Accumulation of Werner protein at DNA double-strand breaks in human cells

Abstract

Werner syndrome is an autosomal recessive accelerated-aging disorder caused by a defect in the WRN gene, which encodes a member of the RecQ family of DNA helicases with an exonuclease activity. In vitro experiments have suggested that WRN functions in several DNA repair processes, but the actual functions of WRN in living cells remain unknown. Here, we analyzed the kinetics of the intranuclear mobilization of WRN protein in response to a variety of types of DNA damage produced locally in the nucleus of human cells. A striking accumulation of WRN was observed at laser-induced double-strand breaks, but not at single-strand breaks or oxidative base damage. The accumulation of WRN at double-strand breaks was rapid, persisted for many hours, and occurred in the absence of several known interacting proteins including polymerase beta, poly(ADP-ribose) polymerase 1 (PARP1), Ku80, DNA-dependent protein kinase (DNA-PKcs), NBS1 and histone H2AX. Abolition of helicase activity or deletion of the exonuclease domain had no effect on accumulation, whereas the presence of the HRDC (helicase and RNaseD C-terminal) domain was necessary and sufficient for the accumulation. Our data suggest that WRN functions mainly at DNA double-strand breaks and structures resembling double-strand breaks in living cells, and that an autonomous accumulation through the HRDC domain is the initial response of WRN to the double-strand breaks.

Full text

Introduction
People with Werner syndrome display several clinical signs
and symptoms associated with early aging, including graying
of hair, cataracts, osteoporosis, atherosclerosis, type II diabetes
mellitus and a high incidence of malignant neoplasms (Chen
and Oshima, 2002). The gene defective in Werner syndrome,
WRN, encodes a protein of the RecQ family of DNA helicases
(Hickson, 2003; Opresko et al., 2003; Yu et al., 1996), which
possesses DNA-dependent ATPase, 3⬘r5⬘helicase activities at
the central region and the 3⬘r5⬘exonuclease activity at its
amino (N) terminus. The substrate specificity of WRN helicase
and exonuclease activities has been determined by in vitro
studies and includes a variety of intermediates produced during
DNA replication, recombination and repair (Shen et al., 1998;
Xue et al., 2002). The carboxyl (C) terminal region of WRN
protein contains the conserved RQC (RecQ conserved)
domain, including a nucleolar targeting sequence (NTS)
(Marciniak et al., 1998; von Kobbe and Bohr, 2002) and an
HRDC (helicase and RNaseD C-terminal) domain, whose
function is still unclear but has been shown to play a role in
DNA binding in the Saccharomyces cerevisiae homologue
Sgs1 (Liu et al., 1999). The nuclear targeting of the WRN
protein is due to the presence of a classical nuclear localization
signal (NLS) in the C-terminal region of the protein
(Matsumoto et al., 1997). Full-length WRN binds DNA with
low efficiency in vitro (Orren et al., 1999; Shen and Loeb,
2000), and the exonuclease, RQC, and HRDC regions of WRN
have been shown to be three distinct, structure-specific, but not
sequence-specific, DNA binding domains (von Kobbe et al.,
2003b).
It has been reported that many proteins physically and
functionally interact with WRN. The interacting proteins appear
to function at various levels in the mechanisms for maintaining
the integrity of the genome and in the DNA damage response,
suggesting that WRN plays one or more roles in DNA repair.
WRN has been shown to interact with Ku and DNA-PKcs
(Karmakar et al., 2002a; Karmakar et al., 2002b; Li and Comai,
2000; Orren et al., 2001), and a recent report suggests that WRN
may participate in non-homologous end-joining (Li and Comai,
2002). Werner syndrome fibroblasts transformed with Simian
Virus-40 (SV40) T antigen or immortalized by expressing
human telomerase reverse transcriptase (hTERT) display a mild
but distinct sensitivity to ionizing radiation when compared
with appropriate control fibroblasts and Werner syndrome
fibroblasts expressing exogenous WRN (Cheng et al., 2004;
Yannone et al., 2001). This suggests that WRN may be involved
4153
Werner syndrome is an autosomal recessive accelerated-
aging disorder caused by a defect in the WRN gene, which
encodes a member of the RecQ family of DNA helicases
with an exonuclease activity. In vitro experiments have
suggested that WRN functions in several DNA repair
processes, but the actual functions of WRN in living cells
remain unknown. Here, we analyzed the kinetics of the
intranuclear mobilization of WRN protein in response to a
variety of types of DNA damage produced locally in the
nucleus of human cells. A striking accumulation of WRN
was observed at laser-induced double-strand breaks, but
not at single-strand breaks or oxidative base damage. The
accumulation of WRN at double-strand breaks was rapid,
persisted for many hours, and occurred in the absence of
several known interacting proteins including polymerase ␤␤,
poly(ADP-ribose) polymerase 1 (PARP1), Ku80, DNA-
dependent protein kinase (DNA-PKcs), NBS1 and histone
H2AX. Abolition of helicase activity or deletion of the
exonuclease domain had no effect on accumulation,
whereas the presence of the HRDC (helicase and RNaseD
C-terminal) domain was necessary and sufficient for the
accumulation. Our data suggest that WRN functions
mainly at DNA double-strand breaks and structures
resembling double-strand breaks in living cells, and that an
autonomous accumulation through the HRDC domain is
the initial response of WRN to the double-strand breaks.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/118/18/4153/DC1
Key words: Werner protein, Double-strand breaks, Laser irradiation,
Damage accumulation, HRDC domain
Summary
Accumulation of Werner protein at DNA double-strand
breaks in human cells
Li Lan1, Satoshi Nakajima1, Kenshi Komatsu2, Andre Nussenzweig3, Akira Shimamoto4, Junko Oshima5and
Akira Yasui1,*
1Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1, Sendai 980-8575, Japan
2Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan
3Experimental Immunology Branch, NIH, National Cancer Institute, Bethesda, MD 20892-1360, USA
4GeneCare Research Institute, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan
5Department of Pathology, University of Washington, Seattle, WA 98195-7470, USA
*Author for correspondence (e-mail: ayasui@idac.tohoku.ac.jp)
Accepted 6 June 2005
Journal of Cell Science 118, 4153-4162 Published by The Company of Biologists 2005
doi:10.1242/jcs.02544
Research Article
Journal of Cell Science
JCS ePress online publication date 1 September 2005

4154
in processing ionizing radiation-induced double-strand breaks.
