Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities.
ABSTRACT Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA-mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.
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ABSTRACT: DNA-damage response machinery is crucial to maintain the genomic integrity of cells, by enabling effective repair of even highly lethal lesions such as DNA double-strand breaks (DSBs). Defects in specific genes acquired through mutations, copy-number alterations or epigenetic changes can alter the balance of these pathways, triggering cancerous potential in cells. Selective killing of cancer cells by sensitizing them to further DNA damage, especially by induction of DSBs, therefore requires careful modulation of DSB-repair pathways.Here, we review the latest knowledge on the two DSB-repair pathways, homologous recombination and non-homologous end joining in human, describing in detail the functions of their components and the key mechanisms contributing to the repair. Such an in-depth characterization of these pathways enables a more mechanistic understanding of how cells respond to therapies, and suggests molecules and processes that can be explored as potential therapeutic targets. One such avenue that has shown immense promise is via the exploitation of synthetic lethal relationships, for which the BRCA1-PARP1 relationship is particularly notable. Here, we describe how this relationship functions and the manner in which cancer cells acquire therapy resistance by restoring their DSB repair potential.Nucleic Acids Research 05/2014; · 8.81 Impact Factor
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ABSTRACT: Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.The Journal of Cell Biology 12/2013; · 10.82 Impact Factor
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ABSTRACT: The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.Nucleic Acids Research 08/2014; · 8.81 Impact Factor
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 186 No. 5 655–663
Correspondence to Jiri Lukas: email@example.com
J. Falck’s present address is Novo Nordisk A/S, DK-2880 Bagsvaerd,
Abbreviations used in this paper: BLM, Bloom; DOX, doxycycline; DSB, double-
strand break; dsDNA, double-stranded DNA; EMSA, electrophoretic mobility
shift assay; hFbh1, human Fbh1; HR, homologous recombination; HU, hydroxy-
urea; IR, ionizing radiation; RPA, replication protein A; SCE, sister chromatid
exchange; ssDNA, single-stranded DNA, WT, wild type.
Genome integrity is constantly challenged by DNA damage,
resulting from a range of genotoxic insults. DNA double-strand
breaks (DSBs) represent the most toxic chromosomal lesion,
arising from a variety of sources such as ionizing radiation (IR)
or collapsed replication forks. To counteract the potentially
deleterious effects of DSBs, cells have evolved homologous
recombination (HR)–based repair mechanisms capable of re-
storing genomic integrity in an error-free manner and that rely
on the availability of an undamaged homologous sister chroma-
tid as a template for the repair process.
A key event in HR repair is the formation of a nucleofilament
of the rate-limiting recombinase Rad51, wrapped around single-
stranded DNA (ssDNA) generated in the vicinity of the DSB (San
Filippo et al., 2008). The Rad51/ssDNA nucleofilament catalyzes
a search for a homologous sequence on the sister chromatid and
promotes DNA strand invasion to initiate the repair process.
Despite its importance for preserving genomic integrity, HR repair
must be tightly controlled. Unrestricted HR activity is a hallmark
of genetic disorders such as Bloom (BLM) and Werner syndromes,
both of which display a hyper-recombination phenotype and ge-
nomic instability (Sung and Klein, 2006; Branzei and Foiani, 2007).
To restrict HR, cells harbor proteins termed anti-recombinases. In
budding yeast, the Srs2 helicase has such a function, preventing
spontaneous and unscheduled HR by dismantling Rad51 from
ssDNA (Krejci et al., 2003; Veaute et al., 2003). In humans, the
genes mutated in BLM, Werner, and Rothmund-Thomson
(RecQL4) syndromes also encode helicases belonging to the
RecQ family, all of which exhibit anti-recombinase activity (Wu
and Hickson, 2006). BLM dissociates Rad51/ssDNA nucleofila-
ments, thereby suppressing HR, a function that was also reported
for the helicase RecQL5 (Bugreev et al., 2007; Hu et al., 2007).
nome integrity and lead to premature aging or cancer. To
limit unscheduled HR, cells possess DNA helicases capa-
ble of preventing excessive recombination. In this study,
we show that the human Fbh1 (hFbh1) helicase accumu-
lates at sites of DNA damage or replication stress in a
manner dependent fully on its helicase activity and par-
tially on its conserved F box. hFbh1 interacted with single-
stranded DNA (ssDNA), the formation of which was
omologous recombination (HR) is essential for
faithful repair of DNA lesions yet must be kept in
check, as unrestrained HR may compromise ge-
required for hFbh1 recruitment to DNA lesions. Con-
versely, depletion of endogenous Fbh1 or ectopic expres-
sion of helicase-deficient hFbh1 attenuated ssDNA
production after replication block. Although elevated
levels of hFbh1 impaired Rad51 recruitment to ssDNA
and suppressed HR, its small interfering RNA–mediated
depletion increased the levels of chromatin-associated
Rad51 and caused unscheduled sister chromatid exchange.
