Content uploaded by Hongtae Kim
Author content
All content in this area was uploaded by Hongtae Kim on Jun 24, 2014
Content may be subject to copyright.
CCDC98 is a BRCA1-BRCT domain–binding protein
involved in the DNA damage response
Hongtae Kim
1,2
, Jun Huang
1,2
& Junjie Chen
1
The product of the breast cancer-1 gene, BRCA1, plays a crucial part in the DNA damage response through its interactions with
many proteins, including BACH1, CtIP and RAP80. Here we identify a coiled-coil domain–containing protein, CCDC98, as a
BRCA1-interacting protein. CCDC98 colocalizes with BRCA1 and is required for the formation of BRCA1 foci in response to
ionizing radiation. Moreover, like BRCA1, CCDC98 has a role in radiation sensitivity and damage-induced G2/M checkpoint control.
Together, these results suggest that CCDC98 is a mediator of BRCA1 function involved in the mammalian DNA damage response.
To survive and maintain their genomic integrity, cells are equipped
with the ability to sense and respond to DNA damage1,2.The
importance of this surveillance system has been demonstrated by
the finding that inactivation of the DNA damage response can lead to
cancer-susceptibility syndromes and neoplastic transformation. Many
proteins, including the protein kinase ataxia-telangiectasia mutated
(ATM), phosphorylated histone H2AX (gH2AX) and mediator of
DNA damage checkpoint-1 (MDC1), are involved in sensing, trans-
ducing and responding to DNA damage signals3. The product of
BRCA1 is also a checkpoint mediator, and its BRCT domains function
in this process by interacting with phosphoserine or phosphothreo-
nine motifs4–6. Previous studies have shown that the BRCA1-BRCT
domains are important for BRCA1’s functions in tumor suppression7
and the DNA damage response8–10. In the presence of DNA lesions,
BRCA1 participates in many DNA damage response pathways, includ-
ing cell-cycle checkpoints during S phase and at the G2/M transition,
and DNA repair via homologous recombination8–11. Defects in these
checkpoints and DNA repair may underlie the increased tumori-
genesis observed in patients with BRCA1 mutations.
Although BRCA1 is known to be recruited to DNA breaks and to
participate in checkpoint regulation, it is not yet clear how the
recruitment of BRCA1 is controlled in the cell. To gain further insights
into the regulation of BRCA1 upon DNA damage, we sought to
identify previously unknown BRCA1-BRCT domain–binding proteins
using a tandem affinity-purification approach. Here we report that
human CCDC98 protein associates with BRCA1 and demonstrate
that CCDC98 acts upstream of BRCA1 and regulates the BRCA1-
dependent DNA damage signaling pathway.
RESULTS
CCDC98 is a BRCA1-associated protein
To identify additional BRCA1-associated proteins, we purified BRCA1-
BRCT domain–containing complexes from human embryonic
kidney 293T cells stably expressing a BRCA1-BRCT domain with
an N-terminal triple tag comprising an S tag, a Flag epitope and a
streptavidin-binding peptide (SFB–BRCA1-BRCT). Mass spectro-
metry revealed a number of known BRCA1-associated proteins,
including BRCA1-associated C-terminal helicase (BACH1), CtBP-
interacting protein (CtIP) and receptor associated protein-80
(RAP80)6,12–15. In the same experiment, we also identified several
putative BRCA1-associated proteins (Supplementary Table 1 online).
Among these, we paid special attention to a coiled-coil domain–
containing protein, CCDC98. This protein contains an SPTF motif at
its extreme C terminus; an identical sequence in BACH1 is required
for interaction of BACH1 with BRCA1-BRCT domains6. The physio-
logical function of CCDC98 is unknown. Notably, we also identified
CCDC98 as a RAP80-associated protein in a tandem affinity purifica-
tion of RAP80-containing complexes (Supplementary Table 2
online), confirming that CCDC98 and RAP80 interact. As both
CCDC98 and RAP80 exist in BRCA1-containing complexes (Supple-
mentary Tables 1 and 2), we speculated that these three proteins
might form a complex.
BRCA1 specifically binds the SPTF motif of CCDC98
We confirmed the association of CCDC98 with BRCA1 and RAP80
using coimmunoprecipitation experiments (Fig. 1a). In addition,
bacterially expressed glutathione S-transferase (GST)-tagged BRCA1-
BRCT domain and GST-RAP80 pulled down CCDC98 from cell
extracts (Fig. 1b), again confirming that CCDC98 interacts with
both BRCA1 and RAP80. Notably, although CCDC98 interacted with
the BRCA1-BRCT domain in a phosphorylation-dependent manner,
its associationwith RAP80 was phosphorylation independent (Fig. 1b).
