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HEMATOPOIESIS
FANCJ (BACH1) helicase forms DNA damage inducible foci with replication
protein A and interacts physically and functionally with the single-stranded
DNA-binding protein
Rigu Gupta,1 Sudha Sharma,1 Joshua A. Sommers,1 Mark K. Kenny,2 Sharon B. Cantor,3 and Robert M. Brosh Jr1
1Laboratory
of Molecular Gerontology, National Institute on Aging (NIA), National Institutes of Health (NIH), Baltimore, MD; 2Department of Emergency Medicine,
Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY; 3Department of Cancer Biology, University of
Massachusetts Medical School, Worcester
The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and
hereditary breast cancer. A critical role of
FANCJ helicase may be to restart replication as a component of downstream
events that occur during the repair of
DNA cross-links or double-strand breaks.
We investigated the potential interaction
of FANCJ with replication protein A (RPA),
a single-stranded DNA-binding protein im-
plicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a
direct interaction of FANCJ and the RPA70
subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with
RPA in nuclear foci after DNA damage.
Our data are consistent with a model in
which FANCJ associates with RPA in a
DNA damage-inducible manner and
through the protein interaction RPA stimulates FANCJ helicase to better unwind
duplex DNA substrates. These findings
identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of
the helicase to more efficiently unwind
DNA repair intermediates to maintain
genomic stability. (Blood. 2007;110:
2390-2398)
© 2007 by The American Society of Hematology
Introduction
Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow
1
failure, and high cancer risk. Cells from patients with FA exhibit
spontaneous chromosomal instability and hypersensitivity to DNA
interstrand cross-linking (ICL) agents. Although the precise mechanistic details of the FA/BRCA pathway of ICL repair are not well
understood, progress has been made in the identification of the FA
2
proteins that are required for the pathway. Among the 13 FA
complementation groups from which all the FA genes have been
3
cloned, only a few of the FA proteins are predicted to have direct
roles in DNA metabolism. One of the recently identified FA
proteins, shown to be responsible for complementation of the FA
4-6
complementation group J, is the BRCA1 associated C-terminal
helicase (BACH1, designated FANCJ), originally identified as a
protein associated with breast cancer. Two females among a cohort
of 65 women with early-onset breast cancer were identified
carrying 2 independent germ line sequence changes (P47A or
M299I) in the FANCJ coding region and normal genotypes for
7
BRCA1 and BRCA2. Truncating FANCJ mutations that cause FA in
biallelic carriers confer susceptibility to breast cancer in monoal8
lelic carriers. Clinically relevant mutations exist in the conserved
5,9,10
iron-sulfur cluster of the FANCJ helicase domain.
Interaction of
FANCJ with BRCA1 and the existence of FANCJ mutations in
patients with early-onset breast cancer and patients with FA have
clarified that FANCJ exerts a tumor suppressor function.
FANCJ has been proposed to function downstream of FANCD2
11
monoubiquitination, a critical event in the FA pathway. Evidence
supports a role of FANCJ in a homologous recombination (HR)
6
pathway of double-strand break (DSB) repair. DSB repair by HR
is important in the late steps of processing ICLs. FA-J cells are
6,12
hypersensitive to ICL agents, exhibit diminished BRCA1 foci in
untreated cells, and have delayed ionizing radiation (IR)–induced
13
BRCA1 foci. FANCJ helicase catalytically unwinds duplex DNA
with a 5⬘ to 3⬘ directionality, preferentially binds and unwinds
forked duplexes, and can unwind the invading strand of a 3-stranded
9,14
D-loop structure, a key early intermediate of HR repair.
A viable approach to understanding the role of FANCJ is to
identify and characterize its interactions with other proteins
implicated in HR repair. The BRCA1-FANCJ interaction depends
15-17
on 2 intact BRCA BRCT repeats and FANCJ phosphorylation
18
and is required for DNA damage-induced checkpoint control.
BRCA1 function at sites of DNA damage involves the assembly of
a BRCA1 super complex containing BARD1, FANCJ, BRCA2,
and Rad51 that is critical for the S phase checkpoint by inhibiting
19
DNA synthesis at late-firing sites of replication initiation. FANCJ
probably plays a critical role in HR-mediated DSB repair through
20
its interaction with BRCA1.
To better understand the roles of FANCJ in pathways of DNA
repair, we have investigated its potential interaction with replication protein A (RPA), a single-stranded DNA-binding protein
implicated in DNA replication and repair. Our findings show that
FANCJ and RPA coimmunoprecipitate with each other and colocalize in nuclear foci after DNA damage or replicational stress.
FANCJ and RPA directly bind to each other with high affinity by
Submitted November 14, 2006; accepted June 22, 2007. Prepublished online
as Blood First Edition paper, June 27, 2007; DOI 10.1182/blood-2006–
11-057273.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2007 by The American Society of Hematology
2390
BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
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BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
the RPA70 subunit. Although FANCJ is severely limited in
unwinding even a 47 base pair (bp) forked duplex, the presence of
RPA enables FANCJ to act as a much more processive helicase.
The functional interaction between FANCJ and RPA is specific
because the Escherichia coli single-stranded DNA binding (ESSB)
protein failed to stimulate FANCJ helicase activity. The FANCJRPA complex is probably important for the role of the helicase in
genome stability maintenance through its DNA repair function.
INTERACTION BETWEEN FANCJ AND RPA
2391
anti–rabbit IgG (1:400, Invitrogen) secondary antibodies for 1 hour at 37°C.