Thus, by possessing both pro- and anti-recombinogenic
potential, hFbh1 may cooperate with other DNA helicases
in tightly controlling cellular HR activity.
Human Fbh1 helicase contributes to genome
maintenance via pro- and anti-recombinase activities
Kasper Fugger,1 Martin Mistrik,2 Jannie Rendtlew Danielsen,1 Christoffel Dinant,1 Jacob Falck,1 Jiri Bartek,1,2
Jiri Lukas,1 and Niels Mailand1
1Institute of Cancer Biology and Center for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark
2Laboratory of Genomic Integrity, Palacky University, 783 71 Olomouc, Czech Republic
© 2009 Fugger et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 186 • NUMBER 5 • 2009 656
the kinetics of GFP-hFbh1 focus formation paralleled that of RPA,
becoming apparent 30 min after exposure to the drug (Fig. 1 C).
Similar kinetics of GFP-hFbh1 recruitment was observed in
response to IR (Fig. S1 B). These observations suggest that
hFbh1 is a novel factor involved in the DNA damage response in
human cells, functioning at an advanced stage associated with
hFbh1 interacts with ssDNA
The recruitment of RecQ helicases to DNA lesions can be highly
dependent on specific DNA structures produced during the pro-
cess of HR (Wu and Hickson, 2006). To investigate the require-
ments for hFbh1 recruitment to DNA lesions, we first assessed
the impact of depleting different proteins involved in processing
damaged DNA. Knockdown of CtIP, a factor required for resec-
tion of DSBs into ssDNA (Sartori et al., 2007), severely im-
paired GFP-hFbh1 focus formation in response to IR but not HU
(Fig. 2 A), which generates ssDNA independent of resection.
This suggested that hFbh1 is recruited to ssDNA but not unpro-
cessed DSBs. To determine whether hFbh1 recruitment to dam-
aged DNA required DNA intermediates produced after strand
invasion during HR, we depleted Rad51 or Rad51C, a Rad51
mediator essential for Rad51 focus formation after DNA dam-
age (Rodrigue et al., 2006). Although the Rad51 and Rad51C
siRNAs depleted their targets (Fig. 2 A), such treatments did not
interfere with GFP-hFbh1 recruitment in response to either IR
or HU (Fig. 2 A), suggesting that hFbh1 is directly recruited to
ssDNA rather than recombination intermediates.
To test the idea that hFbh1 recognizes ssDNA, cells were
prelabeled with BrdU before exposure to HU and subjected to
immunostaining under nondenaturing conditions in which BrdU
is only detectable in ssDNA regions (Groth et al., 2007). Under
these conditions, GFP-hFbh1 completely colocalized with the
BrdU-decorated ssDNA compartment (Fig. 2 B), supporting the
notion that hFbh1 associates with ssDNA. To see whether hFbh1
directly binds ssDNA, we performed electrophoretic mobility
shift assays (EMSAs) in which purified GST-hFbh1 was incu-
bated with ssDNA or double-stranded DNA (dsDNA) probes. As
shown in Fig. 2 C, hFbh1 bound to ssDNA but not to dsDNA.
Although hFbh1 recruitment to ssDNA regions generated by IR was
restricted to S and G2 phases, we observed that GFP-hFbh1 ac-
cumulated at UV-damaged DNA in a subset of cyclin A–negative
cells (Fig. S2 A). However, we failed to detect accumulation of
GFP-Fbh1 at UV lesions in early G1 cells after mitotic shake off
(Fig. S2 B). We conclude from these experiments that hFbh1 may
be recruited to ssDNA produced in late G1 but not in early G1.
hFbh1 displaces Rad51 from chromatin to
Evidence from yeast and other organisms suggests that Srs2 and
the RecQ helicases possess anti-recombinogenic activities by
dismantling the Rad51 nucleofilament from ssDNA produced
during normal replication (Sung and Klein, 2006). We tested
whether hFbh1 might similarly prevent Rad51 focus forma-
tion after a replication block using the inducible GFP-hFbh1
cell line. As expected, Rad51 readily accumulated in nuclear
foci when uninduced cells were treated with HU or IR (Fig. 3 A).
