From the Department of Molecular Medicine and Institute of
Biotechnology, The University of Texas Health Science Center at San
Antonio, San Antonio, Texas 78245
Received for publication, October 21, 2002, and in revised form, December 3, 2002
Efficient repair of DNA double-strand breaks
depends on the intact signaling cascade, comprising molecules
involved in DNA damage signal pathways and checkpoints. Budding yeast
Rad9 (scRad9) is required for activation of scRad53 (mammalian homolog
Chk2) and transduction of the signal further downstream in this
pathway. In the search for a mammalian homolog, three proteins in the
human data base, including BRCA1, 53BP1, and nuclear factor with BRCT domains protein 1 (NFBD1), were found to share significant homology with the BRCT motifs of scRad9. Because BRCA1 and 53BP1 are involved in
DNA damage responses, a similar role for NFBD1 was tested. We show that
NFBD1 is a 250-kDa nuclear protein containing a forkhead-associated motif at its N terminus, two BRCT motifs at its C terminus, and 13 internal repetitions of a 41-amino acid sequence. Five minutes after
-irradiation, NFBD1 formed nuclear foci that colocalized with the
phosphorylated form of H2AX and Chk2, two phosphorylation events known
to be involved in early DNA damage response. NFBD1 foci are also
detected in response to camptothecin, etoposide, and
methylmethanesulfonate treatments. Deletion of the forkhead-associated motif or the internal repeats of NFBD1 has no effect on DNA
damage-induced NFBD1 foci formation. Conversely, deletion of the BRCT
motifs abrogates damage-induced NFBD1 foci. Ectopic expression of the BRCT motifs reduced damage-induced NFBD1 foci and compromised phosphorylated Chk2- and phosphorylated H2AX-containing foci. These
results suggest that NFBD1, like BRCA1 and 53BP1, participates in the
early response to DNA damage.
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INTRODUCTION |
Genomic DNA in all living species is continuously assaulted by
exogenous sources, such as environmental mutagens, and endogenous factors, such as reactive oxygen species, that arise during normal cellular metabolism (1). Cells have evolved surveillance mechanisms to
promote faithful transmission of genetic information, which involves
detection of DNA damage and a resulting signal transduction cascade
leading to the induction of cell cycle checkpoints and initiation of
DNA repair (2-5). Defects in DNA damage response pathways may result
in inefficient repair and accumulation of mutations.
Participants in the DNA damage response pathways can be divided into
sensors, proximal kinases, adaptors, transducer kinases, and effectors
(4, 5). In budding yeast, a heterotrimeric complex, Rad17-Ddc1-Mec3,
which shares homology with proliferating cell nuclear antigen and the
replication factor c-like factor Rad24 are thought to be DNA
damage sensors (6). Mec1 and Tel1, members of the phosphatidyl
inositol-3 kinase-like kinase family, are classified as proximal
kinases that activated in response to DNA damage (7, 8). Rad9, an
adaptor protein, is phosphorylated by Mec1, which in turn activates the
downstream transducer kinase Rad53, a serine/threonine kinase (9-12).
Activated Rad53 then further transduces and amplifies signals to
downstream effectors that regulate the cell cycle machinery
(13-15).
Components of this signaling pathway are conserved in mammals (4, 5).
In the initial step of checkpoint activation, DNA damage sensor(s)
Rad9-Rad1-Hus1, which share homology with proliferating cell nuclear
antigen and the reduced folate carrier protein-like factor Rad17, relay
signals to the proximal kinases, ataxia-telangiectasia mutated
(ATM)1 and ataxia
telangiectasia and Rad3-related (ATR) (16-20). Signals are then
amplified by the transducer kinases, Chk1 and Chk2 (21-25). Finally,
activation of the downstream effectors, such as p53 and CDC25, leads to
cell cycle arrest (26-27).