Cells were washed 4 times with PBS containing 0.1% Tween-20, mounted
with Prolong Gold containing DAPI (Invitrogen), and cured at room
temperature in the dark for 24 hours. Immunofluorescence was performed
on a Zeiss LSM 510 META inverted Axiovert 200M laser scan microscope
(Carl Zeiss, Jena, Germany) with a Plan-Apochromat 63⫻/1.4 oil DIC
objective. Images were captured with a CCD camera and analyzed using
LSM Browser software (Carl Zeiss). Physical and functional protein
interaction assays are described in Document S1, available on the Blood
website; see the Supplemental Materials link at the top of the online article.
Materials and methods
Cell lines
Immortalized fibroblasts FA-A (PD220), FA-J (EUFA030), and HeLa were
grown in Dulbecco modified Eagle medium supplemented with 10% fetal
bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine at
37°C in 5% CO2. Immortalized FA-D2 (PD20) fibroblasts were grown in
RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 15% FBS,
1% penicillin-streptomycin, and 1% L -glutamine at 37°C in 5% CO2. HCC
1937 and BRCA1-reconstituted cells were grown in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin. For BRCA1
knockdown, smartpool BRCA1 siRNA (100 nM; Dharmacon, Chicago, IL)
was transfected into HeLa cells using Lipofectamine2000 (Invitrogen), and
cells were used 48 hours after transfection. For FANCJ transfections, 3 ␮g
plasmid encoding Myc-tagged FANCJ-WT, FANCJ-M299I, or FANCJ7
P47A proteins were transfected into FA-J cells with Fugene 6 (Roche,
Indianapolis, IN), and cells were used 36 hours after transfection.
Coimmunoprecipitation experiments
HeLa nuclear extracts (NEs) were prepared from exponentially growing
21
cells as described previously. NE (1 mg protein) was incubated with rabbit
anti-FANCJ polyclonal antibody (1 ␮g; Sigma, St Louis, MO), mouse
anti-RPA70 monoclonal antibody (1 ␮g; Calbiochem, La Jolla, CA), or
normal rabbit IgG antibody (1 ␮g; Santa Cruz Biotechnology, Santa Cruz,
CA) in buffer D (50 mM HEPES [pH 7.5], 100 mM KCl, 10% glycerol) for
2 hours, tumbled with 20 ␮L protein-G agarose (Roche) for 2 hours at 4°C.
Beads were washed with buffer D containing 0.1% Tween-20. Proteins
were eluted by boiling in SDS sample buffer, resolved on 10% polyacrylamide Tris-glycine SDS gels, and transferred to PVDF membranes
(Amersham Biosciences, Piscataway, NJ). Immunoprecipitations from FA
mutant and BRCA1-depleted cells were performed using whole-cell lysates
22
prepared as previously described. Membranes were probed for FANCJ,
RPA, or BRCA1 using rabbit polyclonal anti-FANCJ (1:5000; Sigma),
mouse anti-RPA70 monoclonal (1:100; Calbiochem), or mouse monoclonal
anti-BRCA1 (Ab-4, 1:50; Oncogene, Seattle, WA) antibodies, respectively.
Proteins on immunoblot were detected using ECL Plus (Amersham
Biosciences).
Immunofluorescence cellular localization studies
Cells were grown as monolayer on chamber slides (Nalge Nunc International, Rochester, NY) as described. Mitomycin-C (MMC; 500 ng/mL;
Sigma) or hydroxyurea (2 mM; Sigma) was added to the 50% to 80%
confluent cultures for 16 hours before harvesting. For IR treatment, cells
were irradiated at 10 Gy using Gammacell 40, a 137Cs source emitting at a
fixed dose rate of 0.82 Gy/minute (Nordion International, Ottawa, ON) and
subsequently incubated at 37°C for 6 hours before harvesting. Cells were
fixed with formaldehyde (3.7% Polysciences Inc., Warrington, PA) for
15 minutes, washed with PBS, and treated with 0.5% Triton (Sigma) at
room temperature for 3 minutes followed by washing with PBS and
blocking with 10% goat serum (Sigma) overnight at 4°C. Indirect immunostaining was performed by incubating cells in primary antibodies, rabbit
anti-FANCJ polyclonal (1:250; Sigma), and mouse anti-RPA34 Ab-2
monoclonal (1:100; Calbiochem) for 1 hour at 37°C. After washes in PBS
with 0.1% Tween-20, cells were incubated with Alexa Fluor 488 goat
anti–mouse IgG (1:400; Invitrogen, Eugene, OR) and Alexa Fluor 568 goat
Results
In vivo interaction of FANCJ and RPA
To explore whether endogenous FANCJ and RPA reside in a
complex in vivo, coimmunoprecipitation experiments were performed. Anti-FANCJ antibody precipitated both FANCJ and RPA
from HeLa NE (Figure 1A lane 3). Twenty-two percent of the RPA
from HeLa NE input (Figure 1A lane 1) was coimmunoprecipitated
with FANCJ. Neither FANCJ nor RPA was precipitated when
normal rabbit IgG was used (Figure 1A lane 2). Specificity of the
FANCJ antibody was shown by using FA-J extracts. RPA failed to
be precipitated by the FANCJ antibody (Figure 1A lane 5) despite
its presence in FA-J extracts (Figure 1A lane 4). RPA was
coimmunoprecipitated with FANCJ from the FA-J–corrected cells
by the anti-FANCJ antibody (Figure 1A lane 9), whereas no RPA
was precipitated in the control IgG sample (Figure 1A lane 10). The
specificity of the FANCJ antibody was verified by the immunoprecipitation of BRCA1 as a positive control from the HeLa extract
(Figure 1B). FANCJ and RPA coimmunoprecipitated in the presence of DNaseI or EtBr, albeit slightly reduced with EtBr (Figure
1C), suggesting that DNA is not essential for the interaction.