The existence of several helicases with anti-recombino-
genic properties in mammalian cells suggests a consider-
able degree of complexity and redundancy in HR regulation.
Recently, a functional homologue of Saccharomyces cerevisiae
Srs2, RTEL1, was identified in humans (Barber et al., 2008).
Fbh1, another conserved helicase with similarity to Srs2, has
also been proposed to be a functional homologue of Srs2 in
fission yeast and higher eukaryotes (Chiolo et al., 2007), but
so far little is known about the function of Fbh1. Fbh1 belongs
to the UvrD family of helicases and has 3–5 DNA-unwinding
activity (Kim et al., 2004). Moreover, Fbh1 is a putative E3
ubiquitin ligase by virtue of a conserved F box, enabling it to
potentially function as an adaptor for the Skp, Cullin, F box–
containing complex (Kim et al., 2004). However, at present, its
ubiquitylation targets are unknown. In Schizosaccharomyces
pombe, Fbh1 knockout leads to increased sensitivity to DNA-
damaging agents (Morishita et al., 2005; Osman et al., 2005).
Chicken DT40 cells ablated for Fbh1 exhibit an overall mild
phenotype yet display an elevated rate of sister chromatid ex-
change (SCE), which is a phenomenon associated with hyper
recombination (Kohzaki et al., 2007). In these cells, Fbh1 acts
redundantly with BLM, and their codisruption causes a syner-
gistic increase in DNA damage sensitivity.
In this study, we have performed a detailed analysis of the
functional properties of Fbh1 in human cells. We show that human
Fbh1 (hFbh1) possesses both pro- and anti-recombinogenic
activities, contributing to the regulation of ssDNA production at
replication blocks as well as regulation of Rad51 nucleofilament
formation and HR repair. Our findings reveal evolutionary con-
servancy of Fbh1 function and broaden the spectrum of anti-
recombinogenic helicases in mammalian cells.
Results and discussion
hFbh1 accumulates at sites of
To investigate whether Fbh1 plays a role in the DNA damage
response in human cells, we examined the impact of DNA-
damaging insults on the subcellular localization of hFbh1. Be-
cause high levels of hFbh1 appeared toxic to cells (unpublished
data), we generated a U2OS cell line stably expressing GFP-
tagged hFbh1 in a doxycycline (DOX)-inducible fashion at low
levels, which did not detectably interfere with cell proliferation
(Fig. S1 A). In this cell line, GFP-hFbh1 showed uniform nu-
clear staining upon induction in the absence of exogenous DNA
damage (Fig. 1 A). However, in response to DSBs generated by
IR or laser microirradiation or replication blocks elicited by
treatment with hydroxyurea (HU), GFP-hFbh1 accumulated in
discrete subnuclear foci (Fig. 1 A), which is similar to the be-
havior of a range of DNA damage–responsive proteins. The
GFP-hFbh1 foci completely colocalized with the ssDNA-binding
protein replication protein A (RPA) but only partially over-
lapped with -H2AX, a marker for DSBs (Fig. 1 A). The forma-
tion of GFP-hFbh1 foci in response to IR was restricted to S and
G2 phases, the cell cycle stages permissive for HR, as their for-
mation occurred only in cyclin A–positive cells and included
BrdU-incorporating S phase cells (Fig. 1 B). In response to HU,
REGULATION OF HOMOLOGOUS RECOMBINATION BY hFbh1 • Fugger et al.
Figure 1. hFbh1 accumulates at sites of DNA damage in S and G2 phase cells. (A) U2OS/GFP-hFbh1 WT cells were induced to express GFP-hFbh1 by
addition of DOX for 24 h, and were subsequently left untreated or subjected to laser microirradiation, IR (6 Gy), or HU treatment. 1 h later, the cells were
fixed and coimmunostained with antibodies to RPA and -H2AX. Magnifications of HU- and IR-treated cells are shown (red lines). (B) U2OS/GFP-hFbh1
WT cells induced to express the transgene for 24 h were subjected to IR (6 Gy) for 1 h, pulsed with BrdU for 20 min, and fixed and coimmunostained with
the indicated antibodies. (C) U2OS/GFP-hFbh1 WT cells induced with DOX for 24 h were incubated in the presence of HU for the indicated times and
processed for immunofluorescence as in A. Bars, 10 µm.