Budding yeast Rad9 (scRad9) was the first cell cycle checkpoint protein
discovered. It is essential for arresting division upon DNA damage
(28). When yeast is treated with DNA-damaging agents, scRad9 is
phosphorylated, which promotes binding to the C-terminal forkhead
homology-associated (FHA) motif of Rad53 (12). Therefore, scRad9 plays
a critical role in this signaling pathway by ensuring that the damage
signal is transduced downstream. A mammalian homologue of scRad9,
however, has not been identified. The structural signature motifs of
scRad9 are two BRCT repeats in its C terminus. Based on this structural
similarity, it was predicted that three mammalian proteins, BRCA1,
p53BP1, and an uncharacterized protein, KIAA0170, might be the
candidates for the scRad9 homologue (29-31). Only BRCA1 and 53BP1 have
been significantly characterized to date.
In addition to their structural similarity, BRCA1 and Rad9 both serve
as substrates of ATM and ATR kinases in response to DNA damage (32-34)
and compromised G2/M checkpoint control upon inactivation
(35). However, BRCA1 also has unique functions, including acting as a
substrate of Chk2 (36), directly participating in homologous
recombination, and microhomology-mediated end-joining through
interaction with the Rad50-Mre11-NBS1 complex (37-39). Moreover, BRCA1
contains a ring-finger motif at its N terminus and has ubiquitin ligase
activity (40). BRCA1 also interacts with many transcription factors and
coordinately regulates the critical cell cycle inhibitor p21 and the
G2/M checkpoint protein Gadd45 (41). Because of these
diverse functions, BRCA1 may not be a bona fide homolog of
Rad9. Another protein, 53BP1 also resembles scRad9. It has two
C-terminal BRCT repeats and participates in early DNA damage response
(42-43). It is also a substrate for ATM (44-45) and is required for
G2/M checkpoint arrest in response to
-irradiation (46).
Thus, 53BP1 seems to be even more functionally similar to scRad9 than BRCA1.
KIAA0170 was an anonymous gene identified by random sequencing
of cDNA library derived from a human immature myeloid cell line
(47). The KIAA0170 gene product was later reported to have transactivation activity and was renamed `nuclear factor contains BRCT
domains protein 1' (NFBD1) (48). NFBD1 is predicted to have an
N-terminal FHA motif (49) and two C-terminal BRCT repeats. Besides this
preliminary characterization of secondary structure, however, very
little is known about NFBD1. In particular, nothing has yet been
published about its potential function in the DNA damage response.
Here, we show for the first time that NFBD1 does participate in the
early DNA damage responses. Deletion of BRCT motifs, but not the FHA
motif or the internal repeats of NFBD1, abolished foci formation.
Ectopic expression of the NFBD1-derived BRCT motifs greatly reduced
IR-induced nuclear foci containing NFBD1, Chk2 phosphorylated at
threonine 68 (Chk2T68P), and H2AX phosphorylated on serine 139 (
-H2AX).
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MATERIALS AND METHODS |
Construction of Plasmids--
The NFBD1 full-length cDNA
(kindly provided by T. Nagase, Kazusa DNA Research Institute, Chiba,
Japan) was cloned into the EGFP-C2 expression vector
(Clontech, Palo Alto, CA). Subsequent motif
deletions were made in the EGFP-NFBD1 construct using the QuikChange
site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed
by DNA sequencing. Primers encoding the nuclear localization signal
were synthesized, annealed, and cloned into EGFP-C2. The fragment
containing the BRCT motifs of NFBD1 (codon 1839-2089) was obtained by
polymerase chain reaction using Pfu polymerase (Stratagene)
and cloned in-frame with nuclear localization signal in EGFP-C2.
