The Rockefeller University Press $30.00
J. Cell Biol. Vol. 192 No. 5 735–750
Correspondence to Roland Kanaar: email@example.com; or Jeroen Essers:
Abbreviations used in this paper: DSB, double-strand break; ES, embryonic
stem; FCS, fluorescence correlation spectroscopy; iFRAP, inversed FRAP; RPA,
replication protein A.
To preserve the integrity of their genome, cells have evolved
several pathways to deal with DNA damage that is created by
both endogenous sources, such as some byproducts of cellular
metabolism like oxygen radicals, and exogenous sources, in-
cluding ultraviolet and ionizing radiation (Friedberg et al.,
2004). Among different kinds of lesions, DNA double-strand
breaks (DSBs) present a special challenge to the cells because
both strands of the double helix are affected. If misrepaired,
DSBs can cause genome rearrangements such as transloca-
tions and deletions that can result in development of cancer
(Hoeijmakers, 2001; Bassing and Alt, 2004; Agarwal et al.,
2006). Thus, it is paramount that DSBs are repaired precisely
and in a timely fashion.
Homologous recombination is an error free, high-fidelity
pathway that repairs DSBs by using an undamaged homologous
DNA molecule, usually the sister chromatid, as a template to
repair the broken molecule (Wyman and Kanaar, 2006).
The process is performed by the Rad52 epistasis group proteins,
identified by the genetic analyses of ionizing radiation–sensitive
Saccharomyces cerevisiae mutants (Game and Mortimer, 1974;
Symington, 2002). Several Rad52 group proteins, including
Rad51 and Rad54, are conserved in mammals, as is the core
mechanism of homologous recombination (Wyman and Kanaar,
2004). The central protein of homologous recombination is
Rad51, which mediates the critical step of homologous pairing
and DNA strand exchange between the broken DNA molecule
and the homologous intact repair template. Once a DSB occurs,
it is processed to single-stranded DNA tails with a 3 polarity,
onto which Rad51 promoters assemble into a nucleoprotein
filament. This nucleoprotein filament is the active molecular
entity in recognition of homologous DNA and the subsequent
exchange of DNA strands. An extensive number of mediator
and/or accessory proteins are implicated in assisting Rad51 at
various stages of recombination (Sung et al., 2003), one of
which is Rad54.
bination. Here we demonstrate that Rad54 is required for
the timely accumulation of the homologous recombination
proteins Rad51 and Brca2 at DSBs. Because replication
protein A and Nbs1 accumulation is not affected by
Rad54 depletion, Rad54 is downstream of DSB resec-
tion. Rad54-mediated Rad51 accumulation does not
require Rad54’s ATPase activity. Thus, our experiments
demonstrate that SWI/SNF proteins may have functions
ad54, a member of the SWI/SNF protein family of
DNA-dependent ATPases, repairs DNA double-
strand breaks (DSBs) through homologous recom-
independent of their ATPase activity. However, quantita-
tive real-time analysis of Rad54 focus formation indicates
that Rad54’s ATPase activity is required for the disassocia-
tion of Rad54 from DNA and Rad54 turnover at DSBs.
Although the non–DNA-bound fraction of Rad54 revers-
ibly interacts with a focus, independent of its ATPase sta-
tus, the DNA-bound fraction is immobilized in the absence
of ATP hydrolysis by Rad54. Finally, we show that ATP
hydrolysis by Rad54 is required for the redistribution of
DSB repair sites within the nucleus.
ATP-dependent and independent functions of Rad54
in genome maintenance
Sheba Agarwal,1 Wiggert A. van Cappellen,2 Aude Guénolé,1 Berina Eppink,1 Sam E.V. Linsen,1 Erik Meijering,3,4
Adriaan Houtsmuller,5 Roland Kanaar,1,6 and Jeroen Essers1,6,7
1Department of Cell Biology and Genetics, Cancer Genomics Center; 2Department of Reproduction and Development; 3Department of Medical Informatics and
4Department of Radiology, Biomedical Imaging Group Rotterdam; 5Department of Pathology; 6Department of Radiation Oncology; and 7Department of Vascular Surgery,
Erasmus Medical Center, 3000 CA Rotterdam, Netherlands
© 2011 Agarwal 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 pub-
lication date (see http://www.rupress.org/terms). 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 192 • NUMBER 5 • 2011 736
nature, composition, and requirement for foci formation is not
apparent from a biochemical point, it is clear that the foci, par-
ticularly of Rad51, are biologically relevant because mutant
cells that cannot form them are DNA damage–sensitive and
display spontaneous chromosomal aberrations (Thacker and
Zdzienicka, 2004; van Veelen et al., 2005). The nature of these
foci with respect to protein composition is highly dynamic.
Photobleaching experiments have shown that Rad51 and Rad54
dissociate and associate with foci, with each protein having a
characteristic dwell time (Essers et al., 2002b).
Here, we studied the ATPase function of Rad54 in homol-
ogous recombination in vivo. To this end we compared knockin
mouse ES cell lines that express GFP-tagged wild-type Rad54
protein with cell lines expressing ATP hydrolysis–defective
Rad54 proteins using advanced live-cell confocal microscopy.
Time-lapse imaging and fluorescence correlation spectroscopy
(FCS) of individual cells and foci allowed quantitative analysis
of the Rad54 in live cells. We measured protein concentrations
and discovered that DSB repair foci relocalize to the nuclear
periphery upon DNA damage induction. In addition, we used
fluorescence photobleaching techniques to show that defective
ATPase activity renders a small fraction of the Rad54 molecules
in a focus immobile. This suggests that only a minority of mol-
ecules present in foci are functional in DNA DSB repair and
that the ATPase activity of Rad54 is required for the release of
protein from DNA damage–induced structures on chromatin.
The ATPase activity of Rad54 contributes
to DNA damage resistance and
To study the effect of the ATPase activity of Rad54 at the cellu-
lar level, mouse ES cells were generated that express wild-type
and ATPase-defective versions of GFP-fused Rad54 from the
endogenous Rad54 locus. A targeting construct, consisting of
the human RAD54 cDNA exons IV–XVIII fused to a GFP cod-
ing sequence or containing a point mutation in the Walker A
ATPase domain (Fig. 1 A), was electroporated into ES cells of
the genotype Rad54wt-HA/, in which one Rad54 allele is inacti-
vated (Tan et al., 1999). Two different mutant constructs were
used, one in which the lysine at position 189 was replaced by
arginine, which is indicated by K189R and one in which the ly-
sine is replaced by alanine, the K189A mutation. The ATPase
activity of the purified Rad54K189R and Rad54K189A proteins was
reduced >100-fold in comparison to the wild-type protein
(Swagemakers et al., 1998 and unpublished data). Clones carry-
ing a homologously integrated knockin construct were identi-
fied by DNA blot analysis. A probe that detects exons VII and
VIII was used in combination with genomic DNA digested with
StuI, which yielded the expected doublet of bands 6.5 kb for
the Rad54 knockin allele, whereas a 6.0-kb band was observed
that is diagnostic for the Rad54 knockout allele (Fig. 1 B).
Proper expression of the full-length wild-type or mutant Rad54–
GFP fusion proteins was confirmed by immunoblot analysis
(Fig. 1 C). In the subsequent studies, two independent clones
for Rad54K189R-GFP/ and one for Rad54K189A-GFP/ were used.