JCB • VOLUME 186 • NUMBER 5 • 2009 658
formation by targeting it for proteasomal degradation. Unlike
total levels of Rad51, cellular fractionation experiments demon-
strated that the HU-dependent increase in chromatin-associated
Rad51 was suppressed by induction of GFP-hFbh1 (Fig. 3 B).
Moreover, GFP-hFbh1 led to an increase in chromatin-bound
RPA after HU treatment (Fig. 3 A), indicating that the ability
of GFP-hFbh1 to prevent Rad51 accumulation at DNA damage
sites did not reflect a general propensity to displace ssDNA-
binding proteins. These data indicate that hFbh1 or an hFbh1-
containing complex is capable of actively removing Rad51
Because ectopic GFP-hFbh1 can displace Rad51 from
chromatin, we speculated that depletion of endogenous hFbh1
would lead to chromatin retention of Rad51 during replication.
To test this, we assessed Rad51 focus formation in primary BJ
fibroblasts transfected with hFbh1 siRNAs. Using the nucleo-
side analogue EdU as a marker for replicating cells, we analyzed
Rad51 focus formation in S phase and observed a significant,
roughly threefold increase in the occurrence of spontaneous
Rad51 foci in cells depleted for endogenous hFbh1 using two
independent siRNAs (Fig. 3 C). To directly test whether hFbh1
has anti-recombinase activity, we used an HR reporter assay in
which HR is measured as the ability of cells to repair an I-SceI–
induced DSB in an inactive GFP construct that becomes func-
tional only through HR-mediated repair (Sartori et al., 2007).
We observed a marked 40% decrease in HR events when
hFbh1 was cotransfected with I-SceI (Fig. 3 D), which is com-
parable with the extent of HR suppression observed after siRNA-
mediated depletion of Rad51 (not depicted). Collectively,
these findings suggest that hFbh1 functions as a bonafide anti-
recombination factor in human cells through its ability to dis-
mantle the Rad51 nucleofilament and thus may help to prevent
unscheduled HR during normal DNA replication and in re-
sponse to DNA damage.
hFbh1 facilitates ssDNA generation at
stalled replication forks
To further dissect the mechanism by which hFbh1 displaces Rad51
from chromatin, we introduced point mutations into hFbh1 to
functionally impair its conserved F box (Fig. 4 A, *FB) or helicase
domains (Fig. 4 A, *HL). We verified the effect of the F box muta-
tions by demonstrating that they impaired hFbh1 binding to the
Skp, Cullin, F Box–containing complex core subunit Skp1, which
is the known function of the F box (Fig. S1 C). Surprisingly, hFbh1
helicase domain was also unable to bind Skp1 (Fig. S1 C), which
may suggest that hFbh1 only associates stably with Skp1 in a
chromatin context (see following paragraph). In agreement with
data from fission yeast (Sakaguchi et al., 2008), mutation of the
F box resulted in pancellular distribution of GFP-hFbh1 (unpub-
lished data), and thus, we tagged the GFP-hFbh1 constructs with
N-terminal NLSs to be able to compare their functional properties.
We generated stable cell lines expressing such NLS–GFP-hFbh1
proteins and tested their ability to recruit to ssDNA regions in-
duced by HU treatment. Although mutation of the F box partially
impaired the binding of GFP-hFbh1 to RPA-coated ssDNA, dis-
ruption of the catalytic activity of its helicase domain completely
prevented accumulation of GFP-hFbh1 on ssDNA (Fig. 4 B).
Strikingly, however, induction of GFP-hFbh1 in these cells
strongly impaired Rad51 foci formation in response to HU and
IR (Fig. 3 A). Expression of Rad51 was unaffected by induc-
tion of GFP-hFbh1 (Fig. 3 B), arguing against the possibility
that hFbh1, a potential E3 ubiquitin ligase, inhibits Rad51 focus
Figure 2. hFbh1 interacts with ssDNA. (A) U2OS/GFP-hFbh1 WT cells
were transfected with the indicated siRNAs for 24 h, induced with DOX
for an additional 24 h, and exposed to IR or HU for 1 h. (top) The cells
were fixed, and the GFP signal was visualized by confocal microscopy.
(bottom) Immunoblot analysis of siRNA-mediated knockdown efficiency.
(B) U2OS/GFP-hFbh1 WT cells were incubated in the presence of BrdU
and DOX for 24 h. The cells were treated with HU for 1 h, fixed, and
immunostained with BrdU antibody under native conditions. (C) EMSA is
shown. 32P-labeled ssDNA or dsDNA probes were incubated with increas-
ing amounts (200–1,000 nM) of GST-hFbh1 and subjected to native gel
electrophoresis. Migration of the probe and the GST-hFbh1–ssDNA com-
plex is indicated. Bars, 10 µm.
