Priority Report
DNA Damage–Induced BARD1 Phosphorylation Is Critical
for the Inhibition of Messenger RNA Processing
by BRCA1/BARD1 Complex
1
1
2
1
Ho-Shik Kim, Hongjie Li, Murat Cevher, Alissa Parmelee, Danae Fonseca,
2
1
Frida Esther Kleiman, and Sean Bong Lee
2
1
Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland
and 2Chemistry Department, Hunter College, City University of New York, New York, New York
Abstract
BRCA1-associated RING domain protein BARD1, along with
its heterodimeric partner BRCA1, plays important roles in
cellular response to DNA damage. Immediate cellular
response to genotoxic stress is mediated by a family of
phosphoinositide 3-kinase–related protein kinases, such as
ataxia-telangiectasia mutated (ATM), ATM and Rad3-related,
and DNA-dependent protein kinase. ATM-mediated phosphorylation of BRCA1 enhances the DNA damage checkpoint
functions of BRCA1, but how BARD1 is regulated during
DNA damage signaling has not been examined. Here, we
report that BARD1 undergoes phosphorylation upon ionizing
radiation or UV radiation and identify Thr714 as the in vivo
BARD1 phosphorylation site. Importantly, DNA damage
functions of BARD1 (i.e., inhibition of pre-mRNA polyadenylation and degradation of RNA polymerase II) are abrogated
in T714A and T734A mutants. Our findings suggest that
phosphorylation of BARD1 is critical for the DNA damage
functions of the BRCA1/BARD1 complex. (Cancer Res 2006;
66(9): 4561-5)
Introduction
Protein phosphorylation is critical in the cellular response to
DNA damage, acting as a molecular switch that regulates many
important DNA damage checkpoint responses. The principal
kinases involved in this signaling process are members of the
phosphoinositide 3-kinase–related protein kinase (PIKK) family,
such as ataxia-telangiectasia mutated (ATM) and ATM and Rad3related (ATR; refs. 1, 2). Many of the ATM/ATR kinase substrates,
such as BRCA1, CHK2, NBS1, MRE11, p53, and SMC1, signal cell
cycle checkpoint responses to DNA damage, playing important
roles in cell cycle arrest, apoptosis, and DNA repair (3). The tumor
suppressor BRCA1 is phosphorylated by ATM upon DNA damage,
and together with BARD1, performs multiple functions in the DNA
damage responses, including in DNA repair, in transcription, and in
RNA processing (4, 5). BRCA1/BARD1 form a complex through
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
H-S. Kim, H. Li, and M. Cevher contributed equally to this study.
H-S. Kim is currently at the Department of Biochemistry, College of Medicine, The
Catholic University of Korea, Seoul 137-701, Korea.
Competing interests statement: The authors declared no competing interests.
Requests for reprints: Sean Bong Lee, Genetics of Development and Disease
Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, 9000
Rockville Pike, Bethesda, MD 20892. Phone: 301-496-9739; Fax: 301-480-0638; E-mail:
[email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-3629
www.aacrjournals.org
their respective NH2-terminal RING domains and exhibit significant E3 ubiquitin ligase activity (5). Through its ubiquitin ligase
activity, the BRCA1/BARD1 complex can undergo autoubiquitination (6, 7) and can ubiquitinate substrates, such as p53 (8),
Nucleophosmin/B2 (9), g-tubulin (10), and RNA polymerase II
(RNAP II; refs. 11, 12). BRCA1/BARD1–mediated ubiquitination of
RNAP II targets it for proteasome-mediated degradation and
subsequent inhibition of transcription and RNA processing in
response to genotoxic stress (11). In contrast to BRCA1, whose
function is regulated by phosphorylation in response to genotoxic
stress, how BARD1 activity is regulated and whether BARD1 is
phosphorylated during DNA damage have not been examined.
Materials and Methods
Cell culture, antibodies, and constructs. Ataxia-telangiectasia fibroblasts (GM02052D and GM03487E) and control human fibroblasts
(GM07532A and GM08398) were obtained from the Coriell Cell Repository
(Camden, NJ). Rabbit polyclonal anti-BARD1 and monoclonal anti-CstF64
antibodies were kindly provided by Richard Baer (Columbia University) and
James Manley (Columbia University), respectively. NH2-terminal FLAG
epitope-tagged human BARD1 cDNA was amplified by PCR and cloned into
pCMV-Sport6. Quick Site–directed mutagenesis kit (Stratagene, La Jolla,
CA) was used to generate Ala substitutions at indicated sites. All constructs
were verified by sequencing.