Cell Culture and Treatments with DNA-damaging Agents--
HeLa,
a human cervical carcinoma cell line; IMR90, a human diploid
fibroblast; and LEM, an Epstein-Barr virus-immortalized human B cell
line, were cultured in Dulbeco's modified Eagle's medium supplemented
with 10% fetal bovine serum, 2 mM L-glutamine, 50 units of penicillin, and 50 µg/ml of streptomycin at 37 °C with
10% CO2. Cells grown in log-phase were irradiated in a
137Cs radiation source (Mark I, Model 68A Irradiator; JL
Shepherd & Associates). UV irradiation was performed using a
Stratalinker UV source (Stratagene). Before UV irradiation, the culture
medium was removed and cells were washed once with phosphate-buffered saline. The medium was replaced immediately after irradiation. All the
cells were then cultured at 37 °C and harvested at the indicated
time points. For treatment with genotoxic agents, the cells were
exposed for 1 h at the indicated dose, washed with phosphate-buffered saline, and cultured in fresh medium for another hour before being fixed for immunostaining. Untreated or mock-treated cells were treated identically with phosphate-buffered saline washes
and medium changes but no irradiation.
Antibodies--
The fragment of the NFBD1 gene
encoding amino acids 2-224 was amplified by polymerase chain reaction
using Pfu polymerase (Stratagene) and was cloned into the
pGEX vector (Amersham Biosciences). The recombinant protein was
expressed in BL21-LysS cells and purified with glutathione
S-transferase beads. The purified recombinant protein was
resolved by SDS-polyacrylamide gel electrophoresis and stained with
imidazole-zinc. The polypeptide band was excised, electroeluted from
the gel, and subsequently used as an antigen to immunize mice and
rabbits. Rabbit antibodies were purified using antigen-coupled affinity
chromatography. Rabbit
-
-H2AX was purchased from Upstate
Biotechnology (Lake Placid, NY), rabbit
-Chk2T68P from Cell
Signaling Technology (Beverly, MA), and mouse
-GFP from Roche
Applied Science.
Immunoprecipitations and Western
Blotting--
[35S]Methionine labeling,
immunoprecipitations, and immunoblotting were performed as described
previously (50, 51).
-NFBD1 and
-GFP monoclonal antibodies were
used for antigen detection in the immunoblots.
Immunostaining--
Procedures for immunostaining were adapted
from Durfee et al. (52). Briefly, cells grown on coverslips
to 60-70% confluence were treated with various DNA-damaging agents
and fixed in 3% formaldehyde with 0.1% Triton X-100. Cells on
coverslips were permeabilized by 0.05% saponin and blocked with 10%
normal goat serum. All primary antibodies were used at a dilution of
1:100 to 1:1000 in 10% goat serum. The secondary antibodies, including anti-rabbit or mouse Alexa 555 and anti-rabbit or anti-mouse Alexa 488 (Molecular Probes, Eugene, OR) were used at a dilution of 1:1000. Cells
were further stained with 4',6-diamidino-2-phenylindole for detection
of DNA and mounted in Permafluor (Lipshaw-Immunonon, Pittsburgh, PA).
Immunofluorescence images were captured using a Zeiss Axiplan2
fluorescence microscope.
Transfections--
Transfections were performed using Lipofectin
(Invitrogen) according to the manufacturer's instructions. The
transfected cells were supplemented with fresh medium and irradiated at
36-38 h after transfection.
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RESULTS |
Identification of Cellular NFBD1 Protein with Anti-NFBD1
Antibodies--
We found that NFBD1, in addition to its N-terminal FHA
motif and two C-terminal BRCT motifs, had 13 unique internal repeats, each containing 41 amino acids enriched with serine, threonine, and
proline residues (Fig. 1). Interestingly,
potential ATM/ATR and cyclin-dependent kinase
phosphorylation sites are scattered throughout these repeats. To
identify the encoded protein of NFBD1, we used the recombinant GST
fusion protein containing the N-terminal 223 amino acids of NFBD1 as an
antigen (named GST-FHA) to raise mouse and rabbit polyclonal
antibodies. Cell extracts prepared from
[35S]methionine-labeled lymphoid cells (LEM) were used
for immunoprecipitation with either mouse NFBD1 antisera or preimmune
serum. As shown in Fig. 1C, a protein with an apparent
molecular mass of 250 kDa, which is near the predicted molecular mass,
was specifically immunoprecipitated by anti-NFBD1 antisera but not
pre-immune sera (lanes 1 and 2). Addition of the
original GST-FHA antigen (lane 4), but not GST alone
(lane 3), blocked the immunoprecipitation of 250-kDa
protein. To further ensure the specificity of the antibodies, the
anti-NFBD1 immunoprecipitates were released and reprecipitated by
anti-NFBD1, as described previously (51). Under these conditions, only
the single 250-kDa band was detected (lane 5), suggesting
that the NFBD1 antisera were specific for the 250-kDa protein. In
addition, protein with the same relative mobility was also
immunoprecipitated by rabbit NFBD1 antisera (lane 6). Mouse
(lane 7) and rabbit (lane 8) NFBD1 antibodies
specifically purified by antigen-coupled affinity chromatography also
detected a major band with the same apparent mobility, although minor
bands with faster mobilities were also detected. The latter may be
degradation products of NFBD1 or yet to be identified proteins. These
results suggest that a protein with an apparent mass of ~250 kDa is
the endogenous NFBD1 gene product.