Prompted by this phosphorylation-dependent interaction between
BRCA1-BRCT and CCDC98, we examined whether the C-terminal
SPTF motif of CCDC98 is required for its interaction with BRCA1-
BRCT. GST–BRCA1-BRCT specifically bound wild-type CCDC98 and
Received 23 April; accepted 26 June; published online 22 July 2007; doi:10.1038/nsmb1277
1
Department of Therapeutic Radiology, Yale University School of Medicine, P.O. Box 208040, New Haven, Connecticut 06520, USA.
2
These authors contributed
equally to this work. Correspondence should be addressed to J.C. (junjie.chen@yale.edu).
710 VOLUME 14 NUMBER 8 A UGUST 2007 NATURE STRUCTURAL & MOLECULAR BIOLOGY
ARTICLES
©2007 Nature Publishing Group http://www.nature.com/nsmb
did not bind CCDC98 lacking the C-terminal SPTF sequence (CCDC
98DSPTF; Fig. 1c). We also generated several point mutations in the
SPTF motif of CCDC98. Whereas GST–BRCA1-BRCT specifically
pulled down wild-type CCDC98, its affinities for the CCDC98 point
mutants were greatly diminished (Fig. 1d). Using a phosphospecific
antibody against the Ser406 residue in the SPTF motif, we confirmed
that this serine residue is indeed phosphorylated in vivo (Fig. 1e). This
phosphorylation and the BRCA1-CCDC98 interaction did not
change after DNA damage (data not shown). Only wild-type
BRCA1, and not a BRCA1 variant lacking the BRCT regions
(BRCA1DBRCT), associated with CCDC98 in vivo (Fig. 1f). Together,
these data suggest that CCDC98 binds BRCA1 in a phosphorylation-
dependent manner through an interaction between BRCA1-BRCTand
the C-terminal SPTF motif of CCDC98.
Figure 1 Identification of CCDC98 as a BRCA1-
binding protein. (a) The interaction between
endogenous CCDC98 and BRCA1 or RAP80.
Immunoprecipitation (IP) reactions were
done using preimmune serum (prebleed) or
anti-CCDC98. Western blotting analyses (W)
were done with indicated antibodies.
(b) Phosphorylation-dependent interaction
between BRCA1-BRCT and CCDC98. GST,
GST–BRCA1-BRCT or GST-RAP80 was incubated
with cell lysates containing exogenously
expressed Flag-tagged wild-type CCDC98, with
or without phosphatase. Bound CCDC98 was
analyzed by anti-Flag immunoblotting. Lower gel
shows amounts of proteins used in these
experiments. (c,d) Beads with GST–BRCA1-BRCT
were incubated with cell lysates containing
exogenously expressed SFB-tagged wild-type
CCDC98, CCDC98DSPTF or SPTF point mutants
with the C-terminal sequences indicated in their
names (CCDC98APTF, CCDC98SATF,
CCDC98SPAF and CCDC98SPTA). Bound
CCDC98 proteins were analyzed by anti-Flag
immunoblotting. (e) CCDC98 is phosphorylated
at Ser406. IP reactions using anti-CCDC98 were
followed by mock or phosphatase treatment. Western blotting was done with indicated antibodies. (f) 293T cells were transfected with plasmid encoding
Myc-BRCA1 or Myc-BRCA1DBRCT and with plasmid encoding SFB-CCDC98. Cell lysates were subjected to immunoprecipitation and immunoblotting with
indicated antibodies (upper blots). Lower blot shows amounts of SFB-tagged CCDC98 in lysates.