WRN also interacts physically with the Mre11-Rad50-NBS1
complex, which functions in homologous recombination for
double-strand break processing (Cheng et al., 2004). Other
reports suggest that WRN may play a role in base excision
repair because of a physical interaction between WRN and
polymerase ␤(POL ␤) involved in base excision repair and the
repair of single-strand breaks (Harrigan et al., 2003).
Furthermore, p53 has been shown to interact with the C-
terminus of WRN and to inhibit WRN exonuclease activity in
vitro (Blander et al., 1999; Brosh et al., 2001). Consistent with
possible roles of WRN in DNA repair and genome stability,
Werner syndrome cells show an attenuated p53-mediated
apoptosis (Spillare et al., 1999) and display extensive deletions
at non-homologous joined ends as well as non-homologous
chromosome exchanges (Oshima et al., 2002). With regard to
the localization of WRN in cells, various distribution patterns
of the protein have been reported. WRN foci have been
observed as diffuse nuclear, nucleolar or nuclear foci depending
on the stage of the cell cycle (Gray et al., 1998; Opresko et al.,
2003). Although the number of WRN-containing nuclear foci
increases after replication fork arrest and upon DNA damage
(Sakamoto et al., 2001; Szekely et al., 2000), the significance
of the formation of these foci remains to be elucidated.
Furthermore, in spite of many reports describing that WRN
modified the enzymatic activity of the interaction partners and
vice versa, it has not be shown whether WRN responds to DNA
damage and whether WRN is involved in base excision repair,
repair of single-strand breaks or double-strand breaks in living
cells.
We recently established a laser micro-irradiation system for
the localized production of single-strand breaks, double-strand
breaks and oxidative base damage in a cell nucleus (Lan et al.,
2004). Using this system we have analyzed the accumulation
of endogenous and green-fluorescent-protein (GFP)-tagged
WRN at various types of DNA damage and found that WRN
accumulated via its HRDC domain at double-strand breaks
within 1-2 minutes after irradiation and remained for a longer
period at the site. Our results showed, for the first time, an
immediate accumulation of WRN at double-strand breaks and
suggest important roles for WRN in genome stability of living
cells.
Materials and Methods
Plasmid construction for GFP-fused genes
Oligonucleotides containing XhoI, SmaI and NotI sites were
introduced into the cloning sites of pEGFP-N1 or C1 vectors
(Clontech), and cDNA for WRN and WRN containing a helicase
mutation (K577M), NBS1, BRCA1 were introduced into the vectors
in frame. Deletion fragments of WRN (a.a. 152-1432) and (a.a. 1012-
1432) were obtained by digestion of the whole WRN cDNA by EcoRV
and NotI, PvuII and NotI, respectively, and the each fragment was then
introduced into the SmaI and NotI sites of the vector. WRN (a.a. 1-
1150) and (a.a. 1229-1432) fragments were constructed by PCR, and
WRN (a.a. 1229-1432) was introduced into the XhoI and NotI site of
pEGFP vector, while WRN (a.a. 1-1150) was cloned into a vector
harboring an extra nuclear localization signal.
Cell lines and transfection
Cell lines of HeLa, Parp1- (a cell line from mouse embryonic
fibroblasts, a generous gift of Mitsuko Masutani), Susa/T-n cells
(p53+/+, telomerase expressed, a generous gift of Kanji Ishizaki),
1022QVA (NBS1-deficient Nijmegen patient cells), CHO9 (WT
cells), XR-1 (XRCC4 deficient cell line), XR-C1 (DNA-PKcs-
deficient cell line), XRV15B (Ku80-deficient cell line), and H2AX-
deficient MEF (Celeste et al., 2003) were used. All the above cell lines
were propagated in D-MEM (Nissui) supplemented with 10% fetal
bovine serum at 37°C and 5% CO2.38⌬(mouse Pol
␤
–/– cell line)
and MB36.3 (mouse Pol
␤
–/–cell transfected with wild-type human
POL
␤
), generous gifts from Samuel H. Wilson, were grown at 34°C
and 10% CO2. Cells were plated on glass bottom dishes (Matsunami
Glass) at 50% confluence 24 hours before the transfection (Fugene-
6, Life Technology) and irradiated by laser under a microscope 48
hours after transfection.