FANCJ forms a direct complex with RPA
Enzyme-linked immunoabsorbent assays (ELISAs) were used to
test for a direct protein-protein interaction. Increasing concentrations of FANCJ were incubated in the presence of 3% BSA with
RPA immobilized on microtiter wells, and the bound FANCJ
protein was detected immunologically. Colorimetric signal was
both dose dependent and saturable (Figure 1D). Specificity of this
interaction was shown by the absence of color in wells precoated
with ESSB (Figure 1D) or BSA (data not shown). The colorimetric
signal from the FANCJ-RPA interaction was resistant to pretreatment of both FANCJ protein and RPA with DNase I (2 ␮g/mL) or
EtBr (10 ␮g/mL) (Figure 1E), suggesting that a contaminating
DNA bridge is not responsible for the signal. Specific binding of
FANCJ to RPA was analyzed according to Scatchard binding
theory. Data were analyzed by a Hill plot and found to be linear,
indicating a single set of binding sites for RPA with FANCJ. The
apparent dissociation constant (Kd) was determined to be 9.4 nM.
FANCJ directly binds to RPA70
To identify the subunit(s) of RPA that mediates the interaction
with FANCJ, Far Western analysis was performed. RPA was
immobilized on a nitrocellulose filter and incubated with
purified FANCJ protein. The filter was washed to remove
unbound protein, and FANCJ was detected by conventional
Western blotting. As controls, the membrane also contained
BSA and ESSB. The anti-FANCJ antibody detected a band at the
position of the 70-kDa subunit RPA, whereas no band was seen
at the positions of either BSA- or ESSB-negative controls or the
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2392
GUPTA et al
BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
Figure 1. FANCJ and RPA are associated with each other in vivo and directly interact. (A) Coimmunoprecipitation of RPA with FANCJ. FANCJ antibody coprecipitates
FANCJ and RPA from HeLa or FA-J–corrected cells but not from the FA-J extracts. The blot was probed with rabbit anti-FANCJ (top) and mouse anti-RPA (bottom) antibodies.
(Lane 1) HeLa nuclear extract (15% of input), (lane 2) control immunoprecipitate from HeLa nuclear extract using normal rabbit IgG, (lane 3) immunoprecipitate from HeLa
nuclear extract using rabbit anti-FANCJ antibody, (lane 4) FA-J whole-cell extract (WCE; 15% of input), (lane 5) immunoprecipitate from FA-J WCE using rabbit anti-FANCJ
antibody, (lane 6) control precipitate from FA-J WCE using normal rabbit IgG, (lane 7) WCE from HeLa included as control for Western detection of FANCJ and RPA, (lane 8)
FA-J corrected WCE (15% of input), (lane 9), immunoprecipitate from FA-J–corrected WCE using rabbit anti-FANCJ antibody, and (lane 10) control immunoprecipitate from
FA-J–corrected WCE using normal rabbit IgG. (B) Coimmunoprecipitation of BRCA1 with FANCJ. FANCJ antibody coprecipitates FANCJ and BRCA1 from HeLa nuclear
extract. The blot was probed with rabbit anti-FANCJ (top) and mouse anti-BRCA1 (bottom) antibodies. (Lane 1) HeLa nuclear extract (15% of input), (lane 2) immunoprecipitate
from HeLa nuclear extract using rabbit anti-FANCJ antibody, and (lane 3) control immunoprecipitate from HeLa nuclear extract using normal rabbit IgG. (C) FANCJ antibody
coprecipitates FANCJ and RPA from HeLa nuclear extracts in the presence of ethidium bromide or DNaseI. The blot was probed with rabbit anti-FANCJ (top) and mouse
anti-RPA (bottom) antibodies. (Lane 1) HeLa nuclear extract (15% of input), (lane 2) immunoprecipitate from HeLa nuclear extract using rabbit anti-FANCJ antibody, (lanes 3
and 4) immunoprecipitate from HeLa nuclear extracts in the presence of 2 ␮g/mL DNaseI or 10 ␮g/mL ethidium bromide using rabbit anti-FANCJ antibody, and (lane 5) control
immunoprecipitate from HeLa nuclear extract using normal rabbit IgG. (D,E) FANCJ and RPA form a complex by direct physical interaction. (D) RPA (96 nM heterotrimer, 䡺) or
ESSB (96 nM homotetramer, f) was coated onto the ELISA plate. After blocking with 3% BSA, the wells were incubated with increasing concentrations of purified recombinant
FANCJ protein (0-150 nM) for 60 minutes at 37°C. Wells were aspirated and washed 3 times, and bound FANCJ-WT protein was detected by ELISA with a rabbit polyclonal
antibody against FANCJ. (E) Same as described for panel D except 2 ␮g/mL DNase I or 10 ␮g/mL ethidium bromide (EtBr) were incubated with RPA (96 nM) and FANCJ
(77 nM) during the binding step in the corresponding wells. The values represent the mean of 3 independent experiments performed in duplicate with standard deviation (SD)
indicated by error bars. (F) FANCJ and RPA interact by the 70-kDa subunit of RPA. Purified RPA, ESSB, and BSA (as indicated above the lanes) were subjected to
SDS–polyacrylamide gel electrophoresis on 3 identical gels. The proteins bound to membranes were stained with Ponceau S or transferred to nitrocellulose membrane and
incubated with either purified FANCJ (⫹FANCJ) or no protein (⫺FANCJ). Western blotting with anti-FANCJ antibody was then used to detect the presence of FANCJ on each
membrane. The positions of the 70-, 32-, and 14-kDa subunits of RPA are indicated by asterisks. The positions of the molecular mass standards running parallel are shown on
the left.