In vivo labeling of 32P-orthosphosphate. U2OS cells stably transfected
with different BARD1 mutant constructs were starved for 30 minutes with
phosphate-free DMEM, and one set of plates was treated with 10 Gy
ionizing radiation. Phosphate-free DMEM containing 200 ACi/mL of 32Porthophosphate was added immediately, and cells were incubated for 2.5
hours. Cells were lysed and immunoprecipitated using anti-FLAG antibody
M2 (Sigma, St. Louis, MO) and Protein G-Sepharose (Amersham, Arlington
Heights, IL). Samples were resolved by SDS-PAGE and transferred to
nitrocellulose membrane followed by autoradiography. The membrane was
subsequently immunoblotted with anti-FLAG M2 antibody.
Generation of phospho-specific p-T714 BARD1 antibody. Phosphopeptide p-T714 [CKPKPDSDVT(PO3)QTINTVA] was synthesized, conjugated to keyhole limpet hemocyanin (KLH), and used to immunize rabbits
(Bethyl Laboratories, Montgomery, TX). The same phospho-peptide was
used for the affinity purification of phospho-specific antibody.
Results
To determine whether BARD1 is modified following DNA
damage, U2OS cells were irradiated with ionizing radiation, and
BARD1 was examined by immunoblotting. As early as 30 minutes
after g-irradiation (10 Gy), appearance of a slower migrating form
of BARD1 in SDS-PAGE was observed (Fig. 1A). Treatment of HeLa
or U2OS cells with doxorubicin, a chemotherapeutic agent that
causes double-stranded DNA breaks, also led to the slower
migration of BARD1 protein (Fig. 1B). Interestingly, only the
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Figure 1. BARD1 phosphorylation in response to DNA damage.
A, U2OS cells were g-irradiated at 10 Gy, and nuclear extracts were
prepared at indicated times and immunoblotted with anti-human
BARD1 antibody. B, nuclear and cytoplasmic extracts from either
U2OS or HeLa cells, treated with either 1 or 5 Ag/mL doxorubicin
(Dox ), were prepared and immunoblotted with anti-BARD1 antibody.
C, U2OS cells were either treated with 1 Ag/mL doxorubicin or with
UV (100 J/m2), and nuclear extracts were prepared at indicated
times were immunoblotted with anti-BARD1 antibody. D, nuclear
extracts from U2OS cells, either untreated or treated with 1 Ag/mL
doxorubicin, were incubated with either tyrosine phosphatase (Y ) or
E phosphatase (k ; New England Biolabs, Ipswich, MA) for 30
minutes and immunoblotted with anti-BARD1 antibody. IR, ionization
radiation.
BARD1 in the nuclear fraction was modified following DNA
damage (Fig. 1B). To examine whether single-strand DNA damage
can also result in BARD1 modification, U2OS cells were exposed to
UV radiation, and the mobility of BARD1 was examined. As shown
with ionizing radiation and doxorubicin treatments, the slow
migrating form of BARD1 can be detected shortly after UV
treatment (Fig. 1C).
To determine whether the observed BARD1 modification was
due to phosphorylation, nuclear extracts of cells either untreated or
doxorubicin-treated were incubated with either E phosphatase or
tyrosine phosphatase followed by immunoblotting. As shown in
Fig. 1D, the slowly migrating forms of BARD1 disappeared when
the extract was treated with E phosphatase, which removes
phosphate groups from serine, threonine, and tyrosine residues.
Treatment with tyrosine phosphatase did not affect BARD1
modification, suggesting that majority of BARD1 phosphorylation
is on serine and threonine residues.