Microscopy and laser irradiation
Fluorescence images were obtained and processed using a FV-500
confocal scanning laser microscopy system (Olympus). A 365 nm
pulse laser micro-irradiation apparatus combined with the confocal
microscope was used as previously described (Lan et al., 2004). We
used two irradiation doses, a lower dose (0.75 ␮J) or a higher dose
(2.5 ␮J), which were obtained by passing lasers through either an F20
or an F25 filter, respectively, in front of the lens. By using this system,
various types of DNA damage, such as single-strand breaks (produced
by lower dose and higher dose irradiation), double-strand breaks and
oxidative base damage (produced by higher dose irradiation), were
produced at restricted nuclear regions of mammalian cells. We also
used a 405 nm pulse laser system (Olympus) for irradiation of cells
in the epi-fluorescence path of the microscope system. The power of
the laser scan can be controlled by the number of scans used or by
laser dose. One scan of the laser light at full power delivers energy of
around 1600 nW. We used only a full power scan from the 405 nm
laser in this study and regulated the dose by changing the number of
scans. Both 365 nm and 405 nm lasers were focused through a 40⫻
objective lens. Cells were incubated with Opti-medium (Gibco) in
glass-bottom dishes, which were placed in chambers to prevent
evaporation, on a 37°C hot plate. The energy of fluorescent light at
the irradiated site was measured with a laser power/energy monitor
(Orion, Ophir Optronics, Israel). The mean intensity of each focus was
obtained after subtraction of the background intensity in the irradiated
cell. Each experiment was performed at least three times and the data
presented here are mean values obtained in a given experiment. Local
UVC-light irradiation were performed as previously described (Okano
et al., 2003).
Cell-cycle synchronization
Cell synchronization was performed by the double thymidine block
method. In all, 5⫻104 cells were seeded in a 3.5 cm dish and grown
for 2 days. Thymidine was then added to 2.5 mM final concentration
and cells were further incubated for 22-24 hours. Thymidine-
containing medium was removed and cells were washed three times
with Hank’s buffer and fresh medium was added. After 10 hours, cells
were treated with hydroxyurea at 1 mM final concentration and
incubated for 14-16 hours. Under these conditions, cells accumulate
at the G1/S border. Cells were then washed three times with Hank’s
buffer. Synchronization of the cell cycle was analyzed by a
FACSCalibur (Becton Dickinson). Cells were incubated for 3 hours,
8 hours and 16 hours in fresh medium to obtain S-phase, G2-M-phase
and G1-phase cells, respectively.
Immunocytochemistry and chemicals
Cells were irradiated and stained with antibodies raised against
human ␥H2AX, WRN and XRCC1. Cells were fixed 15 minutes after
irradiation. Immunofluorescence was performed as described
previously (Okano et al., 2003). Anti-phosphorylated H2AX
Journal of Cell Science 118 (18)
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Werner protein accumulates at DNA double-strand breaks
Fig. 1. Laser irradiation systems and accumulation of WRN. (A) Laser irradiation systems. The left column shows the 365 nm pulse laser
irradiation system, producing the lower dose and the higher dose irradiation, which are regulated by the filter in front of the mirror. The right
column shows the 405 nm laser system. (B) Three types of damage induced by 365 nm and 405 nm laser irradiation. HeLa cells irradiated with
the lower dose or the higher dose of 365 nm laser, or with 405 nm laser of different scan times, were stained with anti-poly(ADP)ribose (left,
for single-strand breaks) or by ␥H2AX antibody (middle, for double-strand breaks), respectively, or accumulation of GFP-tagged OGG1 at
irradiated sites (right, for base damage) is shown. The amounts of accumulated molecules for poly(ADP)ribose, ␥H2AX and GFP-OGG1 after
lower and higher dose irradiation with the 365 nm or 405 nm laser were quantified in the graphs. (C) Time-dependent accumulation of GFP-
tagged WRN after higher dose irradiation with the 365 nm laser in HeLa cells. (D) Accumulation of GFP-tagged XRCC1 and LIGIII␣(upper
panels) and no accumulation of GFP-tagged WRN (lower panels) after lower dose irradiation with the 365 nm laser in HeLa cells. Arrows
indicate the sites of irradiation.
Journal of Cell Science

4156
(␥H2AX) (1:200 dilution; Upstate Biotechnology), anti-WRN (1:20)
and anti-XRCC1 (1:200 dilution, Abcam) were used. For
photosensitization treatment of cells, Ro-19-8022 (Roche) at a final
concentration of 250 nM was added into the medium and cells were
incubated at 37°C for 5 minutes. For 1,5-dihydroxyisoquinoline
(DIQ) treatment, cells were incubated with DIQ (Sigma) in the
medium at a final concentration of 500 ␮M for 1 hour before
irradiation. Camptothecin (Sigma) at a final concentration of 1 ␮M
was added to the medium and cells were incubated at 37°C for 6
hours. 5-Bromo-2⬘-deoxyurine (BrdU; Roche) at a final
concentration of 10 ␮M was added to the medium 8 hours before
laser irradiation.