659REGULATION OF HOMOLOGOUS RECOMBINATION BY hFbh1 • Fugger et al.
Figure 3. hFbh1 acts as an anti-recombinase by displacing Rad51 from chromatin. (A) U2OS/GFP-hFbh1 WT cells were induced or not with DOX for
24 h and subjected to HU for 2 h or IR for 1 h. (left) The cells were preextracted to remove soluble proteins, fixed, and coimmunostained with RPA and
Rad51 antibodies. (right) Quantification of the results of three independent experiments is shown. (B) U2OS/GFP-hFbh1 WT cells subjected to DOX
and HU treatment as in A were harvested and processed for subcellular fractionation. Chromatin-enriched fractions and whole cell extracts (WCE) were
immunoblotted with the indicated antibodies. Relative intensity of the Rad51 signal is indicated. (C) BJ fibroblasts were transfected with control or two
independent hFbh1 siRNAs for 48 h. (top) Cells were incubated with EdU for 30 min, preextracted, fixed, and immunostained with Rad51 antibody and
EdU. Two representative fields from each transfected population are shown. (bottom) The intensity of chromatin-bound Rad51 staining was quantified
by image analysis software and depicted in a box plot. SID, signal-integrated density. At least 50 EdU-positive cells were analyzed in each experiment.
(D) U2OS/DR-GFP cells were transfected with plasmids encoding RFP, I-SceI, and, where indicated, empty vector or Flag-hFbh1 for 48 h. Cells were pro-
cessed for flow cytometric analysis of RFP and GFP, and the extent of HR was scored as the GFP/RFP ratio. The experiment was performed in triplicates.
Error bars indicate the standard deviation. Bars, 10 µm.
JCB • VOLUME 186 • NUMBER 5 • 2009 660
Figure 4. The helicase activity of hFbh1 facilitates ssDNA generation at replication blocks. (A) Schematic depiction of the hFbh1 protein, showing localiza-
tion of conserved domains. Positions of residues mutated to generate hFbh1 F box (*FB) and helicase domain (*HL) mutants are indicated in red.
(B) U2OS/NLS–GFP-hFbh1 cell lines were induced or not with DOX for 24 h, treated with HU for 1 h, and fixed. Where indicated, cells were preextracted
before fixation. The cells were coimmunostained with the indicated antibodies. (C) U2OS/NLS–GFP-hFbh1 cell lines were treated with DOX and HU as in
B and processed for subcellular fractionation. Chromatin-enriched fractions (CHR) or whole cell extracts (WCE) were immunoblotted with the indicated anti-
bodies. (D) U2OS/GFP-hFbh1 cells were transfected with control or hFbh1 siRNA for 48 h and incubated in the presence of BrdU for an additional 24 h.
Cells transfected with hFbh1 siRNA were either left uninduced or induced with DOX for the last 24 h. Cells were treated with HU for 2 h and processed for
native fixation and immunostaining with BrdU antibody. BrdU staining was quantified by image analysis software and depicted in a box plot. SID, signal-
integrated density. At least 1,000 cells were analyzed in each experiment. Bars, 10 µm.
661REGULATION OF HOMOLOGOUS RECOMBINATION BY hFbh1 • Fugger et al.
dominant-negative factor to prevent further ssDNA production in
response to replication blocks, likely blocking the access of ac-
tive Fhh1 (and perhaps even other ssDNA-producing helicases) to
DNA lesions. As has been found for other UvrD helicases (Maluf
et al., 2003), hFbh1 formed homodimers (Fig. S2 D), potentially
explaining the ability of the overexpressed helicase domain mutant
to act in a dominant-negative fashion in the presence of endoge-
nous hFbh1. Thus, the lack of Rad51 focus formation observed in
HU-treated cells expressing GFP-Fbh1 helicase domains (Fig. 4 B)
likely reflects their inability to produce sufficiently long stretches
of ssDNA required for Rad51 recruitment.