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Fig. 1.
Protein structure of NFBD1 and
characterization of anti-NFBD1 antibodies. A, human
NFBD1 protein has a single FHA motif, 13 internal repeats, and two BRCT
motifs. B, alignment of 13 internal repeats shows high
homology of the 41 amino acid repeats. C,
[35S]methionine-labeled cell extracts were used for
immunoprecipitation (IP) in the presence of mouse anti-NFBD1
serum (lane 2), serum preincubated with GST protein
(lane 3), or serum preincubated with GST protein fused
antigen (lane 4). Pre-immune serum was used as a negative
control (lane 1). To remove nonspecific binding, the
immunoprecipitation products were dissociated and immunoprecipitated a
second time with mouse anti-NFBD1 serum (lane 5). Rabbit
NFBD1 anti-serum was also used for immunoprecipitation
(lane 6). Affinity-purified mouse (lane 7) and
rabbit (lane 8) anti-NFBD1 were used for
immunoblotting.
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NFBD1 Forms Nuclear Foci upon Treatment with Various DNA-damaging
Agents--
Proteins involved in DNA damage response pathway,
including 53BP1 and BRCA1, form subnuclear foci after DNA damage (42, 43, 53). To determine whether NFBD1 has similar properties, we examined
its subcellular localization by immunostaining with anti-NFBD1
antibodies. In actively growing HeLa cells, purified rabbit NFBD1
antisera but neither pre-immune nor GST-FHA antigen block demonstrated
immunoreactivity confined to nuclei (Fig.
2A). HeLa cells treated with
ionizing radiation (IR) showed distinct nuclear foci positive for
anti-NFBD1 immunoreactivity (Fig. 2B). To confirm that these
signals are attributed to NFBD1, full-length NFBD1 protein was tagged
with GFP and expressed in the same HeLa cells. GFP-NFBD1, like
endogenous NFBD1, localized exclusively in nuclei and was present in
subnuclear foci after IR (Fig. 2C). Furthermore, when HeLa
cells expressing the GFP-NFBD1 were immunostained with anti-NFBD1
antibodies, the IR-induced nuclear foci derived from GFP signals
(exogenous) colocalized with the foci detected by immunostaining with
anti-NFBD1 antibodies (endogenous) (Fig. 3D). These results suggest
that NFBD1 is present in nuclear foci upon IR.

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Fig. 2.
NFBD1 forms nuclear foci in response to
IR. A, actively growing HeLa cells were fixed and
stained with preimmune serum, purified rabbit anti-NFBD1, anti-NFBD1 in
the presence of GST protein, or GST-fused antigen (GST-FHA)
respectively. B, HeLa cells irradiated (8 Gy)
were stained with preimmune serum, purified rabbit anti-NFBD1,
anti-NFBD1 in the presence of GST protein, or GST-fused antigen
(GST-FHA) for different panels. NFBD1, NFBD1
immunofluorescence; DAPI, 4',6-diamidino-2-phenylindole
staining of cell nuclei. C, relocalization of EGFP-tagged
NFBD1 in transiently transfected cells in response to IR (8 Gy), EGFP alone as a negative control. D, transiently
transfected GFP-tagged NFBD1 (exogenous) in HeLa cells colocalizes with
NFBD1 (anti-NFBD1) after IR (8 Gy).