32- and 14-kDa RPA subunits (Figure 1F). Immunoreactivity at
the position of the 70-kDa RPA subunit was not due to
cross-reactivity of the anti-FANCJ antiserum with RPA, becausethis band was absent from the control blot that had been
incubated with buffer alone (Figure 1F). We conclude that
FANCJ binds to the RPA70 subunit.
FANCJ associates with RPA in a DNA damage-inducible manner
FANCJ and RPA strongly colocalized after DNA damage induced
by IR or MMC or replicational stress induced by hydroxyurea
(Figure 2A). Approximately 80% of FANCJ and RPA foci colocalized with each other after treatment with IR, MMC, or hydroxyurea
(Figure 2B). Specificity of FANCJ antibody was shown by the
absence of FANCJ staining in FA-J cells (Figure 2C). RPA foci
were observed in FA-J cells after IR or MMC treatment, suggesting
that the ability to form RPA foci after DNA damage was intact in
the FA-J cells. FANCJ and RPA colocalized after MMC or IR
treatment in the corrected FA-J cells (Figure 2C).
To determine the effect of DNA damage on FANCJ-RPA
interaction, we performed coimmunoprecipitation experiments.
The results show that RPA and FANCJ can be reciprocally
immunoprecipitated (Figure 3). There was an increased signal for
FANCJ in the RPA immunoprecipitates from cells exposed to IR or
MMC compared with untreated cells (lanes 5 and 6 versus lane 4).
Similarly, there was an increased signal for RPA in the FANCJ
immunoprecipitates from damage-treated cells compared with
untreated cells (lanes 8 and 9 versus lane 7). These results indicate a
DNA damage-inducible association of the two proteins, consistent
with their colocalization.
FANCJ-RPA interaction is intact in FA mutant cells
We next asked whether FANCJ-RPA interaction was defective in
FA-deficient cells. Because FANCJ is implicated in a downstream
event of FANCD2 monoubiquitination, we investigated the possibility that a mutation in an upstream member (core complex protein
FANCA) of the FA pathway might affect the interaction of FANCJ
and RPA. FANCJ and RPA were coimmunoprecipitated from
whole-cell lysate of the FA-A mutant cell line (Figure 4A),
suggesting the ability of FANCJ and RPA to be associated with
each other is intact in cells that are defective in an event upstream
of FANCD2 monoubiquitination. We also examined FA-D2 mutant
cells and their corrected counterpart for FANCJ-RPA interaction
and show that the two proteins could be coimmunoprecipitated
from the whole-cell lysates (Figure 4B).
FANCJ-RPA interaction in BRCA1-deficient cells
We next asked whether a deficiency in the FANCJ-interacting
partner BRCA1 might affect the interaction of FANCJ and RPA.
Coimmunoprecipitation experiments were performed by using
control or BRCA1 siRNA-depleted HeLa cells (Figure 5A).
FANCJ and RPA were coimmunoprecipitated with each other in the
absence or presence of exogenous DNA damage (IR, MMC) under
conditions when BRCA1 is depleted (Figure 5B).
We examined the breast cancer cell line HCC 1937 and its
corrected counterpart for FANCJ and RPA staining. HCC 1937
cells are characterized by a mutant BRCA1 species with a truncated
C-terminal region affecting the integrity of the second BRCT motif
23
and defective in the interaction with FANCJ. In untreated cells as
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BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
INTERACTION BETWEEN FANCJ AND RPA
2393
Figure 3. Coimmunoprecipitation of FANCJ and RPA after DNA damage. Nuclear
extracts were prepared from HeLa cells that were untreated or exposed to IR (10 Gy)
or MMC (500 ng/mL) and immunoprecipitated with anti-FANCJ or anti-RPA antibody
as indicated. The blot was probed with rabbit anti-FANCJ (top) and mouse
anti-RPA70 or anti-RPA32 (bottom) antibodies.
detected in the HCC 1937 cells (Figure 5C), consistent with an
24
earlier observation. These results suggest that BRCA1 status
affects FANCJ foci formation with its protein partner RPA after
DNA damage. FANCJ and RPA colocalized in the reconstituted
HCC 1937 cells after IR or MMC (Figure 5C,D).
Effect of duplex length on FANCJ helicase activity
Figure 2. DNA damage-inducible colocalization of FANCJ helicase and RPA.