Collectively, these data suggest that in addition to its anti-
recombinogenic effect, hFbh1 may also possess prorecombi-
nase activity by facilitating ssDNA production after replication
blocks to promote the loading of RPA, which is in turn required
for proper checkpoint signaling and HR repair. Consistently,
cells depleted for endogenous hFbh1 displayed markedly re-
duced ssDNA production in response to HU treatment, an effect
that could be fully rescued by induction of siRNA-insensitive
GFP-Fbh1 in these cells (Fig. 4 D). Such a dual role of hFbh1 in
terms of pro- and anti-recombination activities is analogous to
that of BLM helicase, which functions in ssDNA production
after DNA damage (Gravel et al., 2008; Zhu et al., 2008) as well
as displacement of Rad51 from ssDNA (Bugreev et al., 2007).
hFbh1 is a suppressor of SCE
A prominent feature of unrestricted HR is the hyper-recombination
phenotype, a hallmark of BLM syndrome, which is characterized
Surprisingly, overexpression of both F box and helicase do-
main mutants was still able to prevent accumulation of Rad51 at
the sites of stalled replication forks (Fig. 4 B). In case of the F box
mutant, this can be explained, at least in part, by its residual capa-
bility to bind damaged DNA, which might be sufficient to displace
Rad51 from ssDNA. Such a scenario also indicates that the po-
tential ubiquitin ligase activity of hFbh1 (the function of which is
unknown) may not be required per se for its anti-recombinogenic
function. The impact of the helicase domain mutant on Rad51
focus formation appeared more complex, and to elucidate this issue,
we set out to measure the production of ssDNA after replication
stress. Interestingly, we observed that cells overexpressing GFP-
hFbh1 helicase domains failed to produce detectable HU-induced
ssDNA stretches, which is indicated by a quantitative loss of RPA
foci and reduced BrdU immunostaining under native conditions
(Fig. 4 B). We obtained similar results using biochemical isolation
of chromatin-enriched fractions from NLS–GFP-hFbh1–inducible
cells in which the helicase domain mutant but not the wild-type
(WT) or F box alleles of GFP-hFbh1 suppressed the increased
chromatin loading of RPA after HU treatment (Fig. 4 C). Induction
of WT or mutant GFP-hFbh1 had little impact on cell cycle dis-
tribution (Fig. S1 D), ruling out cell cycle effects as a major cause
of the observed phenotypes. Using EMSA, we found that the
hFbh1 helicase domain mutant retained the capability to bind to
ssDNA (Fig. S2 C) even though it was not stably recruited to
ssDNA sites (Fig. 4 B). Thus, it appears that by binding short
stretches of ssDNA at stalled replication forks while being unable
to unwind DNA, the hFbh1 helicase domain mutant may act as a
Figure 5. Depletion of hFbh1 increases SCE.
(A) BJ cells were transfected with control or
hFbh1 siRNAs for 48 h and subjected to SCE
analysis. Metaphase chromosome spreads
were prepared, and the number of SCE events
was scored on a per cell basis and subjected
to statistical analysis. The image shows a rep-
resentative metaphase chromosome spread
from hFbh1-depleted cells. The inset shows a
magnification of an SCE event in the boxed
region. Efficiency of hFbh1 knockdown is
shown in Fig. S3. Bar, 10 µm. (B) Box plot
showing results of the experiment in A. The
increase in SCE events in hFbh1-depleted
cells was reproduced in an independent ex-
periment. At least 25 metaphase spreads were
analyzed in each experiment. (C) SCE assay
of cells as in B. 2.5 nM camptothecin (CPT)
was present in the medium throughout the
course of the experiment. (D) A hypothetical
model of the pro- and anti-recombinase func-
tion of hFbh1 at stalled replication forks (left)
and resected DSBs (right). See Materials and
methods for further details.
JCB • VOLUME 186 • NUMBER 5 • 2009 662
maintained as described previously (Mailand et al., 2007). IR (6 Gy) was
delivered using an x-ray generator (150 kV; 15 mA; dose rate, 2.18 Gy/min;
HF160; Pantak). To induce local UV damage, cells grown on coverslips
were irradiated with 100 J/m2 UV-C light in a Stratalinker (Agilent Technol-
ogies) through a polycarbonate filter with 5-µm pores (Millipore). Drugs
used in this study included 1 µg/ml DOX (EMD), 1 mM HU (Sigma-Aldrich),
and 10 µM BrdU (Sigma-Aldrich).