a
W : Anti-BRCA1
W : Anti-Flag
(CCDC98)
No PPase
GST-RAP80
GST–BRCA1
BRCT
GST
PPase
treatment
Prebleed
Anti-CCDC98
10% input
GST
GST
GST
–BRCA1-BRCT
GST-RAP80
IP
W : Anti-RAP80
W : Anti-Flag
(CCDC98)
W : Anti-Flag
(CCDC98)
(Cell lysates)
W : Anti-Flag
(CCDC98)
W : Anti-Flag
(CCDC98)
(Cell lysates)
SFB-CCDC98
IP : Anti-Myc
W : Anti-Flag
(CCDC98)
W : Anti-Flag
(CCDC98)
(
Cell l
y
sates
)
W : Anti-Myc
(BRCA1)
W : Anti-CCDC98
c
d
b
SFB-CCDC98
SFB-CCDC98
∆SPTF
Myc-BRCA1∆
BRCT
Pull-down :
GST–BRCA1-BRCT
Pull-down :
GST–BRCA1-BRCT
SFB-CCDC98
SFB-CCDC98APTF
SFB-CCDC98SATF
SFB-CCDC98SPAF
SFB-CCDC98SPTA
IP : Anti-CCDC98
PPase :
W : Anti-pS406
W : Anti-CCDC98
–+
fe
Myc-BRCA1
c
d
CCDC98 DAPI
γH2AX
ab
CCDC98
0 Gy
10 Gy
DAPI
γH2AX CCDC98 DAPIBRCA1
CCDC98 siRNA
BRCA1 DAPI
γH2AX
RAP80 DAPI
γH2AX
CCDC98 DAPI
γH2AX
Control siRNA
BRCA1 DAPI
γH2AX
RAP80 DAPI
γH2AX
CCDC98 DAPI
γH2AX
RAP80 siRNA
BRCA1 DAPI
γH2AX
RAP80 DAPI
γH2AX
CCDC98 DAPI
γH2AX
BRCA1 siRNA
BRCA1 DAPI
γH2AX
RAP80
Control siRNA
BRCA1 siRNA
RAP80 siRNA
CCDC98 siRNA
DAPI
γH2AX
W : Anti-BRCA1
W : Anti-RAP80
W : Anti-CCDC98
W : Anti–β-actin
Figure 2 Localization of CCDC98 in cells exposed to ionizing
radiation. (a,b) DNA damage–induced RAP80 focus formation
and colocalization with gH2AX (a) and BRCA1 (b). Mock-
treated or irradiated 293T cells were fixed and stained with
monoclonal antibody to gH2AX or BRCA1, or polyclonal
antibody to CCDC98. (c) Requirement of CCDC98 for
damage-induced BRCA1 focus formation. U2OS cells were
transfected with indicated siRNAs, exposed to ionizing
radiation (10 Gy) and immunostained with monoclonal
antibody to gH2AX or polyclonal antibody to CCDC98,
BRCA1 or RAP80. (d) Western blotting analysis (W) of
BRCA1, RAP80 and CCDC98 expression levels in cells
transfected with indicated siRNAs.
ARTICLES
NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 14 NUMBER 8 AUGUST 2007 711
©2007 Nature Publishing Group http://www.nature.com/nsmb
CCDC98 and BRCA1 form foci after DNA damage
As BRCA1 localizes to sites of DNA breaks in cells exposed to ionizing
radiation, we checked the localization of CCDC98 before and after
DNA damage. Using an antibody to CCDC98, we found the protein to
be evenly distributed in the nucleoplasm of normal cells (Fig. 2a).
After cells were exposed to ionizing radiation, CCDC98 localized to
DNA damage–induced foci and colocalized with gH2AX (a marker of
DNA damage) and BRCA1 (Fig. 2a,b). This indicates that the
localization of CCDC98, like that of BRCA1, is regulated in response
to DNA damage. Notably, we discovered that BRCA1 did not accu-
mulate at DNA breaks in cells where CCDC98 messenger RNA was
depleted using short interfering RNA
(siRNA); however, the localization of RAP80
to damage sites was normal in these cells
(Fig. 2c). Moreover, formation of both
BRCA1 and CCDC98 foci was abolished
in RAP80-depleted cells, but formation of
CCDC98 and RAP80 foci was normal in
BRCA1-depleted cells (Fig. 2c). As a control,
we showed that the expression level of BRCA1
is the same with or without CCDC98 knock-
down (Fig. 2d). In addition, RAP80 knock-
down also does not change the expression of
CCDC98 or BRCA1 (Fig. 2d). Collectively,
these data suggest that CCDC98 acts down-
stream of RAP80 but upstream of BRCA1 in
the DNA damage response pathway.
CCDC98 focus formation depends on its
N terminus
Our results suggested that CCDC98 forms a
complex with RAP80 and BRCA1 and loca-
lizes to sites of damaged DNA. Next, we
attempted to determine which regions of
CCDC98 are important for its localiza-
tion to foci. Full-length CCDC98 and
CCDC98DSPTF mutant localized normally
to nuclear foci in cells with DNA damage, whereas all of the other
CCDC98 deletion mutants we tested did not (Fig. 3a). All three
N-terminal and internal deletion mutants of CCDC98 also did
not bind RAP80 (Fig. 3b), whereas CCDC98DSPTF and a
CCDC98 mutant with a large C-terminal deletion (CCDC98D4)
were defective in BRCA1 binding (Fig. 3c). Because it localizes
to the cytoplasm, it is difficult to interpret the results obtained
with the CCDC98D4 mutant (two putative nuclear localization
sequences, 358-Lys-Arg-Ser-Arg-361 and 368-Lys-Arg-Ser-Lys-371,
are deleted in this mutant). Nevertheless, these data suggest that
CCDC98 mediates the interaction between BRCA1 and RAP80
CCDC98WT
CCDC98D1
CCDC98D1
CCDC98D2
CCDC98D2
CCDC98D3
CCDC98D3
CCDC98D4
CCDC98D4CCDC98WT
DAPI
Pull-down : GST-RAP80 Pull–down : GST–BRCA1-BRCT
Flag
(CCDC98)
γH2AX
CCDC98∆SPTF
CCDC98∆
SPTF
1
100
101 200
201
250
300
405
409
Focus
formation
BRCA1
binding
RAP80
binding
+
+
?
