WRN helicase regulates the ATR–CHK1-inducedS-phase checkpoint pathway in response totopoisomerase-I–DNA covalent complexes

Birija Sankar Patro1, Rikke Frøhlich1, Vilhelm A. Bohr2 and Tinna Stevnsner1,*1Department of Molecular Biology, University of Aarhus, C. F. Mollers Alle 3, DK-8000 Aarhus C, Denmark2Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA

*Author for correspondence (tvs@mb.au.dk)

Accepted 7 July 2011Journal of Cell Science 124, 1–13� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.081372

SummaryCheckpoints are cellular surveillance and signaling pathways that coordinate the response to DNA damage and replicative stress.Consequently, failure of cellular checkpoints increases susceptibility to DNA damage and can lead to profound genome instability. This

study examines the role of a human RECQ helicase, WRN, in checkpoint activation in response to DNA damage. Mutations in WRN lead togenomic instability and the premature aging condition Werner syndrome. Here, the role of WRN in a DNA-damage-induced checkpointwas analyzed in U-2 OS (WRN wild type) and isogenic cells stably expressing WRN-targeted shRNA (WRN knockdown). The results of

our studies suggest that WRN has a crucial role in inducing an S-phase checkpoint in cells exposed to the topoisomerase I inhibitorcampthothecin (CPT), but not in cells exposed to hydroxyurea. Intriguingly, WRN decreases the rate of replication fork elongation,increases the accumulation of ssDNA and stimulates phosphorylation of CHK1, which releases CHK1 from chromatin in CPT-treated cells.

Importantly, knockdown of WRN expression abolished or delayed all these processes in response to CPT. Together, our results stronglysuggest an essential regulatory role for WRN in controlling the ATR–CHK1-mediated S-phase checkpoint in CPT-treated cells.

Key words: WRN, Topoisomerase I, Checkpoint, S-phase, CPT

IntroductionThe BLM, WRN and RECQL4 helicase genes are mutated

in Bloom’s syndrome (BS), Werner’s syndrome (WS) andRothmund–Thomson syndrome (RTS), respectively. Thesesyndromes are autosomal recessive disorders associated withpredisposition to the development of cancer. Cells from BS and

WS patients have a hyper-recombination phenotype and displaysignificant genomic instability (Bohr, 2008). Mutations in theSGS1 gene, the RECQ helicase in Saccharomyces cerevisiae,

cause a similar phenotype (Gangloff et al., 1994; Watt et al.,1995; Watt et al., 1996; Frei and Gasser, 2000) that can bepartially suppressed by expression of human BLM or WRN (Watt

et al., 1996; Yamagata et al., 1998; Heo et al., 1999). Recently,Bernstein and co-workers suggested that the role of SGS1 inDNA replication can be uncoupled from its role in homologousrecombination (Bernstein et al., 2009). Moreover, redundant

roles of RECQ helicases in the resolution of recombinationintermediates is unlikely to explain the link of recombinationwith the clinical symptoms of WS, BS and RTS.

Defects in RECQ helicases cause hypersensitivity to replication-blocking agents (Cobb et al., 2003; Bernstein et al., 2009). The S-phase checkpoint blocks ongoing replication and late replication

firing, and promotes the repair of damaged replication forks(Branzei and Foiani, 2005; Willis and Rhind, 2009a). Two keyproteins, ATM (ataxia telangiectasia mutated) and ATR (ataxia

and Rad related) kinases, play distinct but overlapping roles inDNA-damage induced checkpoints. ATM–CHK2 kinases areprimary players in the response to ionizing radiation, and the

ATR–CHK1-mediated S-phase checkpoint is pivotal for the

response to UV, methyl methane sulfonate (MMS), hydroxyurea(HU) and campthothecin (CPT) (Kastan and Bartek, 2004; Willis

and Rhind, 2009a). SGS1-deficient yeast (S. cerevisiae) cells havea minor but reproducible defect in the intra-S-phase checkpoint

response, allowing spindle elongation and fork progression in thepresence of HU and MMC, respectively (Frei and Gasser, 2000).