Results
Accumulation of WRN at sites of DNA damage induced
by laser micro-irradiation.
To analyze whether WRN responds to DNA damage in vivo,
we used a laser micro-irradiation apparatus combined with a
confocal microscope (Lan et al., 2004). A single pulse of 365
nm laser micro-irradiation in the nucleus of a human cell at the
lower dose (0.75 ␮J) or one scan with 405 nm laser produced
hardly any detectable DNA lesions other than single-strand
breaks (detected with antibody against poly-ADP-ribose, Fig.
1B), whereas irradiation at the higher dose (2.5 ␮J) or 100 and
500 scans with 405 nm laser produced double-strand breaks
(detected by antibody against phosphorylated H2AX; ␥H2AX)
and base damage (detected by accumulated GFP-OGG1 for 8-
oxoGuanine) in addition to single-strand breaks at the
irradiated sites (Fig. 1B) (Lan et al., 2004). For the analysis of
WRN accumulation at the irradiated sites we used GFP-tagged
WRN, which is functional in cells as previously reported
(Opresko et al., 2003), or antibody raised against human WRN
(see below). Using this system, we found that WRN
accumulated at the site irradiated with the higher dose of laser
in a time-dependent manner (Fig. 1C). Thirty seconds after
irradiation the accumulated GFP-WRN was barely visible, but
3 minutes after irradiation the intensity of the fluorescence
reached its maximum (Fig. 1C). GFP-WRN did not accumulate
at irradiated cytoplasm or regions in mitotic cells without DNA
(Fig. S1 in supplementary material), indicating that the
accumulation of WRN is DNA damage-specific. We also
analyzed the accumulation of WRN in cells irradiated with the
low dose of laser, which produced almost single-strand breaks
alone (Fig. 1B). Even though XRCC1 and LIGIII␣,which are
involved in the repair of single-strand breaks, efficiently
accumulated at the site irradiated with the lower dose, WRN
did not accumulate at all at the site irradiated with the lower
dose (Fig. 1D), suggesting that the substrate for the
accumulation of WRN was not single-strand breaks. To
confirm this, we examined the accumulation of WRN at single-
strand breaks produced in a nucleotide excision repair-deficient
xeroderma pigmentosum group A (XP-A) cell line expressing
Neurospora crassa UV damage endonuclease (UVDE), which
introduces a nick 5⬘to UVC-light induced lesions, and thereby
produces 5⬘-blocked single-strand breaks (Okano et al., 2003).
WRN did not accumulate at all at the UVDE-induced single-
strand breaks, whereas XRCC1 accumulated at the single-
strand breaks very efficiently (Fig. S2 in supplementary
material). These results provide further indications that the
substrate for the accumulation of WRN is not single-strand
breaks.
Accumulation of WRN at the irradiated sites was
significantly enhanced by BrdU treatment
Incorporation of BrdU in DNA has been shown to enhance the
production of double-strand breaks induced by UVA light and
Journal of Cell Science 118 (18)
Fig. 2. Enhanced accumulation of WRN by pre-treatment of cells
with BrdU. (A) Accumulation of GFP-tagged WRN indicated by
yellow arrows after higher dose irradiation with the 365 nm laser in
HeLa cells with or without pre-treatment with BrdU.
(B) Accumulation of GFP-tagged WRN after 10 and 100 scans with
the 405 nm laser with or without pre-treatment with BrdU in HeLa
cells. (C) Immunochemical detection of ␥H2AX after laser
irradiation in HeLa cells with or without pre-treatment with BrdU.
The number of scans with the 405 nm laser are indicated in yellow.
(D) Colocalization of GFP-tagged WRN and ␥H2AX at irradiated
sites in HeLa cells after 100 scans with the 405 nm laser.
(E) Immunochemical detection of endogenous WRN after 405 nm
laser irradiation with or without pre-treatment with BrdU in HeLa
cells. The number of scans with the 405 nm laser are indicated in
yellow. Arrows indicate the sites of irradiation.
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4157
Werner protein accumulates at DNA double-strand breaks
laser irradiation (Kim et al., 2002; Limoli and Ward, 1993;
Xiao et al., 2004). To determine whether the accumulation of
WRN with the higher dose laser was at double-strand breaks,
we analyzed the effect of BrdU on the accumulation. We found
that the intensity of accumulated GFP-WRN at the site
irradiated with a higher dose 365 nm laser pulse was
significantly enhanced in cells pre-treated with BrdU compared
with the accumulation without BrdU treatment (Fig. 2A). To
confirm this result and to distinguish fluorescence at the
irradiated sites from the spotted distribution of GFP-WRN
before irradiation, we introduced the 405 nm laser producing
linear irradiation sites in the nucleus (Fig. 1A, right).
