosphorylation of the immunoprecipitated His6-Rad51 was
detected by immunoblotting with the anti-phosphotyrosine
antibody RC20.
I-R-induced Rad51 Tyrosine Phosphorylation Is Dependent on ATM and
c-Abl--
We used glutathione beads with glutathione
S-transferase (GST)-ATM fusion proteins as the ligand to
bind cellular proteins that may associate with ATM. Rad51 and c-Abl
bound to a GST-ATM fusion protein encompassing the leucine zipper and
proline-rich region (LZP) of ATM (residues 980-1, 437, Fig.
1a, lane 3) but not
to GST or a GST-ATM-N (a fusion protein containing residues 249-523 of
ATM) (Fig. 1a, lanes 2 and 4).
Immunoprecipitation was then carried out to further assess the
association between ATM, c-Abl, and Rad51. Each of these three proteins
was detected in immunoprecipitations with anti-ATM, anti-c-Abl or
anti-Rad51 antibodies but not with preimmune IgG (Fig. 1b).
Treatment of the immunoprecipitates with DNase I had no effect on the
association between ATM, c-Abl, and Rad51, indicating that complex
formation among these proteins is probably not mediated by DNA (Fig.
1b). The amount of co-immunoprecipitating proteins did not
change significantly after I-R treatment (data not shown).
Previous studies have demonstrated an ATM-dependent
activation of c-Abl tyrosine kinase following I-R (36, 37). Prompted by
the physical interaction between Rad51 and c-Abl, we examined if Rad51
is tyrosine phosphorylated before and after I-R. Extracts prepared from
T24 cells before and after I-R treatment were immunoprecipitated with
anti-Rad51 antibody followed by sequential immunoblotting with
anti-phosphotyrosine and anti-Rad51 antibodies. Although the levels of
Rad51 did not change, tyrosine phosphorylation of Rad51 increased more
than 3-fold following I-R treatment (Fig. 2a). Tyrosine phosphorylation
of Rad51 was also examined in embryonic fibroblasts established from
wild-type mice, mice with nullizygous c-Abl mutation (38), and also
from ATM knockout mice (14). Induction of Rad51 tyrosine
phosphorylation after I-R was seen in wild-type mouse embryonic
fibroblasts (MEF), but not in c-Abl
Whether Rad51 protein can serve as a direct substrate for c-Abl was
tested using purified Rad51, Myc-tagged c-Abl and kinase-inactive c-Abl
(39). Rad51 (Fig. 3a) was
phosphorylated by active c-Abl but not by the kinase-inactive c-Abl.
Immunoblotting with an anti-phosphotyrosine antibody indicates that
phosphorylation of Rad51 by c-Abl occurred on tyrosine residues (Fig.
3a). To investigate possible Rad51 phosphorylation by c-Abl
in vivo, Rad51 was expressed together with kinase inactive
or active c-Abl in COS-7 cells. Co-expression of Rad51 with wild-type
c-Abl, but not kinase inactive c-Abl, stimulated Rad51 tyrosine
phosphorylation (Fig. 3b). Mutation of Rad51
Tyr315, but not Tyr205, Tyr191, or
Tyr54 to phenylalanine abolished Rad51 tyrosine
phosphorylation by c-Abl (Fig. 3b). These results strongly
suggest that c-Abl phosphorylates Rad51 Tyr315 in
vivo. Interestingly, Rad51 Tyr315 is present in a
YXXP context, a substrate sequence preferred by c-Abl kinase
activity (40), and is a highly conserved residue in Rad51 in all known
species of the animal kingdom.
To determine whether the elevated tyrosine phosphorylation of Rad51 in
c-Abl Tyrosine Phosphorylation of Rad51 by c-Abl Enhances the Interaction
between Rad51 and Rad52--
Genetic and biochemical studies indicate
that Rad51 cooperates with Rad52 in recombinational DNA repair (18,
22-24, 27). Whether phosphorylation of Rad51 by c-Abl modulates its
interaction with human Rad52 was investigated in binding assays
employing purified Rad51 and Rad52 (Fig.