Because a family of PIKKs, such as ATM and ATR kinases, plays
important roles in the DNA damage response (1, 2), we sought to
test whether PIKK is involved in BARD1 phosphorylation upon
DNA damage. Incubation of U2OS or HeLa cells with increasing
amounts of caffeine, an inhibitor of PIKKs, effectively abolished
BARD1 phosphorylation induced by doxorubicin, suggesting that
phosphorylation of BARD1 is PIKK dependent (Fig. 2A). To test
directly whether ATM is responsible for BARD1 phosphorylation,
two independently derived primary human fibroblast cell lines
from ataxia-telangiectasia patients were treated with either
doxorubicin or ionizing radiation along with two control primary
cell lines. In both ataxia-telangiectasia cells, BARD1 phosphorylation is reduced in response to either treatment compared with
controls (Fig. 2B), showing that ATM is at least partly responsible
for BARD1 phosphorylation. Residual phosphorylation seen in the
ataxia-telangiectasia cells, however, indicates that other kinases
can also phosphorylate BARD1.
ATM kinase phosphorylates BRCA1 at multiple sites in response
to genotoxic stress (3). Because BARD1 exists in a complex with
BRCA1 (5), we wished to test whether the DNA damage–induced
phosphorylation of BARD1 is dependent on the presence of BRCA1.
Thus, we examined DNA damage–induced BARD1 phosphorylation
in BRCA1-deficient cell line HCC1937 (13). HCC1937 cells treated
with either ionizing radiation or doxorubicin completely lacked
phosphorylation of BARD1 (Fig. 2C). The phosphorylation of T68
CHK2 in response to ionizing radiation was readily detected in
HCC1937 cells (data not shown), indicating that the ATM was
functional. This result indicates that BRCA1 is required for BARD1
phosphorylation after DNA damage.
CHK2 is an effector kinase downstream of ATM, which can also
phosphorylate ATM-substrates, such as BRCA1 and p53, in
Figure 2. BARD1 phosphorylation is mediated by ATM
and requires BRCA1. A, nuclear extracts prepared from
U2OS or HeLa cells treated with 1 Ag/mL doxorubicin
(Dox ) in the absence or presence of 2 or 5 mmol/L
caffeine were immunoblotted with anti-BARD1 antibody.
B, nuclear extracts prepared from normal human fibroblast
cells (N1 and N2) or A-T cells (A-T1 and A-T2) treated with
or without 1 Ag/mL doxorubicin or g-irradiated at 10 Gy
(ionization radiation, IR) were immunoblotted with
anti-BARD1 antibody. Nuclear extracts from U2OS were
used as a control. C, U2OS or HCC1937 (BRCA1 mutant)
cells were treated with either 1 Ag/mL doxorubicin for
16 hours or ionization radiation (10 Gy). Nuclear extracts
were prepared at indicated times and immunoblotted with
anti-BARD1 antibody. D, HCT15 cells were treated with
either 1 Ag/mL doxorubicin or 10 Gy ionization radiation.
Nuclear extracts were prepared and immunoblotted with
anti-BARD1 or anti-phospho-p53 (Ser15) antibodies
(Cell Signaling, Danvers, MA).
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DNA Damage Induced Phosphorylation of BARD1
response to DNA damage (3). To determine whether BARD1
phosphorylation is also mediated by CHK2 kinase, we examined
BARD1 phosphorylation in human colon cancer HCT15 cell line,
which has extremely low levels of endogenous CHK2 kinase activity
due to compound mutations in CHEK2 that lead to unstable
proteins (14). In HCT15 cells treated with either doxorubicin or
ionizing radiation, BARD1 phosphorylation was readily observed
(Fig. 2D). As expected, we observed the Ser15 phosphorylation of
p53 by ATM. These results suggest that CHK2 is dispensable for the
observed DNA damage–induced phosphorylation of BARD1.