(A) HeLa cells were treated with the DNA-damaging agents MMC (500 ng/mL) or
hydroxyurea (2 mM) or exposed to 10 Gy IR as described in “Immunofluorescence
cellular localization studies” After fixation and permeabilization, cells were incubated
with anti-RPA (green) and anti-FANCJ (red) antibodies. After treatment with MMC,
hydroxyurea, or IR, RPA localizes in nuclear foci that coincide with FANCJ foci as
shown in the overlapped images. The yellow color results from the overlapping of the
red and green foci in the merged images. In control untreated cells, RPA and FANCJ
staining is diffuse. (B) Quantitation of FANCJ-RPA colocalizing foci as described in
panel A. Percentages of RPA foci colocalizing with FANCJ are represented 䡺, and
percentages of FANCJ foci colocalizing with RPA are represented (u). Experimental
data are the mean of at least 3 independent experiments with standard deviations
indicated by error bars. (C) FA-J vector and FA-J corrected cells were treated with the
indicated DNA-damaging agents and processed for immunofluorescence as described above.
well as HCC 1937 cells exposed to IR or MMC, most cells
displayed poor immunofluorescent staining for FANCJ, whereas
the reconstituted BRCA1-positive cells displayed normal FANCJ
staining (Figure 5C), consistent with an earlier observation by
7
Cantor et al. Thus, although RPA foci form well after DNA
damage in the breast cancer HCC 1937 cells, FANCJ foci
formation is defective. DNA damage-inducible RPA foci were
To elucidate whether RPA may serve as an auxiliary factor for
FANCJ, we examined the ability of FANCJ to unwind preferred
forked duplexes of either 22 bp or a longer 47 bp. FANCJ-WT
unwound a proportionately greater percentage of the 22-bp forked
duplex with increasing protein concentration, achieving approximately 55% of the substrate unwound at 2.4 nM FANCJ-WT
(Figure 6A). In comparison, very little (⬃1%) of the 47-bp forked
duplex was unwound by FANCJ-WT (4.8 nM) and only approximately 6% was unwound by 19.2 nM FANCJ. These results
indicate that FANCJ-WT poorly unwinds the 47-bp forked duplex
substrate under multiple turnover conditions and suggests that the
helicase is not processive.
A naturally occurring FANCJ-M299I sequence variant with an
amino acid substitution in the conserved iron-sulfur cluster of the
10
helicase domain displayed a significantly elevated ATPase activ25
ity compared with FANCJ-WT. We tested whether the elevated
ATPase activity of FANCJ-M299I enabled it to unwind longer
DNA substrates. FANCJ-M299I unwound a proportionately greater
percentage of the 22-bp forked duplex with increasing protein
concentrations; however, FANCJ-M299I was more active than
FANCJ-WT at all concentrations tested (Figure 6A). For example,
approximately 55% of the 22-bp forked duplex substrate was
unwound at 0.6 nM FANCJ-M299I, whereas only 27% of the 22-bp
Figure 4. FANCJ-RPA interaction is intact in FA mutant cells coimmunoprecipitation of FANCJ and RPA in FA-A and FA-D2 cells. Coimmunoprecipitation of RPA
with FANCJ in FA-A vector and FA-A corrected cells (A) or FA-D2 vector and
FA-D2–corrected cells (B) with the use of FANCJ antibody. The blot was probed with
rabbit anti-FANCJ (top) and mouse anti-RPA (bottom) antibodies. Input represents
15% of WCE input for coimmunoprecipitation experiments. Control immunoprecipitate from WCE of FA-A–corrected or FA-D2–corrected cells with the use of normal
rabbit IgG is shown in lane 5.
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BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
Figure 5. Effect of BRCA1 deficiency on the DNA damage-inducible association of FANCJ and RPA in BRCA1-deficient cells. (A) siRNA depletion of BRCA1. WCE of
HeLa cells transfected with control or BRCA1 siRNA were probed using an antibody against BRCA1. Actin serves as a loading control. (B) WCEs were prepared from control
siRNA or BRCA1 siRNA HeLa cells that were untreated or exposed to IR (10 Gy) or MMC (500 ng/mL) as indicated and immunoprecipitated with FANCJ antibody. The blot was
probed with rabbit anti-FANCJ (top) and mouse anti-RPA70 (bottom) antibodies. (C) HCC 1937 cells form RPA but not FANCJ DNA damage-inducible foci. BRCA1 mutant HCC
1937 or reconstituted cells were treated with the DNA-damaging agent MMC (500 ng/mL) or exposed to 10 Gy IR as described in “Immunofluorescence cellular localization
studies.” After fixation and permeabilization, cells were incubated with anti-RPA (green) and anti-FANCJ (red) antibodies. (D) Quantitation of FANCJ-RPA colocalizing foci as
described in panel C. Percentages of RPA foci colocalizing with FANCJ are represented (䡺). Percentages of FANCJ foci colocalizing with RPA are represented (u).
Experimental data are the mean of at least 3 independent experiments with SD indicated by error bars.
substrate was unwound by 0.6 nM FANCJ-WT, consistent with our
previous studies that FANCJ-M299I is more active than
25
FANCJ-WT.
We next evaluated the ability of FANCJ-M299I to unwind
the 47-bp forked duplex. Only 2% of the substrate was unwound
at 0.6 nM FANCJ-M299I (Figure 6A). Nonetheless, FANCJM299I was clearly able to unwind a greater percentage of the
47-bp substrate than FANCJ-WT, achieving approximately 9%
substrate unwound at 4.8 nM FANCJ-M299I (Figure 6A). The
47-bp forked duplex was efficiently wound by E coli DNA
TraI helicase (R.G. and R.M.B., unpublished data, June 2006),
indicating that the substrate can be unwound under FANCJ
helicase reaction conditions. These results suggest that FANCJM299I unwinds both short and long duplex tracts better than
FANCJ-WT; however, even a relatively short duplex length of
47 bp prevents FANCJ-M299I from efficiently unwinding a
great percentage of the forked DNA substrate.
Effect of single-stranded DNA-binding proteins on FANCJ
helicase activity
We tested FANCJ-WT helicase activity on the 47-bp forked duplex
in the presence of increasing RPA concentrations (Figure 6B-D).
FANCJ-WT was stimulated by RPA to unwind the 47-bp substrate.