4a). To avoid interference
from possible un-phosphorylated Rad51, anti-phosphotyrosine antibody was used to isolate tyrosine phosphorylated Rad51 after kinase reaction
with wild-type c-Abl (Fig. 4b). Approximately 4-fold more
Rad52 was associated with tyrosine phosphorylated Rad51 than with Rad51
treated with K290R c-Abl (Fig. 4b), indicating that tyrosine
phosphorylation of Rad51 by c-Abl enhances its interaction with Rad52
in vitro.
Because I-R induces Rad51 tyrosine phosphorylation in wild-type cells,
but not in ATM Because A-T cells arrested in the G1 phase of the cell
cycle are radiosensitive, it appears that defective checkpoint control is not the only cause of radiosensitivity of A-T cells (4-6). Moreover, the aberrant mitotic recombination and elevated chromosomal breaks in A-T cells after I-R suggest that ATM plays a direct role in
the repair of DNA DSB (1, 4, 7, 9-11). In this study, we report that
ATM and ATM-mediated signaling are required for both Rad51
phosphorylation and enhanced assembly of recombination complexes. Our
data support the hypothesis that defective modulation of repair
proteins may underlie the DNA repair deficiency in A-T cells.
Genetic and biochemical studies have assigned a central role for Rad51
in homologous recombination. The other recombination factors, Rad52,
Rad54, and Rad55·Rad57 stimulate the strand exchange activity of
Rad51 (22-27). This stimulation likely requires direct interaction
between the recombination proteins because mutant Rad52 lacking the
Rad51 binding site failed to stimulate Rad51-mediated strand exchange
(23). Our study has identified an I-R-induced phosphorylation of Rad51
on tyrosine residues and a concomitant increase in association between
Rad51 and Rad52, which may lead to increased DNA repair proficiency.
The enhanced association of Rad51 and Rad52 after I-R is likely because
of Rad51 tyrosine phosphorylation mediated by ATM and c-Abl since the
enhanced binding was also observed after phosphorylation of Rad51 by
c-Abl in vitro. It is not yet known whether phosphorylation
of Rad51 affects its binding with other members of the recombination complex.
While this study was in progress, Yuan et al. (41) reported
that phosphorylation of yeast and human Rad51 by c-Abl inhibited the
binding of Rad51 to DNA. Furthermore, the strand exchange activity of
yeast Rad51 also decreased upon treatment with c-Abl (41). The authors
concluded that c-Abl might have inhibitory effects on Rad51 activities
in vivo. In contrast, our studies of Rad51·Rad52 complex
formation in vitro and in vivo suggest that the
ATM and c-Abl-mediated signaling is likely to promote repair given the
biochemical evidence that Rad51 acts in concert with Rad52 in
homologous recombination (22-24, 27). Future biochemical studies are
needed to address whether in the presence of Rad52, Rad51 would indeed
exhibit higher recombinase activity upon phosphorylation by c-Abl.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin antibody, and anti-Myc antibody 9E10 were from Upstate Biotechnologies, Transduction Laboratories, Sigma, and Santa Cruz Biotechnologies, respectively. 8E9
and P6D are monoclonal antibodies that recognize human c-Abl.
-mercaptoethanol, 50 mM NaF, and 1 mM
Na3VO4) plus protease inhibitors.