–
–
–
+
+
+
+
–
–
+
+
+
–
–
–
SFB-CCDC98D4
SFB-CCDC98
SFB-CCDC98D2
SFB-CCDC98D3
SFB-CCDC98D1
SFB-CCDC98∆
SPTF
SFB-CCDC98D4
SFB-CCDC98
SFB-CCDC98D2
SFB-CCDC98D3
SFB-CCDC98D1
SFB-CCDC98∆
SPTF
SFB-RAP80WT
SFB-RAP80D1
SFB-RAP80D2
SFB-RAP80D3
SFB-RAP80D4
SFB-RAP80D5
SFB-RAP80D6
SFB-RAP80WT
SFB-RAP80D1
SFB-RAP80D2
SFB-RAP80D3
SFB-RAP80D4
SFB-RAP80D5
SFB-RAP80D6
a
bc
W : Anti-Flag
(CCDC98)
W : Anti-Flag
(CCDC98)
(Cell lysates)
W : Anti-Flag
(CCDC98)
W : Anti-Flag
(CCDC98)
(Cell lysates)
dRAP80WT UIM UIM ZF ZF
719
CCDC9
8
binding
RAP80D1
RAP80D2
RAP80D3
RAP80D4
RAP80D5
RAP80D6
1
+
+
+
–
+
+
+
71
93
127
200
235 337
336 491
470 583
582
IP :
Anti-Flag
W : Anti-Myc
(CCDC98)
W : Anti-Flag
(RAP80)
W : Anti-Myc
(CCDC98)
W : Anti-Flag
(RAP80)
Whole-cell
lysates
Whole–cell
lysates
W : Anti-Flag
(RAP80)
W : Anti-Myc
(CCDC98)
W : Anti-Flag
(RAP80)
W : Anti-Myc
(
CCDC98
)
IP :
Anti-Myc
Figure 3 Focus localization of CCDC98 depends
on its N-terminal RAP80-binding region.
(a) 293T cells were transfected with SFB-tagged
wild-type (WT) CCDC98 or deletion mutants
shown in diagram. After 24 h, cells were exposed
to 10 Gy of ionizing radiation. Eight hours after
irradiation, cells were fixed and stained with
monoclonal anti-Flag or polyclonal anti-gH2AX.
(b,c) Mapping of the RAP80- and BRCA1-
interacting domains in CCDC98. Beads coated
with GST-RAP80 (b) or GST–BRCA1-BRCT (c)
were incubated with cell lysates containing
exogenously expressed SFB-tagged WT CCDC98
or deletion mutants. After extensive washing,
bound RAP80 was analyzed by western
blotting (W) with anti-Flag. (d) SFB-tagged WT
RAP80 and its internal deletion mutants were
used to map the CCDC98-interacting domain in
RAP80. 293T cells were transfected with
plasmids encoding Myc-CCDC98 and the
indicated SFB-RAP80 proteins. Cell lysates were
subjected to immunoprecipitation (IP) and
immunoblotting with indicated antibodies
(top blots). Bottom blots show amounts of
SFB-RAP80 and Myc-CCDC98 in these lysates.
ARTICLES
712 VOLUME 14 NUMBER 8 A UGUST 2007 NATURE STRUCTURAL & MOLECULAR BIOLOGY
©2007 Nature Publishing Group http://www.nature.com/nsmb
and that the localization of CCDC98 to foci depends on its interaction
with RAP80.
We confirmed a strong interaction between CCDC98 and RAP80
using a baculovirus-insect cell system (Supplementar y Fig. 1 online).
Using a series of deletion mutants of RAP80, we identified a region
(residues 235–337) on the C-terminal side of the ubiquitin-interacting
motifs (UIMs) that is required for its interaction with CCDC98
(Fig. 3d). The same region of RAP80 is also important for its
association with BRCA1 in vivo (Supplementary Fig. 2 online), an
observation which agrees with our proposal that CCDC98 bridges the
interaction between RAP80 and BRCA1.
CCDC98 is required in the G2/M checkpoint
The loss of BRCA1 leads to defects in the DNA damage response—in
particular, impaired cell-cycle checkpoints and increased sensitivity to
DNA damaging agents16,17. We therefore examined whether the loss
of CCDC98 results in similar defects in the DNA damage response.