The fission yeast (Schizosaccharomyces pombe) RECQ homologRQH1, plays a similar role, inducing the S-phase checkpoint in

response to MMS (Willis and Rhind, 2009b) and HU (Stewart

et al., 1997). In Caenorhabditis elegans, the loss-of-functionmutation in HIM-6 (BLM ortholog) results in a partially defective

cell cycle arrest in response to HU (Wicky et al., 2004).Interestingly, RNAi-mediated depletion of WRN-1 or CHK1 in

C. elegans leads to accelerated progression through S phase in thedevelopmental stage. A similar phenotype is also observed in

double RNAi knockdown of WRN-1 and CHK1, suggesting that

WRN and CHK1 are involved in the same S-phase checkpointpathway (Lee et al., 2004).

The evidence that human RECQ helicases play a role incheckpoint activation is limited. After release from HU arrest, BS

cells recover normally but with a slight delay compared with wild-type cells, which indicates that BLM is not required for the

recovery from S-phase arrest (Davies et al., 2004). A recent studyalso suggests a role for RECQL4 in S-phase checkpoints (Sangrithi

et al., 2005). Previous studies documented that WS cells, which are

hyper-sensitive to CPT, HU and PUVA (psoralen plus UVA),arrest and resume DNA replication normally in response to CPT

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JCS ePress online publication date 8 December 2011

(Poot et al., 1992; Pichierri et al., 2001). It has been reported

that WRN-depleted human fibroblasts show a marked delay in

completing the cell cycle after treatment with MMS or HU

(Sidorova et al., 2008). The authors of this study suggested that

WRN might be a downstream target of ATR–CHK1 and/or ATM–

CHK2 checkpoint signaling, rather than an upstream sensor

(Sidorova et al., 2008). However, it was recently revealed that in C.

elegans, WRN-1 acts upstream of ATM and ATR to facilitate RPA

foci formation and CHK1 phosphorylation upon HU stress (Lee

et al., 2010). It has also been suggested that the upstream functions

of WRN-1 are likely to be conserved, but might be cryptic in

human systems as a result of functional redundancy. The precise

role for human WRN in replication-stress-induced checkpoint is

not yet known (Lee et al., 2010).

We have shown previously that WRN-deficient cells have a

defective intra-S-phase checkpoint response to PUVA, HU or

CPT (Cheng et al., 2008). This study provided detailed evidence

that WRN is recruited to DNA double-strand breaks (DSBs) at

replication forks, activating an ATM-dependent checkpoint in

response to PUVA, but not in response to c-irradiation (Cheng

et al., 2008). This suggests a differential role of WRN in

checkpoint activation in response to DSBs. Alternatively, the

paused DNA polymerase and other damage could also be targets

for WRN to mediate the S-phase checkpoint.

CPT is a topoisomerase 1 (TOPI) inhibitor that generates

transient replication-blocking TOPI–DNA covalent complexes,

which undergo rapid reversal after removal of the drug (Covey

et al., 1989; Shao et al., 1999). CPT treatment rapidly induces a

strong S-phase checkpoint through the ATR–CHK1 pathway (Wan

et al., 1999; Cliby et al., 2002; Wang, 2002; Seiler et al., 2007),

which can be reversed by repair of TOPI-mediated DNA lesions.

Consistent with this, depletion of ATR, CHK1 or WRN, but not

ATM, causes hypersensitivity to CPT (Flatten et al., 2005). CPT-

treated WS cells accumulate DSBs and unresolved recombination

structures during S-phase (Pichierri et al., 2001). Nevertheless, the

exact role of WRN, if any, in the ATR–CHK1 pathway in CPT-

treated cells is not known. The goal of this study was to examine

the role of WRN in CPT-induced checkpoint activation, and test

the possibility of its involvement in the ATR–CHK1 pathway. The

results indicate that CPT-treated WRN-depleted human fibroblasts

complete S phase more quickly and activate the ATR–CHK1-

dependent response more slowly than is observed in wild-type

cells. Evidence presented in this study suggests that WRN delays

replication fork elongation in CPT-treated cells.