ATM/ATR kinases phosphorylate serine or threonine residues,
which are immediately followed by a glutamine residue (SQ/TQ;
ref. 3). Examination of primary sequence revealed that there are
four potential ATM/ATR phosphorylation sites (SQ/TQ) in the
human BARD1 (Thr165, Ser244, Thr714, and Thr734). Sequence
comparison with other BARD1 orthologues revealed that the last
two TQ motifs located in the second BRCT domain (Thr714 and
Thr734) are evolutionarily conserved (Fig. 3A). To determine which
of these ATM/ATR phosphorylation sites are modified in response
to genotoxic stress, simultaneous or individual mutations of the
four Thr/Ser to Ala were analyzed. When cells were transfected
with the BARD1 mutant that contains AQ substitutions at all four
SQ/TQ sites (Quad), the BARD1 mutant was not phosphorylated
after DNA damage (Supplementary Fig. S1A). To define the
phosphorylation sites more precisely, U2OS cells were transfected
with FLAG-tagged BARD1 expression vectors containing individual
substitutions at each phosphorylation site and labeled with 32Porthophosphate after ionizing radiation treatment. Transient
overexpression of the different BARD1 mutants in cells led to
Figure 3. BARD1 Thr714 and Thr734 are evolutionarily conserved and
phosphorylated in vivo. A, amino acid sequence alignment near the
COOH-terminal BRCT domain of different BARD1 orthologues. Boxes indicate
conserved PIKK phosphorylation (TQ) sites. T714 and T734 refer to the human
BARD1 residues. B, U2OS cells stably transfected with different BARD1
expression constructs were either untreated or ionization radiation treated
(10 Gy) and then metabolically labeled with 32P-orthophosphate (200 ACi/mL).
Cell extracts were immunoprecipitated with anti-FLAG antibody M2 (Sigma),
resolved by SDS-PAGE, and transferred to nitrocellulose membrane followed by
autoradiography (P32-IP). Subsequently, membrane was immunoblotted with
anti-FLAG M2 antibody (a-FLAG). C, nuclear extracts from U2OS cells treated
with 1 Ag/mL doxorubicin (left) or increasing amounts of doxorubicin (1, 5, or
10 Ag/mL; right ) were immunoblotted with phospho-specific p-T714 BARD1
antibody. Where indicated, nuclear extracts were treated with E phosphatase for
30 minutes before immunoblotting (top left ). The same membrane was stripped
and blotted with anti-BARD1 antibody (bottom left).
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in vivo phosphorylation of the wild type (WT), T165A, S244A, and
T714A versions of BARD1 but not in T734A and Quad mutants
(Supplementary Fig. S1B). However, the observed phosphorylation
was irrespective of DNA damage, prompting us to examine BARD1
phosphorylation under a physiologic condition. Thus, we next
examined DNA damage–induced phosphorylation of BARD1 in
cells stably expressing BARD1 mutants. As expected, WT BARD1
showed enhanced phosphorylation in response to ionizing
radiation, but T714A, T734A, and Quad BARD1 mutants were not
efficiently phosphorylated in response to DNA damage (Fig. 3B).
This result suggests that Thr714 and Thr734 may be important DNA
damage phosphorylation sites. We also note that BARD1 is a
phospho-protein in the absence of DNA damage.
To further examine the role of Thr714 and Thr734 phosphorylation
in vivo, we raised phospho-specific rabbit polyclonal antibodies
that recognize Thr714- or Thr734-phosphorylated human BARD1.
Phospho-specific p-Thr714 antibody specifically recognized endogenous Thr714 phosphorylated BARD1 (Fig. 3C, left). Surprisingly,
Western blot analysis with the phospho-specific p-Thr714 antibody
showed that Thr714 residue of BARD1 was already phosphorylated
in the absence of any genotoxic stress and gradually increased with
DNA damage (Fig. 3C, right). These results suggest that a subset
of BARD1 may be constitutively phosphorylated at Thr714 by the
PIKK family of kinases during normal cell growth, and Thr714
phosphorylation is increased with DNA damage. Unfortunately,
phospho-specific antibody raised against p-Thr734 peptide did not
recognize BARD1 and was not further characterized. This may be
due to the presence of a well-conserved cysteine residue preceding
Thr734 (Fig. 3A), which may have been used during cross-linking
to KLH carrier and thus hinder with proper presentation of the
p-Thr734 peptide.
Recent reports have shown that in response to DNA damage
BRCA1/BARD1 complex ubiquitinates RNAP II (11, 12) and
subsequently leads to a rapid degradation of RNAP II by the
proteasome. Thus, we next examined whether the DNA damage–
induced BARD1 phosphorylation is important for the degradation
of RNAP II after DNA damage. Consistent with previous results,
UV treatment reduced the accumulation of both hypophosphorylated (RNAP IIA) and hyperphosphorylated (RNAP IIO) forms of
RNAP II in cells stably transfected with empty vector or WT
BARD1 (Fig. 4A). In contrast, cells stably transformed with the
T714A or the T734A versions of BARD1 showed a stabilization of
both RNAP II isoforms following UV treatment, especially of the
RNAP IIO isoform. This is consistent with a previous observation
that RNAP IIO is the target of BRCA1/BARD1 ubiquitination
(11, 12). This result suggests that phosphorylation of BARD1 at
Thr714 and Thr734 is important for the preferential degradation of
RNAP IIO mediated by the BRCA1/BARD1 complex in response to
DNA damage.