Accumulation of WRN at 405 nm-laser-irradiated sites was
significantly enhanced by BrdU pre-treatment after scanning
10 as well as 100 times (Fig. 2B). The intensity of the
fluorescence of GFP-WRN at the irradiated site increased more
than threefold compared with the intensity without BrdU,
suggesting that GFP-WRN accumulated at double-strand
breaks produced by the addition of BrdU. To detect the
production of double-strand breaks, ␥H2AX was
immunostained after irradiation with the 405 nm laser. ␥H2AX
was detected as clear lines in irradiated cells following up to
500 scans, whereas scanning 750 and 1000 times produced
dispersed signals of ␥H2AX in the whole nucleus (Fig. 2C).
This result corresponds with the previous report using 337 nm
laser irradiation (Lukas et al., 2003) and may be explained by
the transmission of strong ␥H2AX signals to other parts of the
nucleus. ␥H2AX was stained much more brightly after
irradiation with the 405 nm laser following pre-treatment with
BrdU than without pre-treatment (Fig. 2C), showing that
increased numbers of double-strand breaks were produced in
the presence of BrdU compared with those in the absence of
BrdU. As expected, the lines of ␥H2AX were merged with
accumulated GFP-tagged WRN, even though the staining of
␥H2AX was much stronger and wider than the distribution of
GFP-WRN (Fig. 2D), indicating that the accumulation of
WRN is much more limited at the irradiated sites than the
distribution of ␥H2AX.
To exclude the possibility that the accumulation of WRN is
an artifact due to overexpression of WRN tagged with GFP, we
examined the accumulation of native WRN protein using
antibody against WRN. Accumulation of WRN was not detected
at irradiated sites in cells not pre-treated with BrdU (Fig. 2E left),
but was detected 15 minutes after various numbers of scans using
405 nm laser irradiation following pre-treatment with BrdU in
HeLa cells at the narrow irradiated lines (Fig. 2E right). GFP-
tagged WRN accumulated at the irradiated site even without pre-
treatment with BrdU, which may be explained by the fact that
the antibody recognizes the endogenous human WRN with a
very low affinity. Accumulation of WRN was observed by the
antibody only at the irradiated sites in cells, further suggesting
that WRN accumulated only at the region where double-strand
breaks are present.
Accumulation kinetics of GFP-tagged WRN at the sites
of double-strand breaks
Having established the accumulation of WRN at the site of laser-
induced double-strand breaks, we characterized the kinetics of
the accumulation of WRN and compared it with those of other
repair proteins. Fig. 3 depicts the accumulation kinetics of GFP-
tagged WRN and other proteins after the higher dose of 365 nm
laser irradiation. WRN accumulated rapidly at the irradiated sites
and the fluorescence at the sites reached a plateau 3 minutes after
irradiation. NBS1, an early response protein to double-strand
breaks (Celeste et al., 2003), accumulated at irradiated sites with
very similar kinetics as for WRN, whereas BRCA1 accumulated
much more slowly. Once they had accumulated at the irradiated
sites, all these proteins remained there up to 4 hours after
irradiation. In contrast to these proteins, LIGIII␣, which
functions in a final step in the repair of single-strand breaks,
dissociated from the irradiated site around 1 hour after
irradiation (Fig. 3). Like NBS1 and BRCA1, which are involved
in the repair of double-strand breaks via homologous
recombination, WRN remained at the irradiated site for a long
time, suggesting that the response of WRN might also be an
early event in homologous recombination repair.
Accumulation of WRN at irradiated sites is not enhanced
by photosensitization, which increases the production of
oxidative base damage
As WRN has been reported to enhance the repair of oxidative
base damage in vitro (Harrigan et al., 2003), we wanted to
examine the accumulation of WRN in cells pre-treated with
RO-19-8022, a photosensitizer, which increases the production
of oxidative base damage by absorption of light around 400 nm
(Will et al., 1999). We have previously shown that treatment
of cells with RO-19-8022 before laser irradiation enhanced the
accumulation of various glycosylases for oxidized bases, such
as NTH1, OGG1, NEIL1 and NEIL2, as well as other repair
proteins, such as POL ␤and PCNA, involved in base excision
repair at irradiated sites (Lan et al., 2004). This indicates that
the amount of various types of produced base damage is
increased by the photosensitization. However, in contrast to the
glycosylases and the other proteins, the accumulation of WRN
was not enhanced at all in cells pre-treated with RO-19-8022
(Fig. 4A) under the same irradiation condition as that of GFP-
NTH1 shown here (Fig. 4B), suggesting that the observed
accumulation of WRN is different from that of the glycosylases
and that WRN is not directly involved in the repair of oxidative
base damage.
Fig. 3. Comparison of accumulation kinetics of GFP-tagged WRN,
NBS1, BRCA1 and LigIII␣after laser irradiation of HeLa cells.
HeLa cells transfected with the various GFP-tagged genes were
irradiated with high-dose laser irradiation, and accumulation and
dissociation kinetics of the proteins were quantified.
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The accumulation of WRN at irradiated sites is
independent of DNA replication
A previous study showed that, 6 hours after treatment of cells
with camptothecin, WRN formed distinct foci, which partially
colocalized with RPA and RAD51 (Sakamoto et al., 2001). We
therefore investigated the dependence of the accumulation of
WRN at sites of laser irradiation on DNA replication. HeLa cells
were synchronized at the G1/S border by the double thymidine
block method and released by removing HU-containing
medium; they were then irradiated with the 365 nm laser in each
phase of the cell cycle. WRN accumulated at the laser-induced
damage site in all the cells of different cell-cycle phases shown
in Fig. 5. There was no significant difference in the kinetics of
accumulation of WRN at different cell-cycle phases (not shown).