RAD54, first identified in S. cerevisiae, is conserved in
vertebrates (Kanaar et al., 1996; Bezzubova et al., 1997; Essers
et al., 1997). Rad54 is a member of the SWI2/SNF2 family of
double-strand DNA-stimulated ATPases that modulate protein–
DNA interactions (Pollard and Peterson, 1998). Rad54/
mouse embryonic stem (ES) cells are ionizing radiation–sensitive,
display reduced level of homologous recombination, and ex-
hibit defects in repair of DSBs (Essers et al., 1997; Dronkert
et al., 2000). A plethora of biochemical activities of Rad54 have
been uncovered that have the potential to augment the central
function of Rad51 in homologous recombination (Tan et al.,
2003; Heyer et al., 2006). First, Rad54 physically interacts
with Rad51, both in vitro and in vivo (Jiang et al., 1996; Clever
et al., 1997; Golub et al., 1997; Tan et al., 1999). Interestingly,
in mammalian cells, the interaction between the proteins can
only be detected in cells that have been challenged with DNA-
damaging agents, which suggests that Rad54 interacts with
the Rad51 nucleoprotein filament rather than Rad51 protomers
that are not engaged in recombination (Tan et al., 1999; Essers
et al., 2002a). Second, the interaction is not only physical but
also functional, as Rad54 stimulates Rad51 mediated D-loop
formation, i.e., the generation of a joint between homologous
DNA molecules (Petukhova et al., 1998). Third, Rad54 has a
potent ATPase activity that is triggered specifically by double-
stranded DNA (Petukhova et al., 1998; Swagemakers et al.,
1998). Fourth, the protein uses energy gained from ATP hydro-
lysis to translocate along the DNA double helix (Van Komen
et al., 2000; Ristic et al., 2001; Amitani et al., 2006). Fifth,
presumably through its DNA translocase activity, Rad54 can
affect the interaction of proteins with DNA. Specifically, it can
influence the position of histones on DNA and remove Rad51
nucleoprotein filaments from double-stranded DNA (Alexiadis
and Kadonaga, 2002; Solinger et al., 2002; Alexeev et al., 2003;
Jaskelioff et al., 2003; Li and Heyer, 2009; Li et al., 2009).
Sixth, its translocase activity also allows the protein to perturb
DNA structures. Rad54 can promote branch migration, thereby
affecting the processing of the Holliday junction, which is a
four-way DNA junction that can arise as intermediates at sites
where the recombination partners are physically joined (Bugreev
et al., 2006). Many of the biochemical activities of Rad54 are
affected by abrogating its ATPase activity. Hence, the proper
functioning of Rad54 depends on its ability to harness the
energy from ATP hydrolysis, and this in turn is responsible for
augmenting the role of Rad51. However, Rad51 nucleoprotein
filament stabilization by Rad54, which is probably required
before joint molecule formation occurs, turned out to be inde-
pendent of its ATPase activity (Mazin et al., 2003).
A striking characteristic of several proteins involved
in homologous recombination, including Rad51 and Rad54, is
their ability to accumulate at a high local concentration into nu-
clear foci (Haaf et al., 1995; Tan et al., 1999; Essers et al., 2006).
This occurs spontaneously, that is in the absence of exogenously
induced DNA damage, in a low percentage of cells in S phase
(Tashiro et al., 2000; Tarsounas et al., 2003). Upon the induc-
tion of DNA damage to cells, the majority of cells display co-
localizing Rad51 and Rad54 foci at sites of DNA damage (Tan
et al., 1999; Tashiro et al., 2000; Aten et al., 2004). Although the
737In vivo roles of the Rad54 recombination protein • Agarwal et al.
As a positive control for all experiments, knockin Rad54wt-GFP/
ES cells were used; these cells express wild-type Rad54
fused to GFP from the endogenous Rad54 locus. The function
Figure 1. Characterization of mouse ES cells carrying ATPase-defective Rad54–GFP alleles. (A) Schematic representation of the mouse Rad54 locus and
the gene-targeting constructs. The top line represents a 30-kb portion of endogenous Rad54 locus, where black boxes indicate exons I–XVIII. The middle
line shows the linearized targeting construct, containing the human RAD54 cDNA sequence spanning exons IV–XVIII fused to the GFP coding sequence.
The K189R and K189A mutations in the Walker A ATPase domain are indicated by the asterisks. The construct contains a gene encoding for puromycin
resistance as a selectable marker. The targeting construct will replace the regions between exons III and VIII when correctly integrated to generate the
targeted allele, as shown in the targeted locus. Homologous integration results in the expression of full-length, GFP-tagged Rad54 from its endogenous
promoter. (B) DNA blot analysis of ES cells carrying the knockin contructs. DNA blot analysis was performed using genomic DNA purified from puromycin-
resistant clones and digested with StuI. Detection of bands was performed using a probe that recognized exons VII/VIII. Restriction of the wild-type allele
by StuI, (indicated by “+”), yields a 9.0-kb band after hybridization with an exon VII/VIII probe. Diagnostic bands for the neomycin-resistant knockout
alleles, indicated by ““, are 7.6 kb for a hygromycin-resistant allele and 6.0 kb for a neomycin-resistant allele. Knockin alleles are characterized by
a doublet of bands 6.5 kb. (C) Immunoblot analysis of proteins produced by the Rad54–GFP knockin and -out alleles. Whole cell extracts of ES cells
with the indicated genotypes were probed with affinity purified anti–human Rad54 antibodies. The position of Rad54 and Rad54–GFP are indicated. The
arrowhead indicates a nonspecific signal. Probing against Msh6 and actin was used to confirm equal protein loading. (D) Ionizing radiation and mitomycin
C survivals. ES cells of the indicated genotypes were tested for their ability to survive treatments with increasing doses of ionizing radiation ( irradiation)
or mitomycin C using clonogenic survival assays. The assays were performed in triplicate and the error bars indicated the standard error of the mean.
of Rad54 is not affected by its fusion to GFP because
Rad54wt-GFP/ cells are not DNA damage sensitive (unpub-
JCB • VOLUME 192 • NUMBER 5 • 2011 738
protein (Fig. 2 A). It should be noted that the increase in spontane-
ous foci is only observed when all Rad54 molecules in the cell are
ATPase defective, as such an increase is absent in Rad54K189R-GFP/+
and Rad54K189A-GFP/+ cells (unpublished data).
Rad54 is an accessory protein for Rad51 that performs the
core reaction of homologous recombination, homologous DNA
pairing, and DNA strand exchange (Wyman and Kanaar, 2006).
The proteins physically interact and work closely together in sev-
eral biochemical assays (Sung et al., 2003). At the cellular level,
both proteins colocalize in DNA damage–induced foci (Tan et al.,
1999). We analyzed Rad51 foci in the mutant cells to determine
whether the ATPase activity of Rad54 impacted the behavior of
Rad51 in vivo. Unchallenged ES cells were fixed and stained with
an antibody against Rad51, and both Rad51 and Rad54–GFP were
detected by confocal microscopy (Fig. 2 B). Compared with cells
expressing wild-type Rad54–GFP or lacking Rad54, cells ex-
pressing the ATPase-defective variants of Rad54–GFP displayed
a statistically significant twofold increase in the number of “spon-
taneous” Rad51 foci. Furthermore, almost all Rad54 foci detected
(>90%), including those of Rad54K189R-GFP and Rad54K189A-GFP,
colocalized with Rad51 (Fig. 2 B). Interestingly, although the
number of Rad51 foci per confocal slice was elevated in cells
expressing the ATPase-defective Rad54 mutants, it was not
increased in cells completely lacking Rad54 (Fig. 2 B).