Immunoprecipitation was performed as described earlier (28), using
secondary antibodies cross-linked to magnetic beads (Dynal Inc. Oslo,
Norway). To study whether protein complex formation requires DNA,
immunoprecipitation was performed in the presence of ethidium bromide
(20 µg/ml), and the immunoprecipitates were treated with 100 units of
DNase I at 37 °C for 1 h. To examine Rad51 tyrosine
phosphorylation, immunoblots containing Rad51 were incubated with
anti-phosphotyrosine antibody and then horseradish
peroxidase-conjugated secondary antibody. After detection by enhanced
chemiluminescence (Amersham Pharmacia Biotech), bound antibodies in the
blot were stripped off by incubation in 0.1 M glycine, pH
3.0, for 30 min. Total Rad51 amounts in the blot were determined with
anti-Rad51 antibody. In GST pull-down assays, 1 µg of GST or GST
fusion proteins that had been absorbed on glutathione beads was
incubated with target proteins in EBC buffer for 2 h at
4 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
a, Rad51 and c-Abl bind the LZP domain
of ATM. Left panel, Coomassie Blue staining of purified GST,
GST-ATM-LZP, and GST-ATM-N after separation by SDS-PAGE. Molecular
weight markers are shown on the left. Right
panel, immunoblotting of T24 total cell extracts (lane
1), T24 cellular proteins pulled down by GST (lane 2),
GST-ATM-LZP (lane 3), and GST-ATM-N (lane 4) with
anti-c-Abl and anti-Rad51 antibodies. b,
Co-immunoprecipitation of ATM, c-Abl, and Rad51. Raji cells (5 × 106) were lysed in EBC buffer and subjected to
immunoprecipitation (IP) using anti-ATM, anti-c-Abl, or
anti-Rad51 antibodies. Proteins in the immunoprecipitates were detected
by immunoblotting (IB) using the indicated antibodies.
/
or
ATM
/
MEF (Fig. 2b). Thus, the induced
tyrosine phosphorylation of Rad51 is dependent upon both ATM and c-Abl.
An elevated basal level of Rad51 tyrosine phosphorylation was detected
in c-Abl
/
MEFs (Fig. 2b). The reason for
this result is unknown, but it could be because of the presence of a
compensatory mechanism that results in an elevated basal level of
c-Abl-like activity in c-Abl
/
MEF, which also leads to
increased tyrosine phosphorylation of other c-Abl substrates (37).
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Fig. 2.
I-R-induced tyrosine phosphorylation of Rad51
in vivo depends on ATM and c-Abl. a,
tyrosine phosphorylation of Rad51 in T24 cells before and 60 min after
I-R. The cell lysates were subjected to immunoprecipitation
(IP) with anti-Rad51 antibody, and immunoprecipitates were
separated by SDS-PAGE followed by immunoblotting (IB) with
anti-phosphotyrosine antibody. The filter was re-probed with Rad51
antibody after removal of the anti-phosphotyrosine antibody.
b, tyrosine phosphorylation of Rad51 in wild-type
(WT), ATM /
, and c-Abl
/
MEF
before and 60 min after I-R. Immunoprecipitation and immunoblotting
were carried out as in panel a.
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Fig. 3.
a, c-Abl phosphorylates Rad51 in
vitro. Left panel, purified Rad51 stained with
Coomassie Blue after SDS-PAGE. Right panel, tyrosine
phosphorylation of Rad51 by c-Abl in vitro. Rad51 was
incubated with wild-type (WT) or K290R c-Abl in kinase
reactions. Phosphotyrosine residues, Rad51, and c-Abl were detected by
immunoblotting using anti-phosphotyrosine, Rad51, and c-Abl antibodies
as indicated. b, c-Abl phosphorylates Tyr315 of
Rad51 in vivo. His6-Rad51, wild-type, or with
the indicated tyrosine to phenylalanine mutations, were co-expressed
with Myc-K290R-c-Abl or Myc-c-Abl in COS-7 cells and affinity-purified.
The relative levels of tyrosine phosphorylated and total
His6-Rad51 were determined by immunoblotting with
anti-phosphotyrosine and anti-Rad51 antibodies, respectively. The
expression of c-Abl in the transfected cells was verified by
immunoblotting with anti-Myc antibody and with anti- -actin antibody
as a control for protein loading. c, tyrosine
phosphorylation of wild-type and Y315F Rad51 in c-Abl
/
MEF. The wild-type and mutant Rad51 were expressed in
c-Abl
/
MEF by transient transfection. The relative
levels of tyrosine phosphorylated and total His6-Rad51 were
determined as in panel b.
/
MEFs is because of phosphorylation on
Tyr315, wild-type and Y315F Rad51 was expressed in these
cells by transient transfection. The levels of tyrosine phosphorylation
of wild-type and Y315F Rad51 were similar (Fig. 3c),
suggesting that the majority of Rad51 tyrosine phosphorylation in
c-Abl
/
MEF does not occur on Tyr315.