Immunoblotting, immunoprecipitation, and immunofluorescence were per-
formed as described previously (Mailand et al., 2006). To visualize gener-
ation of ssDNA upon HU treatment, cells were preincubated with BrdU for
24 h and subjected to BrdU immunofluorescence under nondenaturing con-
ditions. To remove soluble proteins before immunofluorescence, cells grown
on coverslips were preextracted for 5 min with ice-cold CSK buffer (10 mM
Pipes, pH 6.8, 300 mM sucrose, 100 mM NaCl, and 1.5 mM MgCl2) sup-
plemented with 0.5% Triton X-100 before fixation with 4% paraformalde-
hyde. To obtain chromatin-enriched cellular fractions, cells were lysed in
CSK buffer supplemented with 0.5% Triton X-100. Cell pellets were washed
once with CSK buffer, resuspended in 0.2 M HCl, and incubated at 4°C
for 2 h. The supernatant represented the chromatin-enriched fraction. A
rabbit polyclonal antibody to hFbh1 was raised against a peptide span-
ning amino acids 168–182 of hFbh1 (Eurogentec). Rabbit polyclonal
Rad51C antibody was provided by R. Kanaar (Erasmus Medical Center,
Rotterdam, Netherlands). Other antibodies used in this study included
mouse monoclonals to RPA (clone 9H8; NeoMarkers), BrdU (GE Health-
care), and Skp1 (Transduction Laboratories), rabbit polyclonals to Rad51
(H-92; Santa Cruz Biotechnology, Inc.), -H2AX (Millipore), cyclin A
(H-432; Santa Cruz Biotechnology, Inc.), full-length GFP (Santa Cruz Bio-
technology, Inc.), and human autoantibody to proliferating cell nuclear
antigen (Immuno Concepts). EdU (5-ethynyl-2-deoxyuridine) labeling was
performed by incubating cells with 10 µM EdU (Invitrogen) for 30 min fol-
lowed by fluorescent staining according to the manufacturer’s instructions.
Acquisition of confocal images was done essentially as described previ-
ously (Bekker-Jensen et al., 2006) using a confocal microscope (LSM 510;
Carl Zeiss, Inc.) fitted with a PPlan Neofluar 40× NA 1.3 oil immersion
objective (Carl Zeiss, Inc.). Laser microirradiation to generate DSBs in de-
fined nuclear volumes was performed as described previously (Bekker-
Jensen et al., 2006). For three-color imaging, GFP signals were combined
with secondary antibodies coupled to AlexaFluor dyes with excitation
wavelengths of 568 and 647 mm. Image acquisition and processing were
performed with LSM 510 software (Carl Zeiss, Inc.). Quantification of
Rad51 foci and BrdU was performed using custom-made macro routines.
In brief, images were acquired using a fluorescence microscope (Axio-
plan II; Carl Zeiss, Inc.) equipped with a Plan Neofluar 40× 1.3 oil immer-
sion objective. Exposure time, binning, and settings of the microscope and
light source were kept constant for all the samples. Photoshop (Adobe)
was used to select single cells based on either EdU (for Rad51 foci) or
DAPI (for BrdU staining). Each selected cell was subsequently analyzed
using ImageJ software (National Institutes of Health) to measure the signal-
integrated density of either Rad51 or BrdU staining. The numerical data
were further processed in Excel (Microsoft) by mathematical operations,
and statistical analysis of paired datasets was performed using t test
(Prism; GraphPad Software, Inc.). The entire procedure was described
previously in Mistrik et al. (2009).
EMSA was performed essentially as described previously (Modesti et al.,
2007). In brief, bacterially purified GST-hFbh1 constructs were incubated
with 32P-labeled ssDNA or dsDNA probes (2 nM) produced by standard
methods in binding buffer (20 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.1 mg/ml
BSA, and 2 mM DTT) at 30°C for 15 min. Samples were resolved on native
TBE polyacrylamide gels, dried, and visualized by autoradiography. DNA
probes used in EMSA were X0-1, 5-GACGCTGCCGAATTCTACCAGT-
GCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC-3; and X0-1c,
HR rates were measured essentially as described previously (Sartori et al.,
2007). In brief, a U2OS derivative cell line harboring an integrated HR
reporter construct (DR-GFP) was cotransfected with plasmids expressing
RFP, I-SceI, and, where indicated, hFbh1 for 48 h. Transfection of RFP
by an increase in the SCE (Branzei and Foiani, 2007). The simi-
lar functions of hFbh1 and BLM prompted us to explore whether
hFbh1 also regulates SCE. Knockdown of hFbh1 in primary fibro-
blasts led to a small but significant increase in SCE compared
with control cells (5.6 and 4.1 events/cell, respectively; Fig. 5,
A and B), an effect augmented by exposure to camptothecin
(34 vs. 22 events/cell, respectively; Fig. 5 C). This is consistent
with data in Fbh1-deficient chicken DT40 cells, which also ex-
hibit increased SCE (Kohzaki et al., 2007), and further supports
the notion that hFbh1 is a bonafide anti-recombinogenic factor
required to restrain unscheduled HR, slight elevations of which
may be sufficient to compromise genomic integrity. The exis-
tence of several helicases with anti-recombinogenic functions
in human cells may account for a higher degree of redundancy
in this system and could explain the relatively mild SCE pheno-
type observed in hFbh1 knockdown cells.