The increase in spontaneous Rad54 foci
does not represent increased endogenous
Spontaneous foci, including Rad51 and Rad54, that are ob-
served in unchallenged cells are thought to be present at sites
of spontaneous DSBs such as those that might occur at im-
paired DNA replication forks (Cox et al., 2000; Tarsounas
et al., 2003; Budzowska and Kanaar, 2009). Therefore, we
asked whether the increased number of spontaneous foci de-
tected in cells was caused by accumulated unrepaired DNA
damage in these cells by determining whether the foci colocal-
ized with DNA damage. First, we investigated the levels of
H2AX, an early marker for DSBs, in whole cell extracts from
wild-type and mutant cell lines. Anti-H2AX antibodies rec-
ognize a specific phosphorylation on the histone variant H2AX
that is triggered by certain types of DNA damage, including DSBs
(Rogakou et al., 1998). However, no increase in the level of H2AX
phosphorylation was detected by immunoblotting in populations
of unchallenged Rad54K189R-GFP/ and Rad54K189A-GFP/ compared
with Rad54wt-GFP/ ES cells (Fig. 3 A, left). The cells expressing
ATPase-defective Rad54 were able to increase H2AX phosphory-
lation upon treatment with ionizing radiation (Fig. 3 A, right).
In addition, we analyzed H2AX phosphorylation by immuno-
fluorescence experiments (Fig. 3 B). Untreated Rad54wt-GFP/ and
Rad54K189R-GFP/ ES cells displayed similar levels of H2AX
foci, which is consistent with the H2AX immunoblotting re-
sults. In addition to H2AX, we also did not observe an in-
crease in the number of foci for the DNA damage marker 53BP1
(Fig. 3 C). We conclude that the increase in spontaneous Rad54
foci in unchallenged Rad54K189R-GFP/ and Rad54K189A-GFP/ ES
cells is unlikely to be caused by a dramatically increased level
of unrepaired DNA damage.
Mouse Rad54/ ES cells are hypersensitive to ionizing
radiation and the interstrand DNA cross-linker mitomycin C
(Essers et al., 1997). Therefore, we investigated the effect of these
DNA-damaging agents on Rad54K189R-GFP/ and Rad54K189A-GFP/
ES cells. Cells expressing ATPase-defective versions of Rad54
were hypersensitive to ionizing radiation and mitomycin C
compared with isogenic control cells; this hypersensitivity was
similar to that demonstrated by cells lacking Rad54 protein al-
together (Fig. 1 D). Next we tested the effect of Rad54 ATPase
activity on homologous recombination. As a measure of homol-
ogous recombination efficiency, we determined the efficiency
of homologous gene targeting (Niedernhofer et al., 2001; Essers
et al., 2002a). ES cells of the genotypes indicated in Table I
were electroporated with a linearized targeting construct for the
Rb locus that carried a hygromycin-selectable marker gene.
Genomic DNA was isolated from individual clones and ana-
lyzed by DNA blotting to discriminate between homologous
and random integration events. Homologous recombination effi-
ciency was measured as the percentage of clones containing the
homologously integrated targeting construct relative to the total
number of drug-resistant clones analyzed (Table I). The homol-
ogous targeting efficiency of 32% in Rad54wt-GFP/ ES cells
was reduced to 1% in Rad54K189R-GFP/ and Rad54K189A-GFP/
ES cells. A similar reduction in homologous recombination
efficiency was observed in the absence of Rad54. We conclude
that the ATPase activity of Rad54 is essential for its DNA repair
and recombination functions in vivo. In these assays, the physi-
cal presence of ATPase-defective Rad54 or the complete ab-
sence of the protein results in indistinguishable phenotypes.
ATP hydrolysis by Rad54 affects foci
behavior in unchallenged cells
Several proteins involved in the cellular response to DNA damage
and repair are known to accumulate in nuclear foci at sites of DNA
damage (Wyman and Kanaar, 2006). We analyzed the ATPase-
defective Rad54 mutant cells for accumulation of Rad54 foci in
the absence of exogenously induced DNA damage. Observation
of living cells using a confocal microscope revealed an increase
in the amount of foci present in cells containing ATPase-deficient
Rad54K189R and Rad54K189A protein compared with wild-type Rad54
Table I. Efficiency of homologous recombination in Rad54
GenotypeTargeting efficiency at Rb locus
31.9% (16 out of 46)
0.95% (1 out of 105)
1.2% (1 out of 81)
<1% (0 out of 82)
ES cells with the indicated genotypes were electroporated with a linearized
Rb-Hyg construct (Niedernhofer et al., 2001). Hygromycin-resistant clones were
expanded, genomic DNA was purified, and samples were subjected to DNA
blot analysis to distinguish between randomly and homologously integrated
events. Values indicate the percentage of clones that contain the homologously
integrated targeting construct relative to the total number of clones analyzed.
Absolute numbers are indicated in parentheses. The differences in recombina-
tion efficiency between Rad54WT-GFP/ cells and cells of all other genotypes listed
are significant (P > 0.001), whereas the difference between the mutant geno-
types is not.
739In vivo roles of the Rad54 recombination protein • Agarwal et al.
Figure 2. Effect of Rad54 ATPase activity on focus formation. (A) Shown are confocal images of untreated living ES cells expressing wild-type or ATPase-
defective Rad54–GFP. The mean number of spontaneous nuclear foci in cells expressing either version of ATPase-defective Rad54 is considerably greater
compared with cells expressing wild-type Rad54. (B) Rad51 immunostaining in untreated ES cells of the indicated genotypes. The top shows confocal
images of Rad54 as detected by GFP fluorescence. The middle shows the Rad51 staining pattern as detected by anti-Rad51 antibody staining. The merged
images are shown on the bottom. The number of Rad51 foci per cell is indicated (mean ± SD). The difference in number of Rad51 foci per cell between
Rad54wt-GFP/ and Rad54/ ES cells and the difference between Rad54K189R-GFP/ and Rad54K189A-GFP/ ES cells is not significant, whereas the difference
between these two groups is (P < 0.0001), as determined by the one-way analysis of variance (ANOVA) and a Student’s t test. Bars, 10 µm.
JCB • VOLUME 192 • NUMBER 5 • 2011 740
interact with the structures, with residence times ranging from
a couple of seconds for Rad54 to several minutes for Rad51
(Essers et al., 2002b). To determine whether the ATPase activity
of Rad54 influences this mobility, we analyzed Rad54 interaction
with foci using photobleaching techniques. For this purpose, we
Lack of ATPase activity of Rad54 affects
dynamic interaction with nuclear foci
A remarkable feature of DNA damage–induced foci is their highly
dynamic nature. We previously showed that these accumulations
of proteins are not static but that their components dynamically
Figure 3. Analysis of H2AX and 53BP1 in Rad54 ATPase-defective ES cells. (A) Whole cell extracts of ES cells with the indicated genotype were either
not treated (left) or harvested 1 h after irradiation with 8 Gy (right) and analyzed by immunoblotting using an anti-H2AX antibody. Antibodies against
Ku80 were used to confirm equal loading (bottom). (B) Quantification of the mean number of H2AX foci in untreated Rad54wt-GFP/ and Rad54K189R-GFP/
ES cells. Error bars indicates standard error of the mean. (C) Immunofluorescence detection of 53BP1 in untreated ES cells. The top shows 53BP1 staining in
Rad54wt-GFP/, Rad54K189R-GFP/, and Rad54K189A-GFP/ ES cells, the middle shows the GFP staining, and the panel shows the merged images. Bar, 10 µm.
741 In vivo roles of the Rad54 recombination protein • Agarwal et al.
fluorescence was aimed to photobleach the fluorescence of a
complete nucleus with the exception of one individual focus
and a region of similar size in which no focus was present. The
fluorescence intensity in the two regions (indicated in Fig. 4 B,
red circle and blue circle, respectively) in the nucleus was mea-
sured after the bleach pulse. The observed loss of fluorescence
of the unbleached focus (Fig. 4 B, red circle) was then monitored
and compared with the fluorescence level of the unbleached re-
gion without a focus (blue circle). Measurement of the residence
time of Rad54 in the nuclear focus revealed a complete exchange
of wild-type Rad54 molecules in a focus but a stably associ-
ated fraction for the Rad54K189R-GFP mutant, confirming the spot-
FRAP measurements (Fig. 4 C). Thus, although the mobility of
the vast majority is hardly affected, a fraction of RAD54 pro-
teins is permanently immobilized if it cannot hydrolyze ATP.