In addition to RNAP II, BARD1 also interacts with CstF50, a
component of the polyadenylation complex, and thus, the
polyadenylation machinery is inhibited in in vitro functional assays
(15). To examine the role of BARD1 phosphorylation in the DNA
damage–induced inhibition of mRNA 3¶ end formation, we did
in vitro RNA cleavage assays with nuclear extracts isolated from
different cell lines expressing various BARD1 mutants. As shown in
Fig. 4B, the inhibition of 3¶ cleavage after DNA damage was
significantly reduced in cells expressing the T714A and T734A
BARD1 mutants, whereas the vector and WT BARD1 expressing
cells exhibited normal inhibition of RNA cleavage after UV
treatment.
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Figure 4. BARD1 T714A and T734A mutants are defective in
RNAP II degradation and mRNA cleavage inhibitory activity in
response to DNA damage. A, U2OS cells stably transformed with
various BARD1 mutants were untreated or treated with UV
(20 J/m2). After 2 hours, cell extracts were immunoblotted with
indicated antibodies: BRCA1 (C-20, Santa Cruz Biotechnology,
Santa Cruz, CA), RNAP II (8WG16, Covance, Berkeley, CA), and
RNAP IIO (H5, Covance, Berkeley, CA). Anti-actin (A2066, Sigma)
blot shows relatively equal loading in all samples. B, nuclear extracts
were prepared from cells stably transformed with various BARD1
mutants, either untreated or treated with UV (20/m2). In vitro RNA
cleavage assay was done as previously described (15) using SV40
late precursor RNA (SVL). 5¶ cleaved product and SVL precursor
RNA are denoted. C, nuclear extracts prepared from various stable
BARD1 expressing cells, either untreated or treated with UV
(20 J/m2), were immunoprecipitated with anti-CstF64 antibody.
Supernatants and the immunoprecipitated pellets were resolved
by SDS-PAGE and immunoblotted with anti-FLAG M2, anti-CstF64,
or anti-actin antibodies.
The transient inhibition of 3¶ RNA processing following DNA
damage reflects the formation of the BRCA1/BARD1/CstF
complex (15). To test the effect of BARD1 phosphorylation on
the BRCA1/BARD1/CstF complex formation, we analyzed the
complex in nuclear extracts from UV-treated cells expressing
different mutants of BARD1. As the BARD1/CstF-50 interaction
involves the intact CstF complex (15), we used monoclonal
antibodies against CstF-64, another CstF subunit, to immunoprecipitate the complex. As shown in Fig. 4C, T734A BARD1 mutant
did not form a complex with CstF, irrespective of UV treatment,
whereas WT BARD1 was able to form a complex that increased
significantly after the UV damage. Unexpectedly, T714A version of
BARD1 still formed a complex with CstF even in the absence of
genotoxic stress; however, unlike the WT BARD1, this interaction
did not increase with DNA damage (Fig. 4C). The results indicate
that the phosphorylation of BARD1 plays an important role in the
BARD1/CstF interaction and subsequent inhibition of CstF
activity. In contrast, BARD1/BRCA1 interaction was still retained
in the T714A, T734A, and Quad BARD1 mutants (Supplementary
Fig. S2).
Discussion
DNA damage leads to different cellular responses, such as cell
cycle arrest, inhibition of transcription and of RNA processing,
DNA repair, and apoptosis. ATM/ATR kinases are able to control
many aspects of the DNA damage response by phosphorylating
specific substrates important in different cellular pathways (2, 3).
Of the four potential ATM/ATR phosphorylation sites in human
BARD1, only two TQ sites near the tandem BRCT motifs are
evolutionarily conserved (Fig. 3A), suggesting the importance of
BARD1 phosphorylation at these residues. Our study shows that
BARD1 is phosphorylated at Thr714, which increased with DNA
damage, using phospho-specific p-T714 antibody (Fig. 3C). Our
results also revealed that BARD1 exists as a phospho-protein,
which is hyperphosphorylated in response to DNA damage.