These data suggest that accumulation of WRN at sites of DNA
damage is independent of DNA replication.
WRN accumulated at double-strand breaks via its
HRDC domain
WRN contains an exonuclease domain, a DEAH (single-
letter amino acid code) helicase domain, a conserved RecQ
family C-terminal domain (RQC) with NTS, a HRDC
domain and a NLS (Fig. 6A). To determine which domain is
responsible for the accumulation of WRN at laser-irradiated
sites, we examined the accumulation of deletion mutants of
WRN. We found that a GFP-tagged helicase mutant of WRN
(K577M) without the helicase activity (Chen et al., 2003)
and WRN with the deletion of the N-terminal exonuclease
domain (a.a. 152-1432) accumulated at the irradiated site
(Fig. 6B). To our surprise, the C-terminus HRDC domain
(a.a. 1021-1432) lacking exonuclease and helicase domains
accumulated at the irradiated site (Fig. 6B; see also Fig. S3
in supplementary material), indicating the importance of the
C-terminal region for the damage response. To further
address the importance of the HRDC domain for the damage
response, we examined the accumulation of the GFP-tagged
N-terminus WRN (a.a. 1-1150), which lacks the HRDC
domain but has an additional NLS attached at the C-
terminus, and a C-terminal WRN-NLS fragment without the
HRDC domain (a.a. 1229-1432). Both deletion fragments
failed to accumulate at sites of laser-induced double-strand
breaks (Fig. 6B; see also Fig. S3 in supplementary material).
Thus, the minimum region necessary for the accumulation
contains the HRDC domain with the C-terminal NLS
domain. Using the GFP-HRDC (a.a. 1021-1432) we
analyzed its accumulation kinetics. As shown in Fig. 6C, the
accumulation kinetics of the HRDC domain was exactly the
same as that of the full-length WRN, and the HRDC domain
remained at the irradiated site for a long period. Thus, the
HRDC domain is the domain responsible for the
accumulation of WRN at sites of double-strand breaks.
Journal of Cell Science 118 (18)
Fig. 4. Influence of RO-19-8022 on accumulation of GFP-WRN in
HeLa cells. After higher dose irradiation with the 365 nm laser in
HeLa cells, accumulation of WRN was not affected (A), whereas that
of GFP-tagged NTH1 was affected (B) by pre-treatment with RO-19-
8022.
Fig. 5. Replication-independent accumulation of WRN at irradiated
site.Accumulation of GFP-tagged WRN in G1/S, S and G1 phase HeLa
cells after higher dose irradiation with the 365 nm laser is shown. At
least ten cells from each cell-cycle phases were irradiated, and
representative data are shown. Arrows indicate the sites of irradiation.
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Werner protein accumulates at DNA double-strand breaks
Interacting proteins do not influence WRN accumulation
at double-strand breaks
Many proteins involved in DNA repair interact with WRN. To
identify whether the accumulation of WRN at sites of double-
strand breaks is dependent on the double-strand breaks-repair
proteins that have been reported to interact with WRN, we first
examined the accumulation of WRN in cells deficient in the
proteins. Because WRN has been reported to interact with Ku,
DNA-PKcs, and NBS1 (Cheng et al., 2004), we checked the
accumulation of WRN in cells derived from the Chinese
hamster ovary (CHO) cell lines of XR-C1 (DNA-PKcs-
deficient), XR-V15B (Ku80-deficient) and XR-1 (XRCC4-
deficient), as well as in a human cell line 1022QVA (Nijmegen
patient cells, NBS1-deficient). To examine whether the
modification of H2AX, an initial signal of double-strand
breaks, influences the accumulation of WRN, its accumulation
in H2AX–/– MEF cells was also analyzed. WRN accumulated
in all of the cell lines listed above in the same way as it
accumulated in their corresponding wild-type cells after a
higher dose of irradiation with the 365 nm laser (Fig. 7), which
indicates that the accumulation of WRN at double-strand
breaks is independent of these proteins. WRN is
phosphorylated in an ATM/ATR-dependent manner on
production of stalled replication fork (Pichierri et al., 2003).
Because pre-treatment of cells with caffeine has been reported
to interfere efficiently with ATM-dependent events such as
H2AX phosphorylation, we also examined the accumulation of
WRN in cells treated with caffeine. Accumulation of WRN
was not affected by caffeine (not shown), supporting the above
result of H2AX-independent WRN accumulation.
Although we showed that WRN did not accumulate at
single-strand breaks and base damage, WRN has been reported
to interact with poly(ADP)ribose polymerase-1 (PARP1) and
POL ␤(von Kobbe et al., 2003a), which play important roles
in single-strand breaks and base excision repair. Because these
proteins may also influence the accumulation of WRN at
double-strand breaks, we tested the accumulation of WRN in
PARP1–/– or POL ␤–/– MEF cells and found that accumulation
of WRN at the damage site in POL ␤–/– MEF cells and
PARP1–/– MEF cells was the same as that in the parental cells
after the higher dose of 365 nm laser irradiation (Fig. 7A).