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Fig. 4.
Effect of Rad51 phosphorylation on its
interaction with Rad52. a, purified Rad52, 3 µg, was
stained with Coomassie Blue after SDS-PAGE. b, binding of
Rad52 to immobilized Rad51. Rad51, treated with K290R or wild-type
c-Abl, was subject to immunoprecipitation by anti-Rad51 and
anti-phosphotyrosine antibodies, respectively. Rad52 was then added to
the mixture containing free and immunoprecipitated Rad51. The relative
levels of Rad51 and Rad52 in the inputs, free, and antibody-immobilized
(Bound) fractions were determined by immunoblotting.
P, phosphate. Duplicate experiments (Exps. 1 and 2) are
shown. The anti-Rad51 antibody did not interfere with the binding of
Rad51 to His-Rad52 (data not shown).
/
or c-Abl
/
MEF, we
examined the association between Rad51 and Rad52 in cells with these
distinct genetic backgrounds before and after I-R. Extracts prepared
from mock-treated and irradiated T24, wild-type MEF, as well as
c-Abl
/
and ATM
/
MEF were subjected to
immunoprecipitation using anti-Rad51 antibody. The level of Rad52 in
the Rad51 immunoprecipitates was quantitated by immunoblotting with
anti-Rad52 antibody. Significantly more (>3-fold) Rad52 was
co-precipitated after I-R treatment of T24 and wild-type MEF. In
contrast, enhanced association of Rad51 and Rad52 was not seen in
c-Abl
/
or ATM
/
MEF after I-R (Fig.
5a). I-R did not affect the
overall levels of Rad51 and Rad52 proteins (Fig. 5b). Thus,
the I-R-induced association between Rad51 and Rad52 appears to
correlate with the I-R-induced Rad51 tyrosine phosphorylation.
Interestingly, there was an elevated association of Rad51 and Rad52 in
c-Abl
/
MEF before I-R treatment (Fig. 5a),
perhaps because of the increased basal level of Rad51 tyrosine
phosphorylation in these cells.
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Fig. 5.
Increased association between Rad51 and Rad52
in vivo following I-R is dependent on ATM and
c-Abl. a, cell lysates prepared from T24, wild-type
MEF, ATM /
MEF, and c-Abl
/
MEF before
and 60 min after I-R were subjected to immunoprecipitation
(IP) with anti-Rad51 antibody. The immunoprecipitates were
then analyzed by immunoblotting (IB) with anti-Rad51 and
anti-Rad52 antibodies. b, the levels of Rad51 and Rad52 in
the cell lysates (100 µg) were quantitated by immunoblotting with the
indicated antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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Anti-Rad51 antibody used in initial studies was kindly provided by Dr. T. Ogawa at the Osaka University, and the Rad51 expression plasmid was supplied by Drs. W.-H. Lee and P.-L Chen. We thank Drs. Gopal K. Dasika and Hao-Chi Hsu for helping with site-specific mutagenesis and providing GST-c-Abl protein, respectively. Critical reading by Drs. Alan Tomkinson, Steve Skapek, McGreggor Crowley, and John Leppard is greatly appreciated.
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FOOTNOTES |
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* This work was supported by grants from Ataxia Telangiectasia Children's Project and National Institutes of Health Grant 1R01NS378381-01 (to E. L.).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 first two authors contributed equally to this work.
¶ Recipient of National Institutes of Health Postdoctoral Fellowship.
¶¶ To whom correspondence should be addressed. Tel.: 210-567-7326; Fax: 210-567-7324; E-mail: Leee{at}uthscsa.edu.
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ABBREVIATIONS |
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The abbreviations used are: A-T, ataxia telangiectasia; I-R, ionizing radiation; ATM, ataxia telangiectasia mutated; DSB, double-strand break(s); GST, glutathione S-transferase; MEF, mouse embryonic fibroblast(s); PAGE, polyacrylamide gel electrophoresis; NTA, nitrilotriacetic acid; LZP, leucine zipper and proline-rich region of ATM.
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