ResultsDepletion of WRN leads to defective S-phase checkpoint in

the presence of CPT but not HU

To maintain low-level expression of WRN, experiments were

carried out with U-2 OS cells stably expressing scrambled

shRNA (WRN-WT) and WRN-targeted shRNA (WRN-KD),

respectively. WRN-KD cells showed ,80% depletion of WRN

protein under all experimental conditions used in this study

(Fig. 1A). The WRN-KD cells exhibited elevated levels of both

spontaneous DSBs and induced DSBs in response to CPT (1 mM)

and HU (1 mM) compared with WRN-WT cells (Fig. 1B,C). A

similar increase in spontaneous DSBs and hypersensitivity

towards CPT has been reported for WRN-deficient patient cell

lines (Pichierri et al., 2001; Franchitto and Pichierri, 2004; Lowe

et al., 2004).

Fig. 1. Depletion of WRN in U-2 OS cells leads to

increased accumulation of DSBs in response to DNA-

damaging agents. (A) Western blot analysis shows

,80% reduction in the level of WRN protein expression

in WRN-KD cells compared with WRN-WT cells.

Lamin B was used as a protein loading control. Analysis

of DNA DSBs in WRN-WT and WRN-KD cells in

response to (B) CPT and (C) HU, respectively. Cells

were either left untreated or treated with CPT (1 mM)

and HU (1 mM) and DNA DSBs were evaluated by

neutral comet assay after 3 hours and 6 hours of

respective drug treatment. Lower panels in B and C

show the quantification of DSBs in terms of Olive tail

moment in comet assay. Points represent mean ± s.d.

from at least three experiments (200 nuclei in

each experiment).

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CPT rapidly induces an intra-S-phase checkpoint (Seiler et al.,

2007). However, after acute pulsed exposure to CPT (1 mM,

3 hours), WRN-depleted cells progress through S phase, whereas

wild-type cells do not (Cheng et al., 2008). To better understand the

role of WRN in the CPT-induced replication checkpoint, we

examined cell cycle progression in wild-type and WRN-depleted

cells exposed to CPT for 24 hours. Cells were treated with CPT

(10 nM to 1 mM) and nocodazole (0.25 mg/ml) for 24 hours and

analyzed by FACS. The nocodazole treatment arrests all the cells in

G2–M phase, which allows for monitoring S-phase progress within

the time of one cell cycle. Nocodazole arrested the same fraction of

WRN-WT and WRN-KD cells in G2–M (Fig. 2A), indicating that

depletion of WRN does not alter cell cycle progression in the

absence of DNA damage. In WRN-WT cells, 10 and 75 nM CPT

induced late S-phase arrest, and 250 nM and 1 mM CPT induced

predominately early S-phase arrest (Fig. 2A–C). By contrast,

WRN-depleted cells treated with 10, 75 or 250 nM CPT

progressed normally to G2, escaping the CPT-induced S-phase

checkpoint (Fig. 2A–C). The majority of WRN-depleted cells

exposed to 1 mM CPT arrested in S phase, although a small fraction

of the cells escaped arrest and progressed to G2 (Fig. 2A–C).

A similar analysis was performed on WRN-KD and WRN-WT

cells exposed to hydroxyurea (HU), which induces replication

stalling due to nucleotide depletion. The results show that WRN-

WT cells treated with 0.1 mM HU progressed through S phase

and arrested in late S or S–G2 (Fig. 3A–C). Intriguingly, at the

same HU concentration, WRN-depleted cells arrested in early S

or G1–S, with a slightly larger fraction of arrested cells at higher

HU concentration (Fig. 3A–C). Thus, at low concentration of HU

(0.1 mM) the G2–M fraction was higher in WRN-WT than in

WRN-KD cells (Fig. 3C). The results demonstrate that WRN-KD

cells show an intact S-phase arrest or checkpoint after exposure

to HU but not after exposure to CPT. Next, we were interested in

probing the exact role of WRN in elicitation of the intra-S-phase

checkpoint in response to CPT.