Yellow dots indicate the colocalization.
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Fig. 3.
NFBD1 relocalizes to nuclear foci in response
to treatments with various DNA damaging-agents. HeLa cells were
treated with -radiation (1 Gy and 8 Gy) or UV
(50 J/m2). Cells were fixed and immunostained for NFBD1
after 30 min. Cells were also treated with 0.5 µg/ml mitomycin C
(MMC), 2 µM camptothecin (CPT), 40 µg/ml etoposide (VP16), or 0.01% methylmethanesulfonate
(MMS) for 1 h, washed, and refed with fresh medium
(without drugs) and incubated for another hour, then fixed and
immunostained with rabbit anti-NFBD1.
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Next we tried to determined whether the foci that form in response to
other DNA-damaging agents contain NFBD1. Camptothecin, a DNA
topoisomerase I inhibitor, and etoposide, a topoisomerase II inhibitor,
produced single-strand breaks and double-strand breaks (DSBs),
respectively. Treatment with these two drugs induced prominent
formation of foci containing NFBD1. Similar results were also observed
in cells treated with the alkylating agent methylmethanesulfonate or
the cross-linking agent mitomycin C, which both produce synthetic
strand breaks (Fig. 3). Cells treated with UV light, which causes
pyrimidine dimers but not DSBs, showed only a few foci positive for
NFBD1 (Fig. 3). These results suggest damage-specific recruitment of
NFBD1 to subnuclear foci during cellular responses.
NFBD1 Forms Foci Very Early in Response to IR--
DNA DSBs induce
formation of nuclear foci at the sites of breaks containing several
damage response proteins. First, we characterized the kinetics of NFBD1
foci formation induced by IR in a normal human fibroblast line, IMR90.
Cells were fixed at different time points after 1 Gy of
-irradiation
and immunostained with NFBD1 antisera. NFBD1-containing foci were
clearly present within 5 min after IR (Fig.
4A). The average number of
NFBD1-positive foci per cell increased with time and peaked 30 min
after irradiation. Gradually, the number of NFBD1-positive foci
decreased to background levels within 4 h (Fig. 4B).
The percentage of cells with more than five NFBD1 foci in the entire
culture populations were also counted. The percentage of cells with
IR-induced foci reached a peak of 95% within 5 min after IR, then
gradually decreased to the background level after 4 h (Fig.
4B). These results indicate that cells, regardless of cell
cycle stage, responded to form IR-induced foci containing NFBD1. They
further suggest that NFBD1 localizes at sites of DNA breaks and
participates in the early DNA damage response.

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Fig. 4.
The kinetics of NFBD1 focus formation in
response to -radiation. A,
immunofluorescence of IMR90 human fibroblast cells exposed to 1 Gy of
radiation was examined for NFBD1 foci at the indicated time points.
B, the average number of foci per cell was plotted
versus time ( ) and the percentage of cells with more than
five foci was plotted versus time ( ). 50 cells were
counted per data point.
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Colocalization of NFBD1,
-H2AX, and Phosphorylated Chk2 at
IR-induced Foci--
-H2AX and Chk2T68P form nuclear foci at the
sites of DNA DSBs upon IR (54, 55). H2AX is a variant of H2A that
undergoes phosphorylation at serine 139.
-H2AX appears in discrete
nuclear foci at sites of DNA breaks within 1-3 min after irradiation. The kinetics of NFBD1-positive foci formation described above are
similar to those of
-H2AX (54, 56), prompting us to ask whether
NFBD1 and co-localize in IR-induced foci. Indeed, NFBD1 and
-H2AX
immunoreactivity colocalize in foci that formed within 15 min to 4 h after irradiation (Fig.
5A).

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Fig. 5.