Cancer Res 2006; 66: (9). May 1, 2006
Mutation of either T714A or T734A significantly reduced DNA
damage–induced phosphorylation of BARD1 (Fig. 3B) and further
resulted in a dysfunctional BARD1 in mediating inhibition of 3¶
RNA processing and degradation of RNAP II after DNA damage
(Fig. 4). Loss of UV-induced inhibition of RNA processing in T734A
mutant might be due to its inability to form a complex with CstF
(Fig. 4C), suggesting that phosphorylation of Thr734 may be
important for the DNA damage–induced BARD1/CstF interaction.
Surprisingly, unlike the T734A substitution, T714A mutation did
not abolish BARD1/CstF interaction (Fig. 4C). This observation
suggests that the DNA damage–induced inhibition of 3¶ processing
by BARD1 may not simply be due to sequestration of CstF but
implicates a more direct role in the inhibition of CstF complex.
Our study, thus, provides mechanistic insights by which BARD1
activity can be regulated by PIKK-mediated phosphorylation.
Although the half-life of both T734A and Quad BARD1 mutants
was reduced compared with WT or other BARD1 mutants
(Supplementary Fig. S3A), it is unlikely that T734A and Quad
mutants are grossly misfolded because these BARD1 mutants
retained the ability to interact with BRCA1 (Supplementary
Fig. S2). However, we cannot exclude the possibility that the loss
of BARD1 function in T734A mutant may be due to local conformational changes in the BRCT domain because a truncated
BARD1 containing only the NH2-terminal RING domain can still
interact with BRCA1 (5). Degradation of the mutant BARD1 was
delayed with proteasome inhibitor MG132, suggesting that the
observed instability of BARD1 was due to proteasome-mediated
degradation (Supplementary Fig. S3A). These results suggest that
phosphorylation of BARD1 may also be an important determinant
of BARD1 stability.
It is likely that BARD1 has additional DNA damage–induced
phosphorylation sites other than Thr714 and Thr734, and that
additional phosphorylation may also regulate different aspects of
BARD1 function. Consistent with this view, recent reports showed
that BARD1 can be phosphorylated by a cyclin-dependent kinase
(CDK)/cyclin complex in a cell cycle–dependent manner (16, 17),
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DNA Damage Induced Phosphorylation of BARD1
and mutations in the CDK2/cyclin E1/A1 phosphorylation sites of
BARD1 confer increased sensitivity to mitomycin C treatment (16).
The precise mechanisms by which CDK/cyclin– or PIKK-mediated
BARD1 phosphorylation regulate its activity is not known. One
possibility is that the phosphorylation sites of BARD1 may directly
be involved in the binding of other proteins (as in Thr734
phosphorylation, leading to formation of BARD1/CstF complex)
or may indirectly influence protein-protein interaction by inducing
conformational changes. Because the BRCT domain serves as a
phospho-peptide binding module (18, 19), DNA damage–induced
phosphorylation of Thr714 and Thr734 residues of BARD1 (which are
located in the second BRCT domain) may convert the BRCT
domain from a phospho-peptide binding module into a phosphoprotein docking site for other phosphorylation-specific binding
proteins. Additionally, although not mutually exclusively, Thr714
and Thr734 phosphorylation and CDK/cyclin–mediated phosphorylation may also serve to activate or enhance the activity of BARD1
complex, such as its E3 ubiquitin ligase activity or homology-
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Acknowledgments
Received 10/11/2005; revised 3/6/2006; accepted 3/14/2006.
Grant support: National Institute of General Medical Sciences-Score grant S06
60654 (F.E. Kleiman) and Intramural Research Program of the NIH/National Institute
of Diabetes and Digestive and Kidney Diseases (S.B. Lee).
The costs of publication of this article were defrayed in part by the payment of page
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DNA Damage−Induced BARD1 Phosphorylation Is Critical for
the Inhibition of Messenger RNA Processing by
BRCA1/BARD1 Complex
Ho-Shik Kim, Hongjie Li, Murat Cevher, et al.
Cancer Res 2006;66:4561-4565.
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