Early recruitment of WRN to stalled replication forks in

response to CPT but not HU

Because WS cells accumulate collapsed replication forks in

response to CPT (Pichierri et al., 2001), it is tempting to

speculate that WRN might be an upstream sensor, which

facilitates checkpoint activation at the replication fork sites to

Fig. 2. Depletion of WRN causes defective S-phase checkpoint in the presence of CPT. (A) WRN-WT and WRN-KD cells were treated with increasing

concentration of CPT (0–1000 nM) for 24 hours in the presence of nocodazole (0.25 mg/ml) as indicated. After 24 hours cells were harvested to determine cell

cycle distributions by flow cytometry as shown in cell count vs DNA content profiles of the cells. (B,C) Quantification of S-phase population (B) and G2–M-phase

population (C) in the same experiments as described in Fig. 1A (n54). Points represent mean ± s.d.

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prevent its collapse. Alternatively, WRN might be a sensor for

DSBs to elicit an S-phase checkpoint. To test these possibilities,

we asked whether WRN colocalizes with 53BP1, which

accumulates rapidly at DSBs in mammalian cells (Schultz et al.,

2000). As shown in Fig. 4A, WRN preferentially located in the

nucleolus in untreated cells, but is mobilized and distributed,

preferentially in foci, throughout the nucleus in approximately

47% of CPT-treated cells. About 31% of WRN foci colocalize

with 53BP1 foci (Fig. 4A,B), thus only a fraction of WRN

associates with DSBs after CPT treatment. Colocalization studies

were also performed for WRN and cH2AX, another DSB marker.

Similarly, a small fraction of WRN foci colocalize with CPT-

induced cH2AX foci (supplementary material Fig. S1), indicating

that WRN does not selectively interact with DSBs in CPT-treated

cells.

CPT stabilizes TOP1–DNA complexes, which causes

replication stalling and collapse, and resulting replication-

associated DSBs. Therefore, we further tested whether WRN

distribution also associates with stalled replication forks by

studying co-immunofluorescence of WRN and the ssDNA

binding protein RPA32 in the presence of CPT. Interestingly,

most of the WRN foci (,73%) colocalize with RPA32 foci in

CPT-treated cells (Fig. 4C), consistent with a previous report and

the idea that WRN associates with RPA32 in stalled or collapsed

replication forks upon CPT stress (Futami et al., 2008).

Moreover, kinetic studies shown in supplementary material Fig.

S2 indicate that WRN starts to form foci as soon as 30 minutes

after exposure to CPT, but it does not form foci after exposure to

HU (1 mM, 30 minutes). Nevertheless, a 30 minute exposure to

1 mM CPT or 1 mM HU caused ,55% or ,82% inhibition of

[3H]thymidine incorporation during DNA synthesis, respectively

(data not shown). Thus, HU-induced stalled replication forks are

not immediate targets of WRN. These data confirm a differential

role for WRN in the response to HU- and CPT-induced DNA

damage. This might reflect the rapid recruitment of WRN to

CPT-induced stalled replication forks (Fig. 4C) to mediate the

checkpoint response in order to avoid fork collapse.

WRN regulates ATR–CHK1-induced S-phase checkpoint in

response to CPT

The interaction between WRN and the ATR–CHK1 axis was

explored by measuring ATR-dependent phosphorylation of CHK1

in CPT-treated WRN-WT and WRN-KD, WRN defective WS-

KO-375, and ATR-defective Seckel cells. As shown in Fig. 5A,

CPT (1 mM, 3 hours) induced CHK1-ser345 phosphorylation

strongly in WRN-WT cells whereas only minimal or no induced

CHK1 Ser345 phosphorylation was observed in WRN-KD, WS-

KO-375 or Seckel cells. Several reports show that ATR mediates

phosphorylation of chromatin bound CHK1 to facilitate its release,

as required for checkpoint activation (Smits et al., 2006; Niida

et al., 2007). We sought to investigate whether this process might

be affected in WRN-depleted cells.

Consistent with the above result, dissociation of CHK1 from

chromatin and its accumulation in non-chromatin soluble fraction

was increased after exposure to CPT for 3 hours in WRN-WT

cells but not in WRN-KD cells (supplementary material Fig. S3).