Colocalization of NFBD1 with
-H2AX and Chk2T68P in response to
-radiation. A, HeLa cells untreated
or treated with -radiation (8 Gy) were fixed 30 min after
irradiation and costained with anti-NFBD1 (red) and
anti- -H2AX antibody (green). B, after mock
treatment or -radiation (8 Gy), cells were fixed and co-stained with
anti-NFBD1 (red) and anti-Chk2T68P (green).
Yellow dots indicate colocalization.
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In addition to
-H2AX, Chk2, a mammalian homologue of the transducing
kinase Rad53, was also examined. In response to IR, ATM rapidly
phosphorylates Chk2 at threonine 68, which activates Chk2 kinase
activity (23, 57, 58) and its relocation to sites of DNA DSBs (55). To
test whether NFBD1 also co-localizes with Chk2T68P after IR, we
immunostained irradiated cells using antibodies specific for
T68-phosphorylated Chk2. As shown in Fig. 5B,
immunoreactivity to both Chk2T68P and NFBD1 antibodies colocalized at
the IR-induced foci at all time points from 15 min to 4 h after irradiation. Collectively, these results indicate that NFBD1, like
-H2AX and Chk2T68P, is recruited to sites of DNA breaks during the
early stage of the response to DNA damage.
Localization of NFBD1 at IR-induced Foci Requires BRCT
Motifs--
NFBD1 contains three significant signature motifs. Each of
them can be important in mediating the function it plays in DNA damage
response. To determine which of the three motifs of NFBD1 was essential
for the IR-induced foci formation, we generated deletion mutants of
NFBD1 that removed them individually (Fig. 6A). The mutated proteins were
N-terminally tagged with GFP, and their expression was assayed in
transfected HeLa cells by immunoprecipitation with anti-GFP antibodies
followed by Western blotting. As shown in Fig. 6B, each of
the mutated proteins was detected in transfected HeLa cells.
Interestingly, all the mutated fusion proteins were localized in
nuclei, suggesting that none are important for nuclear localization of
NFBD1 (Fig. 6C). Removal of the C-terminal BRCT motifs, but
not the N-terminal FHA motif or the internal repeats, abolished its
localization in IR-induced foci (Fig. 6C). These results
indicate that the BRCT motif is essential for the localization of NFBD1
to IR-induced foci.

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Fig. 6.
BRCT motifs are required for NFBD1 focus
formation in response to IR. A, schematic diagram of
wild-type GFP-NFBD1 and deletion mutants. Boxes indicate the
remaining region of NFBD1. B, protein expression for
wild-type GFP-NFBD1 and GFP-NFBD1 mutants. Cell extracts prepared from
HeLa cells transfected with either wild-type or mutant NFBD1 plasmid
constructs were used for IP with anti-GFP (Roche Applied Science), and
then immunoblotted using anti-GFP. C, GFP vectors carrying
wild-type or different mutants were transiently transfected into HeLa
cells. 24 h later, cells were left untreated or treated with
-radiation (8 Gy) and fixed 30 min after mock treatment or
irradiation. Fixed cells were directly observed using fluorescent
microscopy.
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Expression of the BRCT Motif of NFBD1 Abolishes IR-induced Foci
Formation--
The major function of the BRCT motifs is to establish
protein-protein interactions. Their limited sequence homology indicates that each BRCT motif has a distinct binding partner. To determine the
effect of expression of the BRCT motifs in NFDB1 on NFBD1 subnuclear
partitioning, we generated a plasmid to express a GFP-BRCT fusion
protein that also contains a nuclear localization signal. Cells
transfected and expressing GFP-BRCT, but not GFP alone, demonstrated
significantly reduced NFBD1 immunoreactive foci (Fig. 7A), suggesting that the BRCT
motifs have a dominant-negative effect on NFBD1 localization.

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Fig. 7.