At the highest concentration of RPA tested (96 nM), approximately
60% of the substrate was unwound. In comparison, there was little
to no detectable unwinding of the DNA substrate by FANCJ-WT in
the absence of RPA. To determine whether the stimulation of
FANCJ-WT helicase activity by RPA was specific, we evaluated
the effect of ESSB on the unwinding reaction catalyzed by
FANCJ-WT. ESSB concentrations up to 48 nM homotetramer
failed to stimulate FANCJ-catalyzed unwinding of the 47-bp
forked duplex (Figure 6C,D). In contrast, 50% of the 47-bp
substrate was unwound by FANCJ in the presence of 48 nM RPA
heterotrimer (Figure 6B). The only stimulatory effect of ESSB on
FANCJ helicase activity was observed at the highest concentration
tested (96 nM), which was markedly less than that observed for
RPA (Figure 6C,D). ESSB efficiently bound the 47-bp forked
duplex substrate under the FANCJ helicase reaction conditions in
gel-shift assays (data not shown), indicating that the poor ability of
ESSB to stimulate FANCJ helicase was not simply attributed to its
inability to bind ssDNA.
To gain insight into the mechanism of stimulation of
FANCJ-WT helicase activity by RPA, strand displacement was
expressed as a function of the ratio (R) of SSB-binding units per
DNA-binding site. This analysis takes into account that 1 ESSB
26
homotetramer binds 35 nt, and 1 hRPA heterotrimer binds
27
30 nt. Stimulation of FANCJ helicase activity on the 47-bp
forked duplex substrate was first detectable at a 2-fold excess of
RPA heterotrimer binding units compared with ssDNA-binding
sites for RPA (R ⫽ 2) (Figure 6D inset). Approximately 30% of
the 47-bp substrate was unwound at an R value of 4. At an R
value of 16, the RPA-stimulated FANCJ unwinding reaction
attained a value of 48% substrate unwound. In contrast, ESSB
failed to stimulate FANCJ helicase at a 16-fold excess of ESSB
binding equivalents compared with ESSB-binding sites (Figure
6D inset). An effect of ESSB on FANCJ helicase activity was
only detected at a 32-fold excess of ESSB binding units
compared with ESSB binding site. Because ESSB so poorly
stimulated FANCJ unwinding of the 47-bp duplex substrate
suggests that a specific interaction between FANCJ helicase and
RPA is responsible for the observed unwinding of the DNA
substrate. Furthermore, these results suggest that the stimulation
of FANCJ helicase activity by RPA does not only reflect a
ssDNA-coating effect of RPA because it was observed that at
similar ratios of SSB protein binding equivalents to SSB binding
sites, ESSB failed to stimulate FANCJ-WT helicase activity,
whereas RPA clearly stimulated DNA unwinding by FANCJ-WT.
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BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
INTERACTION BETWEEN FANCJ AND RPA
2395
We measured the effect of RPA on ssDNA-stimulated ATPase
activity of FANCJ. There was no detectable stimulation of FANCJ
ATPase activity at all tested RPA concentrations (24-192 nM) (data
not shown).
Kinetic comparison of DNA unwinding by FANCJ
and sequence variants
We performed kinetic analyses of DNA unwinding reactions
catalyzed by FANCJ-WT and its associated polymorphic variants
in the presence or absence of RPA (Figure 7A). As expected, in the
absence of RPA FANCJ-WT poorly unwound the 47-bp forked
duplex during the entire time course. In the presence of RPA, an
increasing percentage of the substrate was unwound by FANCJ-WT
that was proportional to the time of incubation. When RPA was
present, 60% of the 47-bp substrate was unwound by FANCJM299I at 15 minutes compared with only 22% substrate unwound
by FANCJ-WT. A second naturally occurring polymorphic variant
in the ATPase domain, designated FANCJ-P47A, was seriously
compromised in its ability to unwind the 47-bp forked duplex in the
absence of RPA; however, nearly 10% of the substrate was
unwound by FANCJ-P47A in reactions containing RPA (Figure
7A). Thus, FANCJ-P47A helicase activity is stimulated by RPA,
but not nearly to the level of unwinding exhibited by FANCJ-WT.
However, FANCJ-M299I helicase activity shows an increased rate
of DNA unwinding compared with FANCJ-WT in the absence or
presence of RPA.
To evaluate whether RPA interacts with FANCJ variants, we
performed anti-Myc coimmunoprecipitation experiments from
lysates of FA-J cells transfected with the corresponding Myctagged wild-type or FANCJ variant (Figure 7B). Expression of
the M299I or P47A mutant protein was reduced compared with
FANCJ-WT, as shown by the input. However, RPA was
coimmunoprecipitated in cells transfected with M299I or P47A,
suggesting that RPA is associated with either variant in cell
extracts. To determine whether the FANCJ variants can directly
bind RPA, we performed ELISA assays with the purified
recombinant proteins (Figure 7C). Both M299I and P47A
mutant proteins interacted with RPA similar to FANCJ-WT, with
Kd values of 5.3 and 7.4 nM, respectively.
Discussion
Figure 6. Limited unwinding reaction catalyzed by FANCJ is stimulated by RPA.
(A) FANCJ helicase catalyzes a limited unwinding reaction. Helicase assays were as
described,14 using the indicated concentrations of FANCJ-WT or FANCJ-M299I and forked
duplex DNA substrates of 22 bp and 47 bp. Incubation was at 30°C for 15 minutes.