Fig. 3. WRN is dispensable for HU-

induced checkpoint. (A) WRN-WT

and WRN-KD cells were treated with

increasing concentrations of HU (0.1–

1 mM) for 24 hours in the presence of

nocodazole (0.25 mg/ml) as indicated.

After 24 hours, cells were harvested to

determine cell cycle distributions by

flow cytometry as shown in cell count

vs DNA content profiles of the cells.

(B,C) Quantification of S-phase

population (B) and G2–M-phase

population (C) in the same experiments

as described in Fig. 3A (n53). Points

represent mean ± s.d.

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These results suggest that WRN acts upstream of CHK1 in CPT-

treated cells. A time course experiment showed that CHK1

phosphorylation reaches a maximum after exposure for 3 hours

and is maintained at this level for up to 12 hours in CPT-treated

WRN-WT cells. In CPT-treated WRN-KD cells, CHK1

phosphorylation was delayed and reached its maximum after 6–

12 hours (Fig. 5B). By contrast, the level of CHK1 Ser345

phosphorylation after exposure to HU was comparable in WRN-

WT and WRN-KD cells (Fig. 5C). The slow kinetics of CHK1

phosphorylation in WRN-KD cells after CPT treatment is not

likely to be accounted for by fewer S-phase cells or CHK1

downregulation in the WRN-KD population compared with

WRN-WT cells because only 2–5% fewer S-phase cells and

comparable CHK1 expression were monitored in the WRN-KD

cell population (Figs 2, 3, 5) compared with WRN-WT cells.

Moreover CHK1 phosphorylation was not affected in WRN-KD

cells, which displayed a similar number of S-phase cells before

and after HU stress. Furthermore, unlike WRN-WT most WRN-

KD cells completed S-phase (Fig. 2) after CPT treatment, which

is consistent with a defective S-phase checkpoint. Our data are

therefore consistent with distinct roles of WRN during HU- and

CPT-induced cell cycle arrest (Figs 2, 3).

Fig. 4. DSB-dependent and

-independent localization of WRN in

response to CPT. (A) WRN and

53BP1 colocalize to nuclear foci.

Indirect immunofluorescence of WRN

(green) and 53BP1 (red) in untreated

and CPT (1 mM, 3 hours) treated cells

are shown. In the merged image,

yellow coloration is due to

colocalization of WRN and 53BP1.

(B) Quantification of WRN foci

formation at 53BP1 sites (DSBs) and

other sites (non-DSB) in the same

experiment as described in A. Bars

represent mean ± s.d. (C) Detection of

WRN foci formation at replication sites

by immunostaining. Indirect

immunofluorescence of WRN (green)

and RPA32 (red) in untreated and CPT

treated cells (1 mM, 3 hours) are

shown. In the merged image, yellow

coloration is due to colocalization of

WRN and RPA32. The DAPI staining

(blue) in left panel shows the position

of the nucleus. A minimum of 150 cells

were assessed for each sample in three

independent experiments.

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The results shown in Fig. 5A,B predict that early events

downstream of ATR might be compromised in CPT-treated

WRN-KD cells. To test this possibility, phosphorylation of the

ATR targets RPA32 and H2AX, and monoubiquitylation of

ATR-phosphorylated FANCD2 (Andreassen et al., 2004; Wu

et al., 2005), were examined in CPT-treated WT and WRN-KD

cells. Indeed, all these downstream events were compromised in

WRN-KD cells after exposure to CPT (Fig. 5D). It should be

noted that cH2AX levels in the untreated samples varied

significantly between the cell lines. WS-KO-375, Seckel and

WRN-KD cells exhibited higher levels of H2AX phosphorylation

in untreated cells compared with that in untreated WRN-WT

cells. This is in accordance with previous reports showing that

WRN depletion activates the proteins involved in the DNA-

damage-response pathway (Szekely et al., 2004; Cheng et al.,

2008). Therefore, even though the level of H2AX

phosphorylation is lower in WRN-WT cells after CPT

treatment compared with levels in WRN-KD and WS-KO-375

cells, the level of induced H2AX phosphorylation after CPT

treatment is higher in WRN-WT cells.