Both CCDC98 siRNAs we synthesized efficiently decreased CCDC98
expression in cells (Fig. 4a). Cells treated with these siRNAs showed
defective G2/M checkpoint control after DNA damage (Fig. 4b and
Supplementary Fig. 3 online). The protein kinase CHK1 acts down-
stream of BRCA1 and is required for this G2/M checkpoint control
in response to ionizing radiation18–21. If CCDC98 functions upstream
of BRCA1, a defect in CHK1 activation is expected in cells depleted
of CCDC98. This is indeed the case (Fig. 4c). CCDC98 knockdown
cells were also more sensitive to radiation than cells transfected
with control siRNA (Fig. 4d). These data indicate that CCDC98 is a
key upstream regulator that influences BRCA1 function upon DNA
damage (Fig. 4e).
DISCUSSION
In this study, we identified CCDC98 as a BRCA1-BRCT–binding
protein. Like BRCA1, CCDC98 normally exists in the nucleoplasm but
localizes to DNA breaks after exposure to ionizing radiation. CCDC98
also participates in the BRCA1-dependent G2/M checkpoint control,
suggesting that CCDC98 functions together with BRCA1 in the DNA
damage response.
Besides CCDC98, the ubiquitin-interacting protein RAP80 was also
identified as a BRCA1-associated protein in our biochemical purifica-
tion of BRCA1-containing complexes. Studies from several groups,
including ours, have demonstrated that RAP80 acts upstream of
BRCA1 and regulates BRCA1 localization and function after DNA
damage13–15. Moreover, another group has also identified CCDC98
(called Abraxas in their study) as a BRCA1-interacting protein15.
Similar to our current study, they also showed that CCDC98/Abraxas
interacts with BRCA1 in a phosphorylation-dependent manner via its
C-terminal SPTF motif15. Here, we have expanded on our initial
observations and demonstrated a hierarchy in this DNA damage signal
transduction pathway. We show that although RAP80 is required for
formation of both CCDC98 and BRCA1 foci, CCDC98 is required for
formation of only BRCA1 and not RAP80 foci. Moreover, abolishing
BRCA1 does not affect either RAP80 or CCDC98 focus formation
after DNA damage. Thus, we are able to delineate a signaling pathway
from RAP80 to CCDC98 and then to BRCA1 (Fig. 4e).
Our study also permits a better understanding of CCDC98’s activity
as a mediator in this process. We show that the N terminus of
CCDC98 is required for RAP80 binding, and its C-terminal phos-
phorylation motif is required for BRCA1 binding. In agreement with
the notion that CCDC98 functions downstream of RAP80, only the
N-terminal RAP80-binding domain of CCDC98 is important for its
localization to foci after DNA damage. Putting all these studies
together, we now have a better understanding of the mechanisms
underlying the recruitment of BRCA1 to damaged DNA. RAP80
binds directly to the N terminus of CCDC98. This interaction is not
phosphorylation dependent, but rather allows formation of a
stable complex between RAP80 and CCDC98. After DNA damage,
the RAP80–CCDC98 complex localizes to damage sites. RAP80’s
localization to foci depends on its UIM domain, which probably
binds unidentified ubiquitinated proteins at DNA breaks. Through
its C-terminal phosphorylation motif, CCDC98 then recruits
BRCA1 to the DNA damage sites and regulates BRCA1-dependent
checkpoint control.
ab
c
e
d
W : Anti–β-actin
W : Anti–β-actin
W : Anti-CCDC98
Control
siRNA
Control
siRNA
CCDC98
siRNA2
CCDC98
siRNA2
BRCA1
siRNA
RAP80
siRNA
CCDC98
siRNA1
CCDC98
siRNA1
CCDC98
siRNA1
3.0
2.5
2.0
1.5
Percentage of mitotic cells
1.0
0.5
0
0 Gy
2 Gy
CCDC98 siRNA
Control siRNA
100
80
60
Surviving colonies (%)
40
20
0
0
12345
Radiation (Gy)
Control
siRNA
10 Gy :
W : Anti-pCHK1
W : Anti-CHK1
W : Anti-CCDC98
––++
IR
ATM
pH2AX/MDC1
E3 ligase ?
XUb
RAP80
CCDC98
BRCA1
Y ?
DNA
re
p
air?
Damage
checkpoint
Ub
Figure 4 Requirement of CCDC98 for ionizing radiation–induced DNA
damage response. (a) Western blotting analysis (W) of CCDC98 expression
in cells transfected with indicated siRNAs. (b) G2/M checkpoint control in
CCDC98 knockdown cells. HeLa cells transfected with indicated siRNAs
were exposed to 0 or 2 Gy of ionizing radiation. Cells were fixed and stained
with histone-specific anti-pH3 (a mitotic marker) and propidium iodide.
Percentages of mitotic cells were determined by FACS analysis. Data shown
are averages of three independent experiments; error bars indicate s.d.
(c) Requirement of CCDC98 for CHK1 phosphorylation after DNA damage.