Reaction products were analyzed by nondenaturing gel electrophoresis. Quantitation of
results from helicase assays with standard deviations indicated by error bars. FANCJ-WT,
22 bp, Œ; FANCJ-M299I, 22 bp, ‚; FANCJ-WT, 47 bp, f; FANCJ-M299I, 47 bp, 䡺.
Percentage of displacement is expressed as a function of FANCJ-WT or FANCJ-M299I
protein concentration. (B-D) Stimulation of FANCJ-WT helicase activity by RPA. FANCJ-WT
protein (4.8 nM) was incubated with the 47-bp forked duplex in the presence of the
indicated concentrations of RPA heterotrimer (B) or ESSB homotetramer (C) under
standard helicase reaction conditions. Incubation was at 30°C for 60 minutes.
(D) Quantitation of results from helicase assays with SD indicated by error bars.
Percentage of displacement is expressed as a function of single-stranded DNA-binding
protein concentration (RPA heterotrimer or ESSB homotetramer). (Inset) Quantitation of
results with percentage of displacement expressed as a function of the ratio (SSB binding
unit)/(DNA-binding unit).
Understanding the role of FANCJ in cellular DNA metabolism and
how FANCJ dysfunction leads to tumorigenesis requires a comprehensive investigation of its catalytic mechanism and molecular
functions in DNA repair. In this study, we identified RPA as the first
regulatory partner of the FANCJ helicase. We found that RPA
localizes with FANCJ in a DNA damage-inducible manner, coprecipitates with FANCJ from nuclear extracts, and stimulates FANCJ
helicase activity in a specific manner. A key property of DNA
helicases is their ability to unwind long duplexes. Certain DNA
28
29
helicases (eg, RecBCD, TraI ) can processively unwind DNA
tracts of thousands of base pairs, whereas UvrD helicase only
unwinds long DNA duplexes in a protein concentration-dependent
30
manner. In contrast, the human RecQ helicases WRN and BLM
can only unwind DNA substrates of 100 bp or less in the absence of
31
an auxiliary factor. Even on a preferred forked duplex DNA
substrate, FANCJ acts inefficiently to unwind a 47-bp duplex,
suggesting that FANCJ is specifically tailored to act on short
duplex substrates that it might encounter during a process associated with DNA repair or replication restart. Alternatively, FANCJ
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2396
GUPTA et al
BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
32
Figure 7. Kinetic analyses of DNA unwinding of the 47-bp forked duplex DNA
substrate by FANCJ-WT or FANCJ polymorphic variants in the presence of RPA
or ESSB. (A) FANCJ-WT (4.8 nM) or its associated variants (FANCJ-M299I or
FANCJ-P47A) was incubated with the 47-bp forked duplex in the absence or
presence of 24 nM RPA heterotrimer under standard helicase reaction conditions.
Incubation was at 30°C for the indicated times. Quantitation of results from helicase
assays with SD are indicated by error bars. FANCJ-WT (‚); FANCJ-WT ⫹ RPA (Œ);
FANCJ-M299I (E); FANCJ-M299I ⫹ RPA (F); FANCJ-P47A (䡺); FANCJ-P47A ⫹ RPA
(f).(B) Coimmunoprecipitation of FANCJ (FANCJ) sequence variants with RPA. FA-J
cells were transiently transfected with plasmid DNA encoding Myc-tagged FANCJWT, FANCJ-M299I, or FANCJ-P47A, and WCEs were used for coimmunoprecipitation experiments using FANCJ antibody. The blot was probed with rabbit anti-FANCJ
(top) and mouse anti-RPA (bottom) antibodies. Input represents 15% of WCE input
for coimmunoprecipitation experiments. (C) RPA directly binds to FANCJ variants.
RPA (96 nM heterotrimer) was coated onto the ELISA plate. After blocking with 3%
BSA, the wells were incubated with increasing concentrations of purified recombinant
FANCJ-M299I or FANCJ-P47A protein (0-150 nM) for 60 minutes at 37°C. Wells were
aspirated and washed 3 times, and bound FANCJ protein was detected by ELISA
using a rabbit polyclonal antibody against FANCJ.
could be associated with RPA as an accessory factor to facilitate
FANCJ unwinding of longer DNA duplexes that is required during
cellular DNA metabolic processing events. These two possibilities
are not mutually exclusive. Given the potential involvement of
FANCJ in more than one aspect of ICL repair with FA proteins as
well as DSB repair, it will be of interest to determine the role(s) and
DNA unwinding mode(s) of FANCJ in different subcomplexes
involved in these pathways.
RPA was identified in a multiprotein nuclear complex (BRAFT)
containing 5 FA core complex proteins (A, C, E, F, and G), BLM,
and topoisomerase III␣ ; however, to our knowledge the assignment of FANCJ to this or any other multiprotein complex is not yet
reported. A BRCA1-associated genome surveillance complex containing a number of DNA repair or signaling proteins, including
MSH2, MSH6, MLH1, ATM, BLM, and RAD50-MRE11-NBS1,
33
and DNA replication factor C was identified. It will be of interest
to characterize the functional activities of FANCJ protein complexes. Our results indicate that FANCJ probably resides in a
protein subcomplex with RPA.
The functional interaction and mechanism whereby RPA stimulates FANCJ helicase activity is specific as evidenced by the
inability of ESSB to enhance FANCJ unwinding. A specific
functional interaction between FANCJ and RPA is further supported by our demonstration of their physical interaction mediated
by RPA70, a feature that is also characteristic of the interaction of
34,35
36
37
RPA with human RecQ helicases (WRN, BLM, RECQ1 ).