Overexpression of the BRCT motifs of NFBD1
disrupts formation of foci containing NFBD1,
-H2AX, and Chk2T68P in response to IR. Vectors
containing EGFP alone or the BRCT motifs fused with EGFP were
transiently transfected into HeLa cells. 30 min after -radiation
(8 Gy), cells were fixed and stained for NFBD1 (A
and B), -H2AX (C and D), or
Chk2T68P (E and F). GFP (green fluorescence)
signal indicates the transfected cells.
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Because NFBD1 colocalizes with
-H2AX and Chk2T68P at IR-induced foci
upon irradiation, we tested whether
-H2AX- and Chk2T68P-containing foci would be altered in cells expressing the NFBD1-derived GFP-BRCT fusion protein. We immunostained the transfected cells with
anti-
-H2AX and found that the intensity of
-H2AX staining was
similar to that of the control, indicating that the phosphorylation of
H2AX remained intact (Fig. 7, C and D).
IR-induced foci were substantially reduced, however, in cells
expressing the dominant-negative GFP-BRCT fusion protein (Fig.
7D). These results suggest that NFBD1 may have a role in
recruitment of
-H2AX to sites of DNA breaks but no obvious role in
H2AX phosphorylation at serine 139. Similarly, when we immunostained
the transfected cells with anti- Chk2T68P antibodies, IR-induced
Chk2T68P foci were diminished in the presence of GFP-BRCT (Fig.
7F), suggesting that NFBD1 is also required for recruitment
of Chk2T68P to sites of DNA breaks.
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DISCUSSION |
In a search for a mammalian homolog of scRad9, a critical adaptor
protein in DNA damage signal pathway, we characterized a novel nuclear
protein, NFBD1, the BRCT motifs of which share significant homology
with those of scRad9. NFBD1 was identified as a 250-kDa nuclear protein
with specific antibodies. Within 5 min after IR, NFBD1 was found in
nuclear foci, which also seemed to contain phosphorylated forms of H2AX
(
-H2AX) and Chk2 (Chk2T68P), two proteins known to be involved in
early DNA damage response. NFBD1 foci formation was also detected in
response to camptothecin, etoposide, and methylmethanesulfonate
treatment. Deletion of the BRCT motif of NFBD1 had significant effects
on the formation of IR-induced foci containing NFBD1. Moreover, ectopic
expression of the NFBD1-derived BRCT motif alone not only greatly
reduced IR-induced formation of foci containing NFBD1 but also reduced formation of foci containing either Chk2T68P or
-H2AX. These results
suggest that NFBD1 participates in the early stage of cellular response
to DNA damage, a functional property similar to that of scRad9.
In the DNA damage signal cascade of budding yeast, Rad9 is required for
the activation of the Chk2 homolog Rad53 (10). Phosphorylation of Rad9
by Mec1 upon DNA damage results in the formation of an epitope that
binds to the FHA motif of Rad53. Rad9 self-associates (59) and forms
oligomers to bring two Rad53 molecules into close proximity to
facilitate in trans autophosphorylation of the bound Rad53
molecules (60). Thus, Rad9 serves as an adaptor to recruit Rad53.
In mammalian cells, Chk2 activation in response to IR requires initial
phosphorylation of Thr-68 by ATM and subsequent autophosphorylation (57, 58, 61). How Thr-68 phosphorylation leads to Chk2
autophosphorylation and activation is currently unclear. Because the
DNA damage signaling pathway is highly conserved among different
eukaryotes and because ATM can only phosphorylate Chk2 but fails to
activate it in vitro (58), it is likely that an adaptor
protein for Chk2 activation exists in mammals. 53BP1 and BRCA1 were
speculated as the adaptors for Chk2 activation, but in the absence of
functional BRCA1, Chk2 nonetheless can be efficiently activated upon
IR. On the other hand, depletion of 53BP1 can partially affect the
phosphorylation of Chk2 at T68 (46). Thus, 53BP1, but not BRCA1, may
serve as an adaptor protein to facilitate Chk2 activation. Our
immunostaining results have shown that NFBD1 colocalizes with Chk2T68P
at the site of DNA breaks, and overexpression of the NFBD1-derived BRCT motif diminished the IR-induced foci positive for Chk2T68P. This finding, combined with structural clues, suggests an intriguing possibility that NFBD1 may also function as an adaptor protein, and is
required for the phosphorylation of Chk2 and its localization to DNA
break sites. However, further biochemical and genetic evidence is
required to directly support this possibility.