The Rad54 protein, but not its
ATPase activity, affects Rad51
recruitment to DSBs
Biochemical experiments have demonstrated that Rad54 stim-
ulates D-loop formation by Rad51 (Petukhova et al., 1998).
These and other results have led to the suggestion that Rad54 is
used a spot-FRAP protocol in which a small square encompassing
a single Rad54-containing focus was bleached and subsequently
monitored. We quantified and compared the fluorescence recov-
ery of ATPase-proficient and defective Rad54 foci (Fig. 4 A). The
bleaching protocol led to the irreversible bleaching of 20% of
the total pool of fluorescent RAD54 in the nucleus. In the experi-
ment shown in Fig. 4 A, the final measured fluorescence inten-
sity was normalized to the prebleach pulse fluorescence intensity.
The fluorescence in the bleached area recovered to 80% for the
wild-type Rad54–GFP protein in the bleached focus. In contrast,
ATPase-defective Rad54K189R-GFP was present in foci in two dis-
tinct kinetic pools; a transiently immobile fraction (90%) similar
to wild-type Rad54–GFP (Misteli, 2001), and a permanently im-
mobilized fraction (10%), not observed in wild type. The t1/2 of
ATPase proficient foci was 0.9 ± 0.06 s, which represents a faster
recovery as compared with Rad54K189A-GFP/ and Rad54K189R-GFP/
(1.3 ± 0.10 s).
To obtain independent confirmation of the dynamic be-
havior of the wild-type Rad54 and Rad54K189R-GFP proteins in
the DNA damage–induced structures, we examined them
using inversed FRAP (iFRAP; Houtsmuller and Vermeulen,
2001). In these experiments, the laser pulse used for bleaching
Figure 4. Photobleaching analysis of Rad54 in foci. (A) Spot-FRAP analysis of Rad54 in foci. A small square containing an individual Rad54 focus was
bleached and monitored for fluorescence recovery for each indicated genotype (n = 35). As a control, the fluorescence recovery of non–foci-associated
nuclear Rad54 was quantitated (dark blue line). (B) Visualization of iFRAP analysis of Rad54 in foci. The whole cell was bleached excluding a small
circular area containing an individual Rad54 focus (red circle). Fluorescence depletion of the nonbleached focus was monitored and compared with the
fluorescence level of an unbleached region without a focus (blue circle) for Rad54wt-GFP and Rad54K189A-GFP. Shown here are four frames: before, during,
directly after, and 9 s after bleaching. Bar, 5 µm. (C) Quantification of the iFRAP experiment described in B. Graphs represent the fluorescent depletion
over time and are based on five individual cells.
JCB • VOLUME 192 • NUMBER 5 • 2011 742
was depleted from U2Os cells (Fig. 5 B), and cells were sub-
sequently irradiated with particles. Over time, there was an
increased localization of repair proteins acting early in homolo-
gous recombination, including Nbs1 and replication protein A
(RPA), as well as Rad51 and BRCA2, to the sites of damage
(Fig. 5 C). Upon Rad54 depletion, we observed a transient but
considerable delay or partial impairment in the recruitment of
Rad51 and BRCA2, but not of RPA and Nbs1, indicating at the
cellular level that Rad54 affects a specific step in the progres-
sion of homologous recombination. This delayed accumulation
of Rad51 at DSBs was also observed in ES cells deficient in
Rad54 (Rad54/) but, remarkably, was independent of Rad54
ATPase activity, as both Rad54wt-GFP/ and mutant Rad54K189A-GFP/
knockin ES cells showed similar kinetics for Rad51 accumula-
tion at sites of DNA damage as wild-type ES cells (Fig. 5 D).
These results show that timely accumulation of Rad51 in foci at
sites of DNA damage is dependent on the Rad54 protein but not
on its ATPase activity.
important to target the Rad51 nucleoprotein filament to ho-
mologous duplex DNA, where it will then engage its ATPase
activity to promote repair of the DSB (Heyer et al., 2006). To
test whether Rad54 is involved in targeting of Rad51 to the site
of DSBs in vivo, we subjected a human osteosarcoma cell line
(U2Os) to local particle irradiation using an 241Am source
(Aten et al., 2004; Stap et al., 2008). particle irradiation can
be especially useful to analyze the initial response to DNA dam-
age because it can be used to induce DNA damage locally and
in a defined pattern, allowing a clear distinction between al-
ready existing foci and local protein accumulations caused by
the induced DNA damage. Straight tracks of DSBs due to the inter-
action of the particle with chromatin can be visualized using
antibodies against H2AX (Rogakou et al., 1998) or 53BP1
(Bekker-Jensen et al., 2006). Using this method, we showed that
Rad54 localized to the DSB sites (Fig. 5 A). Next, we wanted to
analyze the effect of the accumulation of other repair proteins
to the tracks in the presence and absence of Rad54. Rad54
Figure 5. The Rad54 protein, but not its
ATPase activity, affects Rad51 recruitment to
sites of DSBs. Accumulation of DSB repair pro-
teins at particle–induced DSB tracks. (A) Local-
ization of Rad54 to the particle–induced
double-stranded break colocalizing with DSB
marker H2Ax. Bar, 5 µm. (B) RAD54 protein
levels in U2Os cells transfected with indicated
siRNAs. Cell lysates were analyzed by immuno-
blotting with antibodies against RAD54.
Equal sample loading was verified by the
equal presence of nonspecific bands. (C) Quanti-
fication of accumulation of Nbs1, RPA, Rad51,
and BRCA2 at particle–induced tracks of
DSBs 0, 5, 15, and 60 min after irradiation
in the presence or absence Rad54. U2Os
cells were stained for either H2Ax (Nbs1
and Rad51) or 53BP1 (RPA and Brca2) as a
DSB marker and for one of the indicated re-
pair proteins at t = 0, 5, 15, and 60 min after
irradiation. t = 0 indicates the first time point
after particle irradiation. Graphs represent
mean percentage of positive DSB tracks with a
repair protein. 100 cells containing particle–
induced tracks were scored per experiment.
Error bars represent the range of percentages
obtained from three independent experiments.
(D) Quantification of Rad51 accumulation
at DSB sites 0, 5, 15, and 60 min after
particle irradiation in Rad54+/+, Rad54/,
Rad54wt-GFP/, and Rad54K189R-GFP/ ES cells.
Graphs represent mean percentage of Rad51-
positive tracks per H2Ax track. 100 cells con-
taining damage induced by particles were
scored per experiment. Error bars represent
the range of percentages obtained from two
In vivo roles of the Rad54 recombination protein • Agarwal et al.
number of Rad54 foci was back at the baseline level (Fig. 6 B).
Interestingly, the kinetics of ATPase-defective Rad54 foci dis-
appearance was reduced compared with wild-type Rad54 foci,
as indicated by a comparison of the time required for a two-
fold reduction in the number of Rad54 foci from their peak
values. This twofold reduction occurred 6 h after the irra-
diation in Rad54wt-GFP/ cells, whereas it took at least 25 h in
Rad54K189R-GFP/ ES cells. The number of foci in Rad54 ATPase-
defective cells also increased upon irradiation, but over time
stayed at much higher levels. This indicates that Rad54’s ATPase
Real-time analysis of Rad54–GFP foci
To investigate whether the ATPase activity of Rad54 influenced
the kinetics of Rad54 foci, we used a global irradiation protocol
using a 137CS source followed by prolonged time-lapse analyses
of irradiated cells. To this end, time-lapse movies were made
of Rad54wt-GFP/ and Rad54K189R-GFP/ ES cells starting 45 min
after irradiation with 2 Gy (Fig. 6 A and Material and methods).