Apart from ATR, ATM also plays a key role in eliciting DNA-

damage-response pathways after CPT stress. To determine the

role of ATM in WRN-mediated regulation of ATR pathway, we

investigated the CPT-induced phosphorylation of H2AX and

monoubiquitylation of FANCD2 in WRN-WT and WRN-KD

cells in the presence of an ATM-specific inhibitor (KU 55933).

In control experiments, KU 55933 (10 mM) inhibited

ATM autophosphorylation efficiently in response to CPT

(supplementary material Fig. S4A). CPT treatment induced

H2AX phosphorylation and FANCD2 monoubiquitylation in

WRN-WT cells whether or not ATM inhibitor was present.

Furthermore, ATM inhibition did not affect the response to CPT

treatment of WRN-KD cells (compare Fig. 5 and supplementary

material Fig. S4B). This suggests that the role for WRN in

activating early events in the ATR pathway is independent of

ATM.

One explanation for these data is that CPT generates fewer

DSBs and hence ATR-mediated activation of downstream

proteins is less pronounced in WRN-depleted cells. However,

as evident from Fig. 1B, WRN-KD cells accumulate more DSBs

Fig. 5. WRN regulates ATR-mediated signaling in response to CPT. (A) CHK1 phosphorylation on Ser345 was detected in WRN-WT, WRN-KD, WS-KO-

375 cells and Seckel cells treated with CPT (1 mM). Cells were harvested at the indicated time point and cell lysates prepared as described for western analyses.

(B) CHK1 phosphorylation was determined at various time points as indicated. (C) CHK1 phosphorylation on Ser345 was detected in control and WRN-KD cells

treated with HU (1 mM, 3 hours). (D) Phosphorylation and ubiquitylation of ATR substrates in control and WRN-KD cells after CPT treatment (1 mM, 3 hours).

Arrow shows phosphorylated form of RPA32; asterisk indicates monoubiquitylated form of FANCD2. Because endogenous levels of cH2AX and

monoubiquitylated FANCD2 are high in WRN-depleted cells at untreated conditions, fold increase (FI) in the levels of induced cH2AX and monoubiquitylated

FANCD2 in response to CPT are given by normalizing the fold increase (FI) in WRN-WT cells to 1.

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in response to CPT than seen in WRN-WT cells. DSBs

were also measured in WRN-WT and WRN-KD cells using

immunostaining for 53BP1. Supplementary material Fig. S5

shows that the 53BP1 foci are significantly higher in CPT-treated

WRN-KD cells compared with CPT-treated WRN-WT cells.

Therefore, the lack of ATR pathway activation in CPT-treated

WRN-KD cells is not a consequence of reduced accumulation of

CPT-induced DSBs.

WRN regulates CPT-induced intra-S-phase checkpoint, but

not the G2 checkpoint

The ATR–CHK1 checkpoint arrests CPT-treated cells in S and

G2 phase (Cliby et al., 2002). As evident from Fig. 3, WRN-

deficient WRN-KD cells did not elicit an intact S-phase

checkpoint after CPT treatment. However, it remains possible

that although WRN-depleted cells, with increased amounts of

DSBs (Fig. 1B; supplementary material Fig. S5), complete their

bulk DNA synthesis (with a defective S-phase checkpoint)

(Fig. 2A, Fig. 5A,B), they might display an intact DSB-mediated

G2–M checkpoint. To investigate the integrity of the G2

checkpoint, the fraction of CPT-treated WRN-WT and WRN-

KD cells in S and G2 was measured by FACS. Because the FACS

analysis does not allow us to distinguish G2 cells from mitotic-

arrested cells histone H3 Ser10 phosphorylation, a marker for

mitosis, was also analyzed. Fig. 6A,B shows that WRN-WT cells

accumulated in late S or G2–M phase, whereas WRN-KD cells

progressed through S phase and arrested in G2–M. Interestingly,

although nocodazole treatment leads to histone H3 Ser10

phosphorylation and mitotic arrest in both WRN-WT and

WRN-KD cell lines, CPT treatment failed to cause any such

Fig. 6. WRN regulates CPT-induced intra-

S-phase checkpoint but not G2 checkpoint.