Control or CCDC98 siRNA–transfected HeLa cells were exposed to 0 or
10 Gy of ionizing radiation, harvested 2 h later and immunoblotted with
indicated antibodies. (d) Radiation sensitivity of cells lacking CCDC98. HeLa
cells were transfected with control or CCDC98 siRNAs. Cells were irradiated
with indicated doses of ionizing radiation. Percentage of colonies surviving
was determined 10–12 d later. Experiments were done in triplicate; results
shown are averages of two or three independent experiments at each dose;
error bars indicate s.d. (e) Model of the DNA damage response pathway that
integrates CCDC98. Our data indicate that CCDC98 operates upstream of
BRCA1 and specifically regulates BRCA1 localization and function after
DNA damage.
ARTICLES
NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 14 NUMBER 8 AUGUST 2007 713
©2007 Nature Publishing Group http://www.nature.com/nsmb
The factors that act upstream of the RAP80–CCDC98 complex and
recruit it to sites of DNA damage remain elusive. What we do know is
that both gH2AX and MDC1 are upstream regulators and are required
for the focus formation of many checkpoint proteins, including
RAP80 and BRCA1. Because the localization of RAP80 seems to
depend on the ability of its UIM domain to bind polyubiquitinated
proteins, we speculate that there is at least one E3 ubiquitin ligase
involved in this process. This unidentified E3 ligase may act after
gH2AX and MDC1 to facilitate protein ubiquitination at sites of DNA
damage, which would in turn serve as a signal to recruit RAP80–
CCDC98 and BRCA1. The identification of this E3 ligase and its
substrates at DNA breaks would provide further insight into the
complex regulation of DNA damage response pathways.
Although RAP80 and CCDC98 seem to function upstream of
BRCA1 in the DNA damage signal transduction pathway, it is
noteworthy that checkpoint defects observed in RAP80-or
CCDC98-deficient cells are not as severe as those observed in cells
with a BRCA1 deficiency. One likely explanation is that there are
proteins other than RAP80 and CCDC98 that also participate in
regulating BRCA1 function after DNA damage. We hope that future
studies will identify this parallel pathway, revealing exactly how the
tumor suppressor BRCA1 is regulated after DNA damage and con-
tributes to the maintenance of genomic stability.
METHODS
Plasmids. Human CCDC98 full-length complementary DNA was obtained
using reverse-transcription PCR. Wild-type human CCDC98 and its point
mutants and deletion mutants were generated by PCR and subcloned into a
modified pIRES-EGFP mammalian expression vector to create constructs
encoding SFB-tagged wild-type or mutant CCDC98. DNA fragments encoding
BRCA1-BRCT domain and RAP80 were also generated by PCR and subcloned
into pGEX-4T-1 vector (Pharmacia) to make constructs for expression of GST–
BRCA1-BRCTand GST-RAP80, respectively. Myc-BRCA1, Myc-BRCA1DBRCT,
and full-length human RAP80 and its deletion mutants were described13.
Cell culture and treatment with ionizing radiation. HeLa, U2OS and 293T
cells were purchased from the American Type Culture Collection and main-
tained in RPMI 1,640 medium supplemented with 10% (v/v) FBS at 37 1C
in 5% CO
2
. Cells were irradiated at the indicated doses using a JL Shepherd
137
Cs radiation source. The irradiated cells were then returned to the same
culture conditions and maintained for the periods of time specified in the
figure legends.
Short interfering RNA. All siRNA duplexes used in this study were purchased
from Dharmacon. The sequences of RAP80 siRNA, CCDC98 siRNA 1, CCDC98
siRNA 2, BRCA1 siRNA and the control siRNA are 5¢-GAAGGAUGUGGAAA
CUACCdTdT-3¢,5¢-CAGGGUACCUUUAGUGGUUUU-3¢,5¢-ACACAAGA
CAAACGAUCUAUU-3¢and 5¢-UCACAGUGUCCUUUAUGUAdTdT-3¢and
5¢-UUCAAUAAAUUCUUGAGGUUU-3¢, respectively. siRNAs were transfected
into the cells using Oligofectamine (Invitrogen) according to the manufac-
turer’s instructions.
Antibodies, transfection and immunoprecipitation procedures. Rabbit anti-
bodies to BRCA1, CCDC98 and RAP80 were raised by immunizing rabbits
with GST-BRCA1 fragments, GST-CCDC98 and GST-RAP80 (residues 1–354)
respectively. Phosphospecific antibody to Ser406 was generated by immunizing
rabbits with KLH-conjugated GFGEYSR-pS-PTF peptide. The resulting rabbit
polyclonal sera were affinity-purified using the SulfoLink or AminoLink Plus
Immobilization and Purification Kit (Pierce). gH2AX antibodies were
described22.AntibodiestoFlagandb-actin were purchased from Sigma.