Because PARP2 is known to act as a backup for PARP1, a
potent inhibitor for both PARPs, 1,5-dihydroxyisoquinoline
(DIQ) was used in HeLa cells. DIQ treatment significantly
suppressed the accumulation of XRCC1 at the irradiated sites
(Lan et al., 2004) but did not influence the accumulation of
WRN (not shown). Thus, PARP1, PARP activation and POL ␤
did not influence the accumulation of WRN at the irradiated
sites. It has also been reported that an interaction between
WRN and p53 may be involved in cellular responses to DNA
damage (Blander et al., 1999; Brosh et al., 2001). Because the
presence of functional p53 may influence the accumulation in
WRN at the irradiated site, we investigated the accumulation
of WRN at laser-induced double-strand breaks in hTERT-
Fig. 6. WRN accumulates at double-strand breaks
via its HRDC domain in vivo. (A) Domains and
mutations introduced in GFP-WRN. (B) Results of
accumulation of the mutants of GFP-WRN at
double-strand breaks. + means positive and – means
negative accumulation. (C) Accumulation kinetics
of GFP-tagged full-length WRN (WRN) and the
HRDC domain (HRDC) after laser irradiation of
HeLa cells. Standard deviations derived from at
least three independent data are indicated.
Fig. 7. Accumulation of WRN after higher dose laser irradiation at
double-strand breaks in the cell lines defective in various putative
WRN-interacting proteins. Accumulation of GFP-tagged WRN
indicated by yellow arrows 3 minutes after higher dose irradiation
with the 365 nm laser in corresponding cell lines. Arrows indicate
the sites of irradiation.
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4160
immortalized human fibroblast cells (Susa/T-n) with functional
p53 (Nakamura et al., 2002). WRN accumulated at double-
strand breaks in Susa/T-n cells as well as in other cell lines,
suggesting that the functional p53 does not influence the
accumulation of WRN at double-strand breaks (Fig. 7).
Discussion
Werner syndrome patients display various clinical symptoms
and signs of early aging. The gene responsible for the disease
encodes WRN, which has helicase and exonuclease activities.
Several proteins that interact with WRN have been identified,
most of which are involved in various types of DNA repair.
However, how WRN functions in cells remains unanswered. In
this report we showed for the first time that WRN accumulates
at sites of double-strand breaks locally produced by laser
micro-irradiation in living human cells. A recent report
indicated that the level of phosphorylation of H2AX at a
double-strand break, measured by a chromatin IP assay, is high
on each side of the double-strand breaks, extending up to
around 60 kb on each side, but is low in the region immediately
adjacent to the double-strand break (Unal et al., 2004). As
shown in Figs 2 and 3, ␥H2AX is dispersed over the whole
nucleus after high-dose irradiation (over 500 scans), whereas
only a restricted accumulation of WRN at sites of double-
strand breaks was observed after pre-treatment with BrdU
under high-dose irradiation (Fig. 2). Furthermore, WRN
accumulated at double-strand breaks even in an H2AX-
deficient mutant cell line as shown in Fig. 7. These results
indicate that WRN accumulates only at sites of double-strand
breaks independently of ␥H2AX.
By comparing the accumulation kinetics of WRN with other
proteins, we found that WRN showed similar kinetics to NBS1,
a protein exhibiting an immediate response to double-strand
breaks and playing a central role in the repair of double-strand
breaks. These results suggest that the response of WRN and
NBS might cooperate with each other in related repair
pathways. Although the interaction between WRN and double-
strand breaks repair-related Ku proteins has been reported
(Cooper et al., 2000; Li and Comai, 2001), our results showed
that deletion of the Ku-interaction domain of WRN (N-
terminal of WRN) did not prevent its accumulation at double-
strand breaks and that WRN accumulated at double-strand
breaks even in Ku86-deficient cells, suggesting that the
accumulation of WRN at double-strand breaks is not dependent
on Ku molecules. A recent study showed that TRF2
accumulated at laser-induced double-strand breaks
independently of the presence of Ku70, DNA-PKcs,
MRE11/RAD50/NBS1 complex and WRN as an early
response to DNA damage (Bradshaw et al., 2005). The
accumulation of WRN at double-strand breaks is quite similar
to that of TRF2, as the accumulation of WRN at sites of
double-strand breaks is also independent of the above proteins
(Fig. 7) and WRN responds to double-strand breaks as early as
TRF2 does. Both proteins associate with telomeres as well as
with double-strand breaks, indicating that they may join and
cooperate in the repair of double-strand breaks as well as in
the processes for protecting telomere ends.