In wild-type Rad54wt-GFP/ cells, the number of Rad54 foci
per cell increased fivefold upon induction of DNA damage
in the first 2 h and then declined. 12 h after irradiation, the
Figure 6. Quantification of Rad54 foci over
time in response to irradiation. (A) Time-lapse
imaging of irradiated Rad54wt-GFP/ and
Rad54K189R-GFP/ ES cells. Cells were treated
with 2 Gy and imaged every 15 min starting
45 min after irradiation. Each picture rep-
resents a frame in the resulting movie at the
indicated time point. (B) Quantification of
the number of foci per cell over time based
on the movies represented in A. Quantification
was performed using ImageJ as described in
the Materials and methods. Error bars indicate
SD. Bars, 10 µm.
JCB • VOLUME 192 • NUMBER 5 • 2011 744
over time. In contrast, the number of molecules also increased
in the Rad54K189R-GFP/ mutant cells but then failed to decrease
Rad54 influences relocalization of DSBs to
the nuclear periphery
In yeast, persistent DNA breaks are fixed over time at the nu-
clear periphery (Kalocsay et al., 2009; Oza et al., 2009; Oza
and Peterson, 2010). Our targeted knockin approach allowed
tracking of the nuclear localization of Rad54 foci over time to
determine their cellular localization in mammalian stem cells.
To this end, we used the time-lapse movies recorded after DNA
damage induction by ionizing radiation described and analyzed
in Fig. 6 (A and B). In cells followed for at least 15 consecu-
tive time points, the relocalization of the DNA damage–induced
foci was analyzed (Fig. 8 A). For each focus, the center of mass
was determined in 2D. Next, the distance of the focus location
to the center of mass of the cell was calculated. The foci were
then classified in three distance classes: “inside,” distance <2 µm
from the center of cell; “middle,” distance between 2 and
4 µm from the center of the cell; and “outside,” the distance
>4 µm from the center of the cell (Fig. 8 B). Dividing the cell
into circles with those radii results in mean volumes of 34 µm3
(inside), 235 µm3 (middle), and 181 µm3 (outside). In wild-type
cells, the number of foci in the outside distance class increases
temporarily during the repair process, as indicated by the
observation that Rad54 foci relocalized to the nuclear periphery
and back to their starting positions over time (Fig. 8 B). In
contrast, ATPase-defective Rad54 foci localized to the nuclear
periphery but persisted at this position, which indicates impaired
affects the chromatin association of the protein and shows that
Rad54 ATPase activity specifically influenced its dissociation
Quantification of the number of
The knockin ES cells expressing the Rad54–GFP fusion protein
from the endogenous Rad54 locus allow accurate quantification
of the endogenous concentration of Rad54 molecules in an indi-
vidual cell by FCS and an estimation of the number of mole-
cules per focus. First, we calibrated the instrument using a
dilution series of purified GFP protein in solution (Fig. 7 A).
We then measured autocorrelation function in cells expressing
GFP itself, Rad54wt-GFP, and Rad54K189A-GFP, and determined
actual concentration by curve fitting (Fig. 7 B). Subsequently,
we determined a mean ES cell nuclear volume and determined
the total number of molecules per cell. Next, we used the time-
lapse movies of Rad54wt-GFP/ and Rad54K189R-GFP/ ES cells, in
which the cells were treated with a dose of 2 Gy and sub-
sequently followed up to 25 h to analyze the mean number of
molecules in DNA damage–induced foci (Fig. 6 A). The ratio
between free molecules and molecules in foci was determined
using a calculated thresholding as described in the Materials
and methods to establish the number of molecules in foci and
nucleoplasm. The mean number of molecules per focus over
time was then plotted against the time period for which the cells
could be followed (Fig. 7 C). This analysis revealed that the
mean number of molecules in an individual focus varies be-
tween 100 and 600 molecules. In Rad54wt-GFP/ cells, the num-
ber of molecules increased after damage induction and declined
Figure 7. FCS concentration measurement.
(A) Autocorrelation function G () measured
by FCS of increasing concentrations of GFP.
Inset indicates GFP concentration measure-
ment by FCS of purified GFP in solution plot-
ted as FCS concentration (nM) versus GFP
concentration (nM). (B) Autocorrelation func-
tion G () measured by FCS in Rad54wt-GFP/,
Rad54K189R-GFP/, and Rad54K189A-GFP/ ES cells.
As a control, cells expressing free untagged
GFP (GFP) were used. (C) Quantification of
the number of Rad54–GFP molecules after
treatment of cells with 2 Gy, either wild type
or K189R mutant, in a single focus over time.
Error bars indicate SD.
745 In vivo roles of the Rad54 recombination protein • Agarwal et al.
Differential cellular behavior of ATPase-
proficient and -defective Rad54
The remarkable feature of Rad54K189R-GFP/ and Rad54K189A-GFP/
cells is the presence of an elevated number of spontaneous, “un-
induced” Rad54 foci in their nuclei compared with Rad54wt-GFP/
cells (Fig. 2 A). A clear phenotypic difference in the cell bi-
ology of cells lacking Rad54 altogether and cells that express
ATPase mutants is the corresponding elevated number of Rad51
foci in ATPase mutant cells (Fig. 2 B). Thus, in addition to
causing an increase in its own foci in unchallenged cells, the in-
ability of Rad54 to hydrolyze ATP effectively also causes an
increase in the foci of its partner protein, Rad51. Thus it is pos-
sible that the ATPase activity of Rad54 is involved in turning
over Rad51 in the foci, which is consistent with biochemical
experiments demonstrating that Rad54 can displace Rad51 from
double-stranded DNA (Solinger et al., 2002). Yet, in the absence
of Rad54, no increase in Rad51 is detected, although its stabil-
ity is affected (Tan et al., 1999; van Veelen et al., 2005). It is
possible that when Rad54 is completely absent, a redundant
protein can act at the site of the DSB, which might take over
Rad54 function with respect to removal of Rad51 but not with
respect to DSB repair, as this is still impaired in knockout cells.
A candidate protein for this function is the Rad54 paralogue
Rad54B (Wesoly et al., 2006). The presence of the ATPase-
defective Rad54 protein would then dominantly affect this as-
pect of Rad54B activity.
The increase in the number of Rad54–GFP foci does
not correlate with an increase in spontaneous DNA damage in
Rad54 ATPase-defective cells, as no increase in the DNA dam-
age marker H2AX can be detected in Rad54 ATPase-defective
cells versus Rad54 ATPase-proficient cells (Fig. 3 A). Consis-
tently, when analyzed by immunofluorescence, no increase in the
number of 53BP1 and H2AX foci can be detected (Fig. 3,
B and C). Thus, within the limitations of these techniques, the
level of DNA damage is not significantly different in mutant,
wild-type, and knockout cells, showing that the elevated num-
ber of spontaneous foci is not caused by an increased number
of unrepaired breaks. We cannot exclude the possibility that
replication-associated DSBs generated because of endogenous
damage are efficiently repaired by Rad51, but the complex of
Rad54 remains attached after the repair has been completed and
therefore Rad54 and Rad51 foci persist. Our data are consis-
tent with the absence of an overt proliferation defect as well
as unaffected genomic stability of the cells expressing ATPase-
defective Rad54 and cells lacking Rad54 (unpublished data).
In addition, the immunofluorescence experiments show that the
ATPase activity of Rad54 is not important for localization of
Rad54 and Rad51 to sites of DNA damage. Untreated ES cells
contain many more H2AX foci than Rad54 foci and therefore
not all H2AX colocalize with Rad54. However, most if not
all Rad54 foci, including those containing ATPase-defective
Rad54, are at sites marked by H2AX (unpublished data).