Antibody to phosphorylated histone H3 (pH3) was purchased from Upstate
Biotechnology. Transient transfection was done using Fugene 6 transfection
reagent (Roche) according to the manufacturer’s instructions. For immuno-
precipitation, cells were washed with ice-cold PBS and then lysed in NETN
buffer (0.5% (v/v) Nonidet P-40, 20 mM Tris (pH 8.0), 50 mM NaCl, 50 mM
NaF, 100 mMNa
3
VO
4
, 1 mM DTT and 50 mgml
–1
PMSF) at 4 1C for 10 min.
Crude lysates were cleared by centrifugation at 14,000 r.p.m. (Micro 240A,
Scientific) and 4 1C for 5 min, and supernatants were incubated with protein
A–agarose–conjugated primary antibodies. The immunocomplexes were
washed three times with NETN buffer and then subjected to SDS-PAGE.
Western blotting was done using the antibodies specified in the figures.
Cell lines and affinity purification of SFB-tagged protein complexes. To
establish cell lines stably expressing various epitope-tagged proteins, 293T cells
were transfected with plasmids encoding SFB-tagged proteins and pGK-Puro.
Forty-eight hours after transfection, the cells were split at a 1:10 ratio and
cultured in medium containing puromycin (10 mgml
–1
) for 3 weeks. The
individual puromycin-resistant colonies were isolated and screened by western
blotting. 293T cells stably expressing tagged proteins were lysed with 4 ml
NETN buffer on ice for 10 min. Crude lysates were cleared by centrifugation at
14,000 r.p.m. (Micro 240A, Scientific) at 4 1C for 10 min, and supernatants
were incubated with 300 ml streptavidin-conjugated beads (Amersham). The
immunocomplexes were washed three times with NETN buffer and then bead-
bound proteins were eluted with 500 ml NETN buffer containing 2 mg ml
–1
biotin (Sigma). The eluted supernatant was incubated with 60 ml S protein
beads (Novagen). The immunocomplexes were washed three times with NETN
buffer and subjected to SDS-PAGE. Protein bands were visualized by silver
staining, excised and digested, and the peptides were analyzed by mass
spectrometry.
Glutathione S-transferase pull-down assay. GST fusion protein was expressed
in Escherichia coli and purified as described23. GST fusion protein or GSTalone
(2 mg) was immobilized on glutathione-Sepharose 4B beads and incubated for
2 h at 4 1C with lysates prepared from cells transiently transfected with
plasmids encoding the indicated proteins. After washing with NETN buffer,
the samples were separated by SDS-PAGE and analyzed by western blotting.
Immunofluorescent staining. Cells grown on coverslips were fixed with
3% (w/v) paraformaldehyde at room temperature for 15 min and then
permeabilized with PBS containing 0.5% (v/v) Triton X-100 at room tempera-
ture for 5 min. The coverslips were blocked with PBS containing 5% (v/v) goat
serum at room temperature for 30 min before incubation with primary
antibodies at room temperature for 20 min. After washing with PBS, cells
were incubated with the secondary antibodies fluorescein isothiocyanate–
conjugated goat anti-mouse IgG, rhodamine-conjugated goat anti-rabbit IgG
or rhodamine-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) at
room temperature for 20 min. Nuclei were counterstained with 4,6-diamidino-
2-phenylindole (DAPI). After a final wash with PBS, coverslips were mounted
with glycerin containing p-phenylenediamine. All images were obtained with a
Nikon ECLIPSE E800 fluorescence microscope.
G2/M cell-cycle checkpoint assay. HeLa cells in a 100-mm plate were
transfected twice with control or CCDC98 siRNAs at 24-h intervals. Forty-
eight hours after the second transfection, transfected cells were mock-treated or
irradiated at indicated doses using a JL Shepherd
137
Cs radiation source. One
hour after irradiation, cells were fixed with 70% (v/v) ethanol at –20 1Cfor
24 h, then stained with rabbit antibody to pH3 and incubated with fluorescein
isothiocyanate–conjugated goat secondary antibody to rabbit immunoglobulin.
The stained cells were treated with RNase A, incubated with propidium iodide
and then analyzed by flow cytometry.
Cell survival assays. HeLa cells in a 60-mm plate were transfected twice with
control or CCDC98 siRNAs at 24-h intervals. Forty-eight hours after the second
transfection, transfected cells were irradiated at the indicated doses using a JL
Shepherd
137
Cs radiation source. Ten to twelve days after irradiation, cells were
washed with PBS, fixed and stained with 2% (w/v) methylene blue, and the
colonies were counted.
Note: Supplementary information is available on the Nature Structural &Molecular
Biology website.