WRN and BLM interact with RPA70 through functionally
conserved domains of the helicases that do not display extensive
31
sequence homology. The WRN-interacting domain and ssDNA31,38
binding domain of RPA overlap with each other,
suggesting
that ssDNA and WRN protein-binding domains of RPA70 are
functionally intertwined. In addition to RPA loading a DNAprocessing protein such as helicase on to ssDNA, RPA interaction with some helicases may enable them to actively place RPA
39
on ssDNA as it emerges from the helicase complex. Protein
partners of RPA, such as helicases, may trade places on ssDNA
by binding to RPA and mediating conformational changes that
alter the ssDNA-binding properties of RPA. The order for
sequential loading of FANCJ and RPA to unwind a DNA
intermediate is particularly interesting and challenging to decipher because the precise role of FANCJ in the multistep FA
pathway is not known.
Formation of the FA core complex and FANCD2 monoubiquitination is intact, suggesting that the FANCJ functions down11
stream in the FA pathway. However, FANCD2 monoubiquitination is required for its association with chromatin and subnuclear
localization with BRCA1, RAD51, MRE11-RAD50-NBS1, RPA,
PCNA, and BRCA2, suggesting a connection of the upstream
and downstream events of the FA pathway in which FANCJ
40
operates. Unwinding of a DNA structure associated with
a stalled replication fork by FANCJ may enable HR or
nonhomologous end-joining repair proteins to access the dam11
aged site and facilitate ICL repair. FANCJ recognizes a D-loop
structure and catalytically unwinds the invading strand, irrespec14
tive of tail status, in vitro. This action of FANCJ may be
interpreted as a potential hindrance to some aspect of recombinational repair. It is likely that the role of FANCJ in terms of its
catalytic function is better understood in the context of its
interacting partners, such as RPA which improves its ability to
unwind longer DNA duplexes. However, RPA does not enable
FANCJ to unwind a Holliday junction (R.G. and R.M.B.,
unpublished data, April 2006), another key HR intermediate,
suggesting that other structure-specific helicases such as the
41
RecQ enzymes, are responsible for resolving these structures.
42
The reported direct binding of FANCD2 to Holliday junctions
may facilitate its subsequent resolution through branchmigrating helicases and resolving enzymes. Understanding how
the classic FA proteins prevent replication fork-associated DSBs
and maintain genomic stability will require the characterization
of functional FA protein interactions and the assembly of models
for the pathways from these biochemical findings.
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BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
INTERACTION BETWEEN FANCJ AND RPA
FANCJ and RPA colocalize in nuclear foci after DNA
damage and replicational stress, suggesting that they may
collaborate with each other and other components of DSB repair
to facilitate recombinational repair of frank DSBs or DSBs
induced by stalled or arrested replication forks. RPA, but not
FANCJ, formed DNA damage-inducible foci in BRCA1 mutant
HCC 1937 cells. Thus, although BRCA1 was reported to be
24
associated with RPA after IR treatment, FANCJ does not form
foci with RPA well after IR or MMC. The importance of BRCA1
for colocalization of FANCJ and RPA after MMC damage is
consistent with the hypersensitivity of BRCA1-deficient mouse
embryonic fibroblasts to MMC accompanied by defective
43
MMC-induced Rad51 foci and aberrant S-phase arrest. Together, these results suggest a role of BRCA1 in HR during
S phase to repair MMC-induced ICLs that involve the concerted
action of FANCJ and RPA.
Although FANCJ and RPA are associated with each other in FA
mutant cell lines, it is possible that FANCJ and RPA function
noncooperatively in DNA repair centers that operate when the FA
pathway is dysfunctional. FANCJ and RPA may be important for
functions independent of the FA core complex. FANCJ helicase
activity was shown to be essential for its function in restoring ICL
12
resistance. RPA is implicated in signaling at stalled replication
forks in which the accumulated ssDNA is bound by RPA, creating a
44
signal for activation of the ATR-dependent checkpoint response.
In the future, it will be important to address the role of FANCJ
interactions with DNA repair factors such as RPA that modulates its
catalytic activity to understand its proposed roles in DNA repair or
2397
DNA damage signaling that are either dependent or independent of
the FA pathway.
Acknowledgments
We thank the Fanconi Anemia Research Fund for FA-A (PD220),
FA-C (PD331), and FA-D2 (PD20) cells and Dr Hans Joenje
(Utrecht Medical Center, the Netherlands) for FA-J EUFA030
cells. We thank Dr Fred Indig (NIA-NIH) for assistance with
microscopy.
This work was supported by the Intramural Research program
of the NIH, National Institute on Aging.
Authorship
Contribution: R.G. performed research, analyzed data, and wrote
the paper; S.S. performed research and analyzed data; J.A.S. and
M.K.K. contributed valuable reagents; S.B.C. contributed valuable
reagents and wrote the paper; and R.M.B. designed the research,
analyzed data, and wrote the paper.
R.G. and S.S. contributed equally to this study.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Robert M. Brosh Jr, Laboratory of Molecular
Gerontology, NIA, NIH, 5600 Nathan Shock Drive, Baltimore, MD
21224; e-mail: [email protected].
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2007 110: 2390-2398
doi:10.1182/blood-2006-11-057273 originally published
online June 27, 2007
FANCJ (BACH1) helicase forms DNA damage inducible foci with
replication protein A and interacts physically and functionally with the
single-stranded DNA-binding protein
Rigu Gupta, Sudha Sharma, Joshua A. Sommers, Mark K. Kenny, Sharon B. Cantor and Robert M.
Brosh Jr
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