ATPase-deficient Rad54 is partially
immobilized in nuclear DNA repair foci
The nature and function of foci is still ambiguous. Many models
of the composition of a focus have been postulated. It is possible
relocalization of DSB repair. However, this could also indi-
cate the persistence of foci in less accessible peripherally local-
Rad54 is a multifunctional protein that possesses several differ-
ent activities that promote the progression of homologous re-
combination, an accurate pathway of repairing DSBs (Tan et al.,
2003). In addition to a close functional interaction with Rad51,
the central protein of homologous recombination, Rad54 also
displays potent ATPase activity, which allows it to translocate
along DNA. This allows the protein to change the conformation
of the template DNA, thereby perturbing DNA structures and
influencing protein–DNA interactions (Heyer et al., 2006).
Here, we have investigated the effect of a mutation in the
ATPase domain of Rad54 on its cellular behavior.
The primary results of this study are as follows. First, the
ATPase activity of Rad54 influences the number of spontaneous
Rad54 foci in unchallenged cells, as ATPase-defective Rad54
cells contain more of them compared with cells expressing
wild-type Rad54. Second, the Rad54 ATPase-defective cells
also display an increase in spontaneous Rad51 foci. However,
the increase of foci containing homologous recombination pro-
teins does not correspond to an increase in DNA damage. Third,
the ATPase activity of Rad54 is not required for the formation
of foci induced by DNA damaging agents. Fourth, Rad54 but
not its ATPase activity is required to accumulate Rad51 at sites
of DSBs in a timely fashion. Fifth, an immobile fraction of
ATPase-defective Rad54 molecules occurs in foci. Sixth, time-
lapse studies revealed that the disappearance of DNA dam-
age–induced foci is delayed when Rad54’s ATPase activity is
attenuated, and these ATPase-defective Rad54 DNA repair foci
are stuck at the nuclear periphery.
The ATPase activity of Rad54 is essential
for its DNA repair function in vivo
To address the importance of the ATPase activity of Rad54, we
generated mouse ES cells that express ATPase-defective mutants
from the endogenous Rad54 locus fused to a carboxy-terminal
GFP tag (Fig. 1), ensuring physiological levels of mutant protein
(Fig. 1 C). The DNA damage sensitivity profiles of the ATPase-
defective Rad54 mutants are similar to cells lacking Rad54
altogether in terms of hypersensitivity to mitomycin C and ion-
izing irradiation (Fig. 1 D). The damage hypersensitivities of
the Rad54 ATPase-defective cells probably result from defective
homologous recombination because Rad54 ATPase-defective and
Rad54/ ES cells are equally impaired in homologous recom-
bination (Table I). This indicates that the Rad54 ATPase activity
is required for the type of recombination that is used to repair
breaks induced by irradiation and mitomycin C, as well to
effectively homologously integrate a linear piece of DNA into
the genome. Furthermore, this indicates that both binding and
hydrolysis of ATP is essential because ATPase mutants (K189A
and K189R) display similar phenotypes. These experiments re-
veal the essential role of the Rad54 ATPase function in mamma-
lian cells and its in vivo importance for DNA repair.
JCB • VOLUME 192 • NUMBER 5 • 2011 746
maintaining Rad54 molecules at the site of the break apparently
continues until the break is repaired, as seen by the damage-
induced increase in the number of molecules over time (Fig. 7).
These foci might simply continue growing because the signal
that locates Rad54 to the site of damage continues to signal.
Furthermore, our FRAP data show that a small fraction of the
molecules in the mutant are stably bound to the chromatin (the
immobile fraction) (Fig. 4), which suggests that only a minority
of molecules present in foci are functional in DNA DSB repair.
Several interesting questions remain. Why are there 10-fold
more molecules present in foci than necessary for repair? What
role do the excess molecules have? Is it simply a mechanism of
trial and error, the higher the local concentration the higher the
efficiency of repair, or is there an underlying reason that re-
mains to be discovered in the future?
The molecular processes required for the accumulation
and disassembly of homologous recombination proteins in foci
at sites of DNA damage are not well understood. Cytological
studies in yeast, chicken DT-40 cells, and mouse ES cells sug-
gest that Rad54 is not necessary for the formation of Rad51 foci
(Shinohara et al., 2000; Takata et al., 2000; Lisby et al., 2004;
Miyazaki et al., 2004; van Veelen et al., 2005). We show that
that, as has been put forth for yeast, the mammalian foci are also
a reflection of the so-called “repair centers” (Lisby et al., 2004),
but this premise lacks clear-cut evidence in mammalian cells.
Foci have also been assumed to represent the one or all of the
various stages of recombination, and are therefore not a clear
method of distinguishing between the different stages of recom-
bination. It is still not clear why such a high local concentration
of protein is required at the site of damage, especially because
in biochemical studies, the optimal ratios of Rad51 and Rad54
are not necessarily 1:1 (Jiang et al., 1996; Clever et al., 1997;
Golub et al., 1997). However, the presence and quantification of
foci have been used as an indication of repair activity because
foci form within a short time in response to DNA-damaging
agents, and decrease in number over time. A focus is a highly
dynamic structure, with active and rapid association and dis-
association of proteins, particularly Rad54 (Essers et al., 2002b).
Our FCS analysis revealed that the mean number of molecules
in an individual focus varies between 100 and 600 molecules
(Fig. 7). This number, however, is higher than expected based
on the few Rad54 molecules predicted to be necessary to repair
a single DSB. These results implicate that not all molecules
present in a foci are necessary to repair the break. Attracting and
Figure 8. Nuclear relocalization of Rad54–
GFP foci in response to DNA damage.
(A) Grayscale representation of the nuclear
redistribution of Rad54 foci in a wild-type or
ATPase-defective Rad54–GFP cell after treat-
ment with ionizing radiation using a 137Cs
source. Shown are a subset of time points in
2 (out of 10) z axes of a single cell. Bar, 5 µm.
(B) Quantification of A. The relative distribution
of foci in distance classes; inside (0–2 µm),
middle (2–4 µm), and outside (> 4 µm) fol-
lowed over time. Graph indicates the best
fitting distribution curve of outside foci in
Rad54wt-GFP/ and Rad54K189R-GFP/ cells.
747 In vivo roles of the Rad54 recombination protein • Agarwal et al.
the homogenously distributed Rad54–GFP in the nucleoplasm
is highly mobile (Essers et al., 2002b). The combination of the
time-lapse and photobleaching experiments provides additional
insight into foci biology. The experiments presented in Fig. 4
reveal that although the ATPase activity of Rad54 slightly
affects its effective diffusion rate, it renders a fraction of 10%
of molecules immobile in Rad54K189A-GFP/ and Rad54K189R-GFP/
cells compared with Rad54wt-GFP/ cells. The result of this is
that it takes about twice as long to repopulate the entire focus.
A DNA damaged–induced Rad54 focus disappears half as fast,
and the time it takes to repopulate a focus takes twice as long
when Rad54 cannot hydrolyze ATP. The net effect is that on
average, the same number of Rad54 molecules associated with
a single focus over time in wild-type and mutant cells, even
though DNA repair is inoperative in the mutant cells.
Our study shows that Rad54’s ATPase activity is impor-
tant for DNA repair and recombination, but, interestingly, that it
also affects its cellular behavior. The ATPase activity is required
for release of the protein from DNA damaged–induced struc-
tures on chromatin, not only of itself but also of Rad51. The fact
that the ATPase activity of Rad54 affects its cellular behavior is
interesting because this activity is only triggered when it is
bound to DNA. Mutations that attenuate the ATPase activity of
Rad54 are likely to be separation-of-function alleles that differ-
entially affect the behavior of the pool of Rad54 in a focus that
is bound to DNA versus the pool that is not bound to DNA.