ACKNOWLEDGMENTS
We thank members of the Chen laboratory for helpful discussions and technical
support. This work was supported in part by grants from the US National
ARTICLES
714 VOLUME 14 NUMBER 8 A UGUST 2007 NATURE STRUCTURAL & MOLECULAR BIOLOGY
©2007 Nature Publishing Group http://www.nature.com/nsmb
Institute of Health (RO1CA089239 to J.C.) and the US Department of Defense
breast cancer Era of Hope Scholar Award (W81XWH-05-1-0470 to J.C.).
AUTHOR CONTRIBUTIONS
H.K., J.H. and J.C. designed experiments and interpreted the data; H.K. and J.H.
performed all experiments; H.K. and J.C. prepared the manuscript.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/nsmb/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
1. Zhou, B.B. & Elledge, S.J. The DNA damage response: putting checkpoints in
perspective. Nature 408, 433–439 (2000).
2. Rouse, J. & Jackson, S.P. Interfaces between the detection, signaling, and repair of
DNA damage. Science 297, 547–551 (2002).
3. Su, T.T. Cellular responses to DNA damage: one signal, multiple choices. Annu. Rev.
Genet. 40, 187–208 (2006).
4. Manke, I.A., Lowery, D.M., Nguyen, A. & Yaffe, M.B.BRCT repeats as phosphopeptide-
binding modules involved in protein targeting. Science 302, 636–639 (2003).
5. Yu, X., Chini, C.C., He, M., Mer, G. & Chen, J. The BRCT domain is a phospho-protein
binding domain. Science 302, 639–642 (2003).
6. Yu, X. & Chen, J. DNA damage-induced cell cycle checkpoint control requires CtIP, a
phosphorylation-dependent binding partner of BRCA1 C-terminal domains. Mol. Cell.
Biol. 24, 9478–9486 (2004).
7. Ludwig, T., Fisher, P., Ganesan, S. & Efstratiadis, A. Tumorigenesis in mice carrying a
truncating Brca1 mutation. Genes Dev. 15, 1188–1193 (2001).
8. Scully, R. & Livingston, D.M. In search of the tumour-suppressor functions of BRCA1
and BRCA2. Nature 408, 429–432 (2000).
9. Deng, C.X. & Brodie, S.G. Roles of BRCA1 and its interacting proteins. Bioessays 22,
728–737 (2000).
10. Venkitaraman, A.R. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell
108, 171–182 (2002).
11. Deng, C.X. Roles of BRCA1 in centrosome duplication. Oncogene 21, 6222–6227
(2002).
12. Cantor, S.B. et al. BACH1, a novel helicase-like protein, interacts directly with BRCA1
and contributes to its DNA repair function. Cell 105, 149–160 (2001).
13. Kim, H., Chen, J. & Yu, X. Ubiquitin-binding protein RAP80 mediates BRCA1-
dependent DNA damage response. Science 316, 1202–1205 (2007).
14. Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA
damage sites. Science 316, 1198–1202 (2007).
15. Wang, B. et al. Abraxas and RAP80 form a BRCA1 protein complex required for the
DNA damage response. Science 316, 1194–1198 (2007).
16. Xu, B., Kim, S. & Kastan, M.B. Involvement of Brca1 in S-phase and G
2
-phase
checkpoints after ionizing irradiation. Mol. Cell. Biol. 21, 3445–3450 (2001).
17. Cortez, D., Wang, Y., Qin, J. & Elledge, S.J. Requirement of ATM-dependent phosphor-
ylation of brca1 in the DNA damage response to double-strand breaks. Science 286,
1162–1166 (1999).
18. Liu, Q. et al. Chk1 is an essential kinase that is regulated by Atr and required for the
G
2
/M DNA damage checkpoint. Genes Dev. 14, 1448–1459 (20 00).
19. Takai, H. et al. Aberrant cell cycle checkpoint function and early embryonic death in
Chk1(/)mice.Genes Dev. 14, 1439–1447 (2000).
20. Yarden, R.I., Pardo-Reoyo, S., Sgagias, M., Cowan, K.H. & Brody, L.C. BRCA1
regulates the G2/M checkpoint by activating Chk1 kinase upon DNA damage. Nat.
Genet. 30, 285–289 (2002).
21. Deming, P.B. et al. The human decatenation checkpoint. Proc. Natl. Acad. Sci. USA
98, 12044–12049 (2001).
22. Lou, Z. et al. MDC1 maintains genomic stability by participating in the amplification of
ATM-dependent DNA damage s ignals. Mol. Cell 21, 187–200 (2006 ).
23. Hofer, B., Backhaus, S. & Timmis, K.N. The biphenyl/polychlorinated biphenyl-
degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic
enzymes. Gene 144,9–16(1994).
ARTICLES
NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 14 NUMBER 8 AUGUST 2007 715
©2007 Nature Publishing Group http://www.nature.com/nsmb