A known function of BRCT motifs is to enable protein-protein
interaction, and many proteins in the DNA damage response pathways contain them (30, 31). However, the sequence homology among BRCT motifs
is limited, although they form a similar three-dimensional structure
(62-65). We have shown that the BRCT motif of NFBD1 is required for
its localization to the site of DNA breaks and that overexpression of
this specific motif abolished IR-induced foci containing NFBD1 and
Chk2T68P. These results suggest that the BRCT motif of NFBD1 has a
dominant-negative effect on NFBD1 function. This notion is consistent
with the potential role of NFBD1 as an adaptor protein.
Several examples of protein-protein interactions important for DNA
damage responses are known. Interaction between BRCA1 and CtIP is
mediated by BRCT motifs (66-68) and has been shown to play an
important role in regulating Gadd45, a critical G2/M
checkpoint protein (69). In addition, the BRCT motifs of 53BP1 interact with p53 (64, 65, 70), a key player in both G1/S and
G2/M checkpoint response, as well as in DNA damage-induced
apoptosis (71). However, our preliminary results show that the
expression of the BRCT motif of BRCA1 or 53BP1 has no influence on
NFBD1 foci formation (data not shown), suggesting that the BRCT motif of NFBD1 has a unique role independent of BRCA1 or 53BP1 in the DNA
damage response signaling pathway.
Phosphorylation of histone H2AX on serine 139 by ATM has been observed
in response to DNA double-strand breaks (72) and by ATR in response to
DNA replicative stress (73).
-H2AX is found in subnuclear foci,
which colocalize with BRCA1 and 53BP1 at the arrested replication fork
in S-phase cells (43, 65). In the absence of histone H2AX, cells
display increased sensitivity to ionizing radiation and increased
genomic instability (74). These observations suggest a critical role
for H2AX in the mammalian DNA damage response. The effects of
NFBD1-derived BRCT motifs we observed on the IR-induced foci formation
of
-H2AX suggest that functional NFBD1 may play a role in the
recruitment of
-H2AX to sites of DNA breaks but very little role in
the phosphorylation of H2AX at serine 139. Although further
confirmation is needed, our data suggest that NFBD1 may play little
role in sensing DNA damage.
The results presented in this study suggest strongly that NFBD1 may
serve as an adaptor protein in the DNA damage response pathway in
mammalian cells. Because mutation of either BRCA1 (75) or Chk2 accounts
for some hereditary cases of breast cancer (76), mutation of NFBD1 may
also be a potential contributing factor to the formation of breast or
other cancers. Further elucidation of the precise role(s) that NFBD1
plays in DNA damage response and in tumor formation will address this
intriguing possibility.
We are grateful to Dr. Takahiro Nagase at
Kazusa DNA Research Institute for providing NFBD1
(KIAA0170) cDNA. We thank Paula Garza and Diane Jones for antibody
preparation, Guikai Wu and Aimin Peng for helpful discussion, Nicholas
Ting, Dave Sharp, and Daniel Riley for critically reading the
manuscript, and Wen-Hwa Lee for his encouragement and support.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M210749200
The abbreviations used are:
ATM, ataxia
telangiectasia mutated;
ATR, ataxia telangiectasia and Rad3-related;
FHA, forkhead-associated;
NFBD1, nuclear factor with BRCT domains
protein 1;
EGFP, enhanced green fluorescent protein;
Chk2T68P, Chk2
phosphorylated at threonine 68;
-H2AX, H2AX phosphorylated at serine
139;
GFP, green fluorescence protein;
GST, glutathione
S-transferase;
IR, ionizing radiation;
DSB, double-strand
break;
Gy, gray;
53BP1, tumor suppressor p53-binding protein 1.
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