Rad54 molecules that are not bound to DNA are not hydrolyz-
ing ATP, are therefore not actively engaged in repair, and can
still reversibly interact with the focus. However, the Rad54 mole-
cules bound to DNA but no longer capable of hydrolyzing ATP
appear to lose the ability to quickly turn over in the focus. Our
observations are of interest in the context of the number of each
homologous recombination protein required for DNA repair,
which is much less than are present in a focus based on bio-
chemical experiments. Thus, our experiments reveal the need to
develop a cellular system that allows for identification and
tracking of individual molecules in a crowd, and the ability to
separate those that do the work from those that do not.
Materials and methods
The mouse ES cells used to generate cells expressing ATPase-defective
Rad54 had the genotype Rad54wt-HA/, where one allele is disrupted and
the other expresses HA-tagged Rad54 from the endogenous Rad54 locus
(Tan et al., 1999). ES cells were cultured on gelatin-coated dishes in a 1:1
mixture of DME and buffalo rat liver (BRL)-conditioned medium, supple-
mented with 10% (vol/vol) FBS (Thermo Fisher Scientific), 0.1 mM non-
essential amino acids (Biowhittaker; Lonza), 50 mM -mercaptoethanol
(Sigma-Aldrich), and 500 U ml–1 leukemia inhibitory factor. U2Os cells
were cultured in a 1:1 mixture of DME and Ham’s F10, supplemented with
10% (vol/vol) fetal calf serum (Thermo Fisher Scientific) and streptomycin/
penicillin at 37°C in an atmosphere containing 5% CO2.
The primary antibodies used in this study were: anti-Rad51 (rabbit poly-
clonal; van Veelen et al., 2005), anti-Rad54 (rabbit polyclonal; Essers
et al., 1997), anti-H2AX (Millipore), anti-53BP1 (rabbit polyclonal;
Novus Biologicals), anti-Rad54 (goat polyclonal, D-18; Santa Cruz Biotech-
nology, Inc.), anti-NBS1 (goat polyclonal, C-19; Santa Cruz Biotechnol-
ogy, Inc.), anti-RPA34 (mouse monoclonal, Ab-2; Oncogene), and
anti-BRCA2 (mouse monoclonal, Ab-1; EMD). The secondary antibodies
the Rad54 but not its ATPase activity is required to accumu-
late Rad51 and BRCA2 into foci in a timely fashion (Fig. 5).
However, Rad54 does not influence the accumulation of Nbs1
and RPA. These results establish the role of Rad54 downstream
of break resection and indicate that Rad54 is mainly involved
in stages of recombination performed by Rad51 and BRCA2.
These results resemble the biochemical properties of Rad54.
We know from biochemical experiments that Rad54 can load
Rad51 on the filaments. This activity is independent of Rad54
ATPase activity, as shown in yeast experiments (Wolner and
Peterson, 2005). Genetic studies have demonstrated that the
ATPase activity is crucial for in vivo Rad54 function, and we
have shown that Rad54 K189R mutants display DNA damage
sensitivities equivalent to the deletion mutant (Fig. 1). In con-
trast, the disassembly process is affected by Rad54-mediated
hydrolysis of ATP (Fig. 7). Clearance of foci induced by DNA
damage takes about twice as long in Rad54K189R-GFP/ cells com-
pared with Rad54wt-GFP/. These foci especially persist close to
the nuclear periphery (Fig. 8). The temporal resolution we used
in these time-lapse experiments was too low to actually track
individual foci. The temporal resolution could not be increased
because of the sensitivity of ES cells to laser irradiation and
bleaching of the fluorescent signal. We can therefore not dis-
tinguish whether DNA damaged–induced relocalization of foci
over time is impaired or whether DNA damage–induced foci
are immobile and persist longer closer to the nuclear periphery
in ATPase-deficient cells (Soutoglou et al., 2007). ATPase-
dependent Rad54 translocation might be required for local
chromatin decondensation observed around DSBs (Kruhlak
et al., 2006; Kim et al., 2007) because compact heterochromatin
structure at the nuclear periphery could inhibit access of repair
proteins to DSBs or DNA strand exchange. However, because
the persisting foci are found in the vicinity of the nuclear border,
it is also possible that the transient increase in Rad54 foci in this
area reflects migration of damaged DNA toward low-density
chromatin, resulting in concentration at the border of condensed
DNA, as is also the case for H2AX foci in ataxia telangiec-
tasia mutated (ATM)-deficient cells (Goodarzi et al., 2008).
This regulation of DSB relocalization to the nuclear periphery
has also been observed for yeast Rad51 (Kalocsay et al., 2009;
Oza et al., 2009; Oza and Peterson, 2010) and indicates that the
prolonged presence of mutant Rad54 at the periphery reflects
the presence of delayed repair of DSBs. It is not unexpected for
a protein in the SWI2/SNF2 family to affect this feature of foci.
Just as genuine chromatin remodeling motor proteins affect his-
tone DNA interactions, Rad54’s motor activity, in conjunction
with its direct interaction with Rad51, might be well suited to
deal with accumulations of homologous recombination proteins
on chromatin after the repair process has been completed.
Mutations that attenuate the
ATPase activity of Rad54 are
The ATPase activity of Rad54 is essential for many of its bio-
chemical activities (Tan et al., 2003), but its effect on the cel-
lular behavior of the protein is unknown. Using photobleaching
experiments in living cells, we determined previously that all of
JCB • VOLUME 192 • NUMBER 5 • 2011 748
and the cells were covered with a coverslip. The piece of Mylar containing
the stained cells was then cut out and was placed, together with the cover-
slip, on a slide. The piece of Mylar with the coverslip on top of it was glued
to the slide using rubber cement. In case of Rad51 staining, preextraction
for 1 min with Triton X-100 buffer (0.5% Triton X-100, 20 mM Hepes-KOH,
pH 7.9, 50 mM NaCl, 3 mM MgCl2, and 300 mM sucrose) was per-
formed (Petrini, 2000). particle tracks were visualized with a confocal
laser scanning microscope (LSM 510 META; Carl Zeiss, Inc.) consisting
of an inverted microscope (Axiovert 100) equipped with an Argon gas
laser (visualizing Alexa Fluor 488, green) and a helium neon laser (visual-
izing Alexa Fluor 543, red). Images were taken with 63× Plan-Apochromat
1.4 NA oil immersion lens (Carl Zeiss, Inc), on a single plane of 1 µm
thickness, through the middle of the cell.
Whole cell extracts were prepared by lysing cells with SDS sample buffer
(2% SDS, 10% glycerol, and 60 mM Tris-HCl, pH 6.8) 48 h after transfec-
tion. After the protein concentration was determined by a Lowry protein
assay, extracts were supplemented with 0.5% -mercaptoethanol and
0.02% bromophenol blue. After fractionation by SDS-PAGE, proteins were
transferred to polyvinylidene fluoride membrane. The blots were blocked
with PBS/3% skimmed milk/0.1% Tween 20 and probed with primary
antibodies. After washing with PBS/0.1% Tween 20, the membranes were
probed with relevant secondary antibodies and developed with ECL West-
ern blotting detection reagents (GE Healthcare).
Live cell imaging, semi-automated foci counting, and foci tracking
ES cells were grown overnight on lethally irradiated MEF feeder layers on
24-mm round coverslips. Cells that grew to an 70% confluent monolayer
were irradiated with 2 Gy and transferred to a specially adapted chamber
fitted to the confocal microscope 45 min after irradiation, where they could
be maintained at 37°C with 5% CO2. Using a macro for automated time-
lapse imaging, the cells were imaged taking 10 z slices (covering 9 µm in
total) every 15 min. Movies were analyzed in the AIM image browser
(Carl Zeiss, Inc.) and exported for detailed analysis using ImageJ software
Time-lapse imaging of individual cells
The low-contrast in the images of the Rad54 knockin cells between green
nuclei and background prohibited automated segmentation of the ES cells.