Domain analysis indicated that the HRDC domain is
essential and sufficient for the accumulation of WRN at sites
of double-strand breaks. The function of the HRDC domain in
mammalian WRN has not been elucidated. The
threedimensional structure of the HRDC domain has been
determined for the Saccharomyces cerevisiae RecQ helicase
Sgs1p, the yeast homologue of WRN, by nuclear magnetic
resonance (NMR) spectroscopy (Liu et al., 1999). Structural
similarities of Sgs1p to bacterial DNA helicases suggest that
the HRDC domain of Sgs1p may function as an auxiliary
DNA-binding domain. However, most of the amino acids in the
basic patch of the HRDC domain in Sgs1p are not conserved
in the HRDC domains of other helicases including human
WRN, and a structural model of the HRDC domain of human
WRN shows different surface properties when compared with
that of Sgs1p (Liu et al., 1999). A previous study indicated that
the HRDC domain of WRN (a.a. 1072-1432) binds to the
forked duplex and Holliday junction with high affinity but to
the 5⬘-overhang duplex with lower affinity, whereas
exonuclease and helicase domain of WRN also contain these
binding activities in vitro (von Kobbe et al., 2003b). In
addition, full-length WRN showed a very low binding affinity
to DNA in a non-sequence-specific, structure-dependent
manner, and the HRDC domain is not a specific DNA-binding
domain in vitro. However, our study showed very clearly that
only the HRDC domain provides the protein with the ability to
accumulate at DNA damage. Although the exonuclease and
helicase domains showed binding activities to specific DNA
structure in vitro (von Kobbe et al., 2003b), these two domains
did not respond to double-strand breaks in our assay. This may
suggest a difference in the DNA damage response between in
vivo and in vitro. Our data showed that the HRDC domain of
WRN is able to assemble at double-strand breaks and may
function in guiding the whole WRN to DNA damage in cells.
Because the HRDC domain was shown to be an independently
folded structural domain, the HRDC domain may either bind
directly to double-strand breaks or interact with other
protein(s) present at double-strand breaks, which remain to be
identified.
All the mutations identified so far in patients with Werner
syndrome result in a truncated WRN protein that lacks the C-
terminus and the NLS. The inability of WRN to be transported
into the nucleus has been thought to be crucial for the
pathogenesis of WRN. Although Werner syndrome has been
associated with mutations in the HRDC domain, the deleted
WRN protein in the patients lost both the complete HRDC
domain and NLS (Moser et al., 2000; Moser et al., 1999;
Oshima, 2000). Therefore, the importance of HRDC might
have been concealed because of the simultaneous loss of NLS,
and it is possible that the response to double-strand breaks via
the HRDC domain of WRN is actually important for the
functions of WRN in cells.
Because camptothecin-induced WRN foci formation was
inhibited by aphidicolin, the formation of foci is thought to be
related to replication (Sakamoto et al., 2001). We have tested
the ability of deletion mutants of WRN to form foci. Although
the helicase mutant formed foci as efficiently as the full-length
WRN in cells treated with camptothecin, all the other deletion
mutants including HRDC domain alone failed to form foci (Fig.
S4 in supplementary material). From previous reports, MRE11-
RAD50-NBS1 complex seems to be a key factor for repair of
DNA double-strand break and blocked replication fork by
modulating related proteins, including BRCA1 and WRN. In
response to ␥-irradiation, BRCA1 and MRE11-RAD50-NBS1
Journal of Cell Science 118 (18)
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4161
Werner protein accumulates at DNA double-strand breaks
cooperate with each other to form irradiation-induced foci
(IRIF) (Wu et al., 2000; Zhong et al., 1999). WRN also interacts
with the complex, and hydrourea-induced foci of WRN was
shown to be dependent on NBS1 (Cheng et al., 2004; Franchitto
and Pichierri, 2004; Pichierri and Franchitto, 2004).
Autonomous accumulation of WRN at laser-induced damage
sites through its HRDC domain suggests that the accumulation
of WRN at double-strand breaks is an initial response of WRN
to double-strand breaks in living cells. Because the
accumulation of WRN at sites of laser-induced double-strand
breaks is independent of replication and other interacting
proteins (Figs 5, 7), the HRDC domain-dependent
accumulation of WRN at double-strand breaks is followed by
interaction with other proteins at the site of replication. Further
analysis is necessary to identify the relationship between
double-strand breaks accumulation and replication-dependent
foci formation of WRN in cells and the steps after accumulation
of WRN at double-strand breaks for forming IRIF.
Werner syndrome cells show replication defects and altered
telomere dynamics leading to the shortening of telomeres. A
mouse model with both Wrn and Ter c (encoding the telomerase
RNA component) deficiencies was shown to exhibit
accelerated replicative senescence of cells with Werner-like
premature aging phenotypes (Chang et al., 2004). Because the
telomere exhibits a double-strand breaks-like structure, it is
tempting to suppose that WRN accumulates via the HRDC
domain at shortened telomeres after replication in late S phase
and may contribute to the recovery of telomere length together
with other proteins. Further studies of the functions of WRN
at double-stand breaks in cells will help us to understand the
molecular basis of the phenotype of WS patients.
We thank M. Satou and Y. Watanabe of KS Olympus and Olympus,
respectively, for setting up the laser equipment. We also thank S. J.
McCready for editing the text. We thank S. H. Wilson, M. Masutani
and K. Ishizaki for providing us with POL ␤–/– MEF, PARP1–/–, and
Susa/T-n cells lines, respectively, and Dik C. van Gent for providing
us with XR-1, XR-C1 and XRV15B cell lines, used in this study. This
work was supported in part by Grant-in-Aid for Scientific Research
(no. 12143201 and 13480162) from the Ministry of Education,
Science, Sports and Culture of Japan and a grant from the National
Institute of Health (CA78088).
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