(A) WRN-WT and WRN-KD cells were

treated with CPT (0.25 mM) and cells were

harvested at indicated time points to

determine cell cycle distributions by flow

cytometry. Arrows indicate arrested cells in S

phase. (B) Quantification of S-phase and G2–

M-phase populations in A (n53). Points

represent mean ± s.d. (C) Histone H3 Ser10

phosphorylation was quantified in WRN-WT

and WRN-KD cells untreated and treated with

CPT and nocodazole alone and together as

indicated. Cell lysates were prepared at the

indicated time point. (D) WRN-WT and

WRN-KD cells were untreated (UT) or

treated with CPT (0.25 mM) and nocodazole

(0.25 mg/ml) for 24 hours in the presence or

absence of UCN 01 (0.1 mM) as indicated.

Cell cycle distribution was analyzed by flow

cytometry as shown in cell count vs DNA

content profiles of the cells. Percentage of

cells in S and G2 phase population is

indicated under respective samples (n53).

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phosphorylation in WRN-WT and WRN-KD cells (Fig. 6C).

CPT treatment in the presence of nocodazole also failed to induce

histone H3 Ser10 phosphorylation in WRN-WT and WRN-KD

cells, indicating that these cells were arrested in G2 phase.

Together, these results show that WRN is required for the CHK1-

mediated intra-S-phase checkpoint, but not for the G2 checkpoint

in the presence of CPT.

To confirm the role of WRN in the regulation of the CHK1-

mediated S-phase checkpoint, WRN-WT and WRN-KD cells

were treated with 250 nM CPT and the CHK1 chemical inhibitor,

UCN 01 (Syljuasen et al., 2005; Seiler et al., 2007). FACS

analysis showed that ,81% of WRN-WT cells were blocked in

S-phase by CPT, but most cells overrode this checkpoint to enter

G2 in the presence of UCN 01 plus CPT (Fig. 6D). However, no

or minimal effect of UCN-01 was observed in WRN-KD cells

(Fig. 6D, 45% G2-cells in CPT vs 52% G2-cells in UCN 01 plus

CPT-treated WRN-KD cells). Control experiments showed that

UCN 01 had a minimal effect on cell cycle progression of WRN-

WT and WRN-KD cells in the absence of CPT (Fig. 6D). This

result supports the hypothesis that WRN regulates ATR–CHK1

function in a common intra-S-phase checkpoint pathway in CPT-

treated cells.

WRN inhibits replication fork elongation in the presence

of CPT

A WRN-regulated ATR–CHK1 pathway could inhibit replicon

initiation and/or chain elongation in CPT-treated cells and, in

turn, prevent collapse of stalled replication forks. To differentiate

between these possibilities, pulse-labeled DNA was analyzed on

alkaline sucrose gradients (Wang et al., 2004; Heffernan et al.,

2002; Willis and Rhind, 2009a) to determine the steady-state

distribution of nascent DNA intermediates in CPT-treated WRN-

WT and WRN-KD cells. Fig. 7A shows the experimental design

for these experiments. The results show that both replicon

initiation intermediates (top of the gradient) and chain elongation

intermediates (bottom of the gradient) were less abundant in CPT

treated (500 nM, 2 hours) compared with untreated WRN-WT

cells (Fig. 7B,C). Interestingly, although the replication initiation

process was reduced similarly, replication elongation was only

minimally reduced in CPT-treated WRN-KD cells (Fig. 7C; fork

elongation ,25% in WRN-WT vs ,60% in WRN-KD cells in

response to CPT). This result indicates that the WRN-regulated

inhibition of fork elongation might account for S-phase delay in

response to CPT.