A custom made ContourJ selection tool was used to select all individual
cells by hand (available at http://www.imagescience.org/meijering/
software/contourj/). Then, the center of mass (x,y location) for each cell
was determined. When the distance of the center of mass in two consecu-
tive time points was <3 µm, the cell was regarded as being the same cell.
In this way cells could be followed in time. Those cells that could be tracked
for at least 15 time points were selected for detailed foci analyses. Each
selected cell was then processed to determine the number of foci in the
3D stack. This number per cell was determined as described previously
(van Royen et al., 2007). In brief, for each cell, the mean intensity and SD
was determined. Foci were selected based on fluorescence intensity values
above mean + 1.5× the SD. Visual inspection of the images showed that
this method detected the majority of the foci. From the resulting image, the
number of foci was counted using the particles analysis function of ImageJ.
Duplicate foci in different z planes were automatically removed from the
counting. The data thus obtained was plotted as number of foci per cell
against the time during which the cells could be followed.
A microscope system (LSM510 confocor 2; Carl Zeiss, Inc.) was used for
the FCS experiments. Data were analyzed with the SSTC data processor
(Scientific Software Technologies Center). Every cell was measured five
times for 20 s. The raw data were autocorrelated, and the autocorrela-
tion curves were analyzed as either a one-component free diffusion trip-
let state model (GFP in solution) or a two-component free diffusion triplet
state model (Rad54–GFP) in cells. The models were used to determine
the diffusion time, i.e., the time it takes a molecule to move through the
confocal laser spot and the total number of molecules in the diffraction
limited spot. To be able to estimate the concentration of RAD54 molecules
in a cell nucleus, the volume of the diffraction limited spot was determined
using standard Rhodamine 6G (Invitrogen) and GFP solutions with known
concentrations (Weisshart et al., 2004). A mean ES cell nuclear volume
was determined by measuring two perpendicular diameters in the confocal
plain with the largest size of the nucleus (n = 37; 9.5 ± 1.2 [mean ±
SD] µm/cell). The volume of individual cells was estimated by regard-
ing the nucleus as an ellipsoid object of which the volume was determined
conjugated with alkaline phosphatase were obtained from Roche.
The horseradish peroxidase–conjugated antibodies were obtained from
Jackson ImmunoResearch Laboratories, and relevant Alexa Fluor secondary
antibodies were obtained from Invitrogen.
Generation of ES cells carrying knockin alleles expressing
ATPase-defective Rad54 protein
The wild-type and ATPase-deficient Rad54–GFP knockin constructs were
designed to obtain expression of tagged Rad54 from the endogenous
promoter upon homologous integration. The wild-type construct was
generated by fusing exons IV–XVII of the hRAD54 cDNA, but with the
omission of the STOP codon, to a DNA encoding a 3-terminal GFP
tag, followed by a poly(A) signal (p(A)) and a phosphoglycerate kinase
(PGK) promoter–driven puromycin selectable marker gene. This fragment
was subcloned into exon IV of a 9-kb EcoRI genomic Rad54 fragment.
Using linkers, a downstream SfuI site was introduced. Digestion of this
construct with SfuI yielded a fragment containing the 3-terminal part of
the Rad54–GFP cDNA spanning exons IV–XVII and the puromycin gene.
This fragment was subcloned into the unique SfuI site in exon IV of a
9-kb EcoRI fragment of the mouse Rad54 genomic sequence containing
exons IV–VII in pBluescript II KS. The Rad54 mutant constructs express-
ing the K189R and K189A mutation were generated in a similar way
after introduction of these mutations in the wild-type hRAD54 cDNA
using the following primers: 5-GGGCCTAGGAAGGACGCT-3 (K189R)
and 5-GGGCCTAGGAGCAACGCT-3 (underlines indicate the mutant
codon in Rad54). The constructs are schematically depicted in Fig. 1 A.
Targeting constructs bearing either the K189A or K189R mutation (Fig. 1)
were purified as plasmids and linearized with PvuI, then purified using
electro-elution, phenol extraction, and ethanol precipitation. These linear-
ized constructs were then electroporated into Rad54wt-HA/ cells, using
a 2-mm cuvette, at 117 V, 1200 F, and left for 10 ms in an ECM 830
electroporator (BTX). Replacement of the Rad54HA locus would generate
ES cells with genotypes Rad54K189A-GFP/ or Rad54K189R-GFP/. 24 h after
electroporation, cells were subjected to puromycin selection (1 µg/ml). 100
puromycin resistant colonies were isolated for each construct and their
DNA was analyzed for homologous integration of the knockin constructs
in the Rad54HA locus by DNA blotting using a probe recognizing exons VII
and VIII. Genomic sequence analysis was performed to confirm the correct
integration and presence of mutations in the Rad54 locus, and protein
expression was subsequently analyzed by immunoblotting. DNA damage
sensitivities of the cells were assessed by performing clonogenic survival
assays as described previously (Essers et al., 1997). For ionizing radia-
tion, dishes were treated right away with the indicated dosage. For the
rest, cells were allowed to attach for 12–16 h before treatment. Mitomycin
C was added for 1 h before washing. Finally, as a measure of homologous
recombination efficiency, the frequency of homologous versus random
integration of gene targeting constructs in the Rb locus was determined as
described previously (Niedernhofer et al., 2001).
Ionizing radiation and immunofluorescence
ES cells were seeded on a feeder layer of lethally irradiated (70–80%) con-
fluent mouse embryonic fibroblasts and left to attach overnight. Cells were
irradiated with the indicated doses of ionizing radiation using a 137Cs
source and left to recover for the indicated amount of time. particle irra-
diation was performed using a 241Am source as described previously (Stap
et al., 2008). In brief, U2Os cells were transfected using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s instructions with siRNA
against luciferase (5-CGUACGCGGAAUACUUCGAdTdT-3) or Rad54
(5-GAACUCCCAUCCAGAAUGAUU-3) for 48 h. After 24 h, cells were
plated on a 1.8 µm-thick polyester membrane (Mylar), and transfection
was repeated. In the case of ES cells, Mylar dishes were coated with car-
bon atoms (SC500 sputter coater; EMscope) and gelatin, and cells were
subsequently plated 24 h before irradiation. Cells were irradiated with
particles and subsequently fixed and stained for immunofluorescence
at indicated time points as described previously (Aten et al., 2004).
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bodies, after which they were stained with Hoechst 33342 in a final
concentration of 5 µg/ml for 10 min. After washing again with TNBS, a
droplet of Vectashield (Invitrogen) was placed on top of the stained cells,
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volume of the nuclei was 453 ± 190 µm3 (mean ± SD, n = 39). Using the
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20 s. 60 cells were monitored for each genotype in three independent ex-
periments. Wild-type ES cells stably transfected with pGK-GFP-p(A) were
used as a positive control for fluorescence recovery, where cells with com-
parable fluorescence level as the knockin cells were chosen and treated as
detailed for the Rad54 knockin cells.
Complementary iFRAP experiments were performed on a confocal
microscope (TCS SP5; Leica) with a 63× oil Plan-Apochromat 1.4 NA oil
immersion lens (Dundr et al., 2002). All fluorescence in an individual nu-
cleus, with exception of a small region containing a single focus, was
bleached, and the decrease in fluorescence in the nonbleached focus was
recorded. For each time point, the relative intensity was calculated as fol-
lows: Irel;t = (It BG)/(I0 BG), where I0 is the mean intensity of the region
of interest before bleaching and BG is the background signal.
We thank Nicole van Vliet for expert technical assistance.
This work was supported by a grant from the Dutch Cancer Society and
a TOP grant from the Netherlands Organization for Scientific Research (NWO)
and the Netherlands Genomics Initiative/NWO.
Submitted: 4 November 2010
Accepted: 1 February 2011
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