In conclusion, our findings provide evidence that hFbh1
functions as a regulator of HR repair in human cells and sug-
gest a model of how hFbh1 exerts such a function (Fig. 5 D).
In the first step, hFbh1 is recruited to genotoxic stress-induced
ssDNA. By means of its helicase activity, hFbh1 may help to
facilitate ssDNA production to the extent required for produc-
tive HR, and up to this stage, hFbh1 can function to promote HR
initiation. However, in later stages, hFbh1 restrains excessive
and/or unscheduled HR through its ability to displace Rad51
from ssDNA. Such a dual role in the multistep process of HR
has also been proposed for the BLM helicase (Bugreev et al.,
2007). hFbh1 may increase the pool of cellular pro- and anti-
recombinase activities and thus contributes to the maintenance
of genomic integrity.
Materials and methods
Plasmids and RNAi
A partial cDNA spanning amino acids 126–1,094 of hFbh1 was provided
by Y.-S. Seo (Korea Advanced Institute of Science and Technology,
Daejeon, Korea). The N-terminal part of hFbh1 cDNA (amino acids 1–125)
was amplified from QUICK-Clone Human Universal cDNA (Clontech Labo-
ratories, Inc.). The full-length hFbh1 was constructed by overlapping PCR of
these fragments and was inserted into pEGFP-C1 (Clontech Laboratories,
Inc.). From this construct, GFP-tagged hFbh1 was amplified by PCR and
subcloned into pcDNA4/TO (Invitrogen) to allow DOX-inducible expres-
sion of GFP-hFbh1. To enforce nuclear expression of GFP-hFbh1, GFP-
hFbh1 constructs were subcloned into pcDNA4/TO harboring three
N-terminal NLSs from pCMV/Myc/nuc (Invitrogen). Introduction of point
mutations to generate the F box (L278A and P279A) and helicase (D698N)
mutants of hFbh1 (Osman et al., 2005) was performed using the Quik-
Change site-directed mutagenesis kit (Agilent Technologies). To produce
GST-tagged hFbh1, full-length versions of WT or mutant hFbh1 were sub-
cloned into pGEX-6P1 (Invitrogen). Plasmid transfections were performed
using FuGene6 (Roche).
The following siRNA sequences were used in this study: hFbh1#1,
5-GAUACAGAGUGAAGAAUGU-3; hFbh1#2, 5-GGGAUGUUCUUU-
UGAUAAAUU-3; Rad51, 5-GAGCUUGACAAACUACUUC-3; Rad51C,
5-GUUCAGCACUAGAUGAUAU-3; CtIP, 5-GCUAAAACAGGAAC-
GAAUC-3; and control, 5-GCGCGCUUUGUAGGAUUCG-3. All siRNA
duplexes (Thermo Fisher Scientific) were transfected at a final concentra-
tion of 100 nM using Lipofectamine RNAiMAX (Invitrogen) according to
the manufacturer’s instructions for U2OS and BJ cells.
Human U2OS osteosarcoma cells and primary BJ fibroblasts were cul-
tured in DME containing 10% fetal bovine serum. U2OS derivative cell
lines expressing NLS)GFP–tagged hFbh1 constructs in a DOX-inducible
fashion from pcDNA4/TO-NLS–GFP-hFbh1 vectors were generated and
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Online supplemental material
Fig. S1 shows characterization of GFP-hFbh1–expressing cell lines,
Fig. S2 shows characterization of hFbh1 mutants, and Fig. S3 shows ef-
ficiency of RNAi-mediated knockdown of hFbh1 in BJ fibroblasts. Online
supplemental material is available at http://www.jcb.org/cgi/content/
We thank Dr. Yeon-Soo Seo for providing reagents.
This work was supported by grants from the Danish Cancer Society, the
Danish National Research Foundation, the Danish Research Council, the Czech
Ministry of Education (MSMT6198959216), the Grant Agency of the Czech
Academy of Sciences (IAA501370902), the Lundbeck Foundation (R13-A1287),
the European Commission (integrated projects “DNA Repair” and “GENICA”),
and the John and Birthe Meyer Foundation.
Submitted: 22 December 2008
Accepted: 7 August 2009
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