WRN acts as an upstream sensor to CPT-induced

DNA lesions

The above results suggest that WRN plays an early role in ATR–

CHK1 activation, thereby inhibiting replication fork progression

in CPT-treated cells. Several reports show that ATR activation

requires single-stranded DNA (ssDNA) (You et al., 2002; Zou

et al., 2003). There are two possible mechanisms by which WRN

could regulate ATR-mediated CHK1 phosphorylation at stalled

replication forks in CPT-treated cells: (1) WRN might facilitate

ssDNA formation, leading to sequential activation of ATR and

CHK1, and/or (2) WRN might play a role in recruiting ATR to

stalled replication forks. To test the first possibility, ssDNA was

quantified by ssDNA-specific BrdU staining in CPT-treated

Fig. 7. Inhibition of chain elongation of DNA replication is impaired in WRN-depleted cells following exposure to CPT. (A) Schematic representation of

[3H]thymidine and CPT treatment. (B) Cells were harvested after CPT treatment as in A, and nascent DNA was separated by velocity sedimentation. Net 3H

radioactivity corrected for 14C spillover was normalized to cell number (total 14C radioactivity). Sedimentation is from right to left (i.e. fraction 1 is at the bottom

of the gradient and fraction 25 at the top). Percentage fork progress in the last panel was calculated by dividing the value of normalized 3H c.p.m. of each CPT-

treated fraction with that of untreated sample and multiplied by 100. The double and single arrows indicate the sedimentation rates of 167 kb and 37 kb DNA

marker, respectively.

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WRN-WT and KD cells. Two types of ssDNA foci were

observed in WT cells: large foci that colocalized with 53BP1

foci, representing resected DSB ends; and small foci that did not

colocalize with 53BP1 (i.e. not associated with DSBs) (Fig. 8A–

C; supplementary material Fig. S6). Additional experiments

showed that the ssDNA foci colocalized well with RPA32,

suggesting that ssDNA is generated at the sites of replication

(supplementary material Fig. S7). Large ssDNA foci at DSBs

(colocalization with 53BP1) were significantly more abundant

in CPT-treated WRN-depleted cells (Fig. 8B), whereas small

ssDNA foci were more abundant in CPT-treated WRN-WT cells

(Fig. 8C), suggesting that WRN facilitates ssDNA formation at

replication forks in CPT-treated cells.

We also analyzed whether WRN and ATR co-

immunoprecipitate in U-2 OS cells exposed to CPT. Fig. 8D

shows that WRN and ATR readily co-immunoprecipitate with

Fig. 8. WRN is involved in the generation of ssDNA and ATR activation in response to CPT. (A) Detection of incorporated BrdU specifically in ssDNA after

CPT treatment. Cells pre-labeled with BrdU were treated with CPT (1 mM, 3 hours) and stained with antibodies against BrdU (green) and 53BP1 (red). The DAPI

staining (blue) in the left panel shows the position of the nucleus. DNase-treated cells are included to show that the whole-cell population was uniformly labeled

with BrdU (Syljuasen et al., 2005; Lee et al., 2006). (B,C) Quantification of ssDNA foci formation at DSBs and non-DSB sites in the same experiment as

described in A at indicated time points. A minimum of 100–200 cells were assessed for each sample in three independent experiments. Points represent mean ±

s.d. (D) WRN co-immunoprecipitated with ATR in human U-2 OS cells. Immunoprecipitations were carried out using anti-ATR antibody, or an immunoglobulin

G control in presence of ethidium bromide (30 mg/ml), on extracts from cells exposed to no treatment or CPT treatment (1 mM, 2 and 4 hours). The

immunoprecipitates were analyzed by western blot for the presence of WRN using anti-WRN antibodies. WS, WS-KO-375 cells.

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anti-ATR antibodies in both untreated and CPT-treated U-2 OScell extracts, but not with immunoglobulin G control antibodies

or in WS-KO-375 (WRN-defective) cells. Reciprocal co-immunoprecipitation with anti-WRN antibodies also confirmedthe co-immunoprecipitation of ATR and WRN. This interactionwas enhanced in CPT-treated cells (supplementary material Fig.

S8A,B), similarly to a previous report showing that WRN-ATRinteraction increases in response to interstrand DNA crosslinks(Otterlei et al., 2006). Also, co-immunofluorescence experiments

demonstrated an increase in colocalization between WRN andATR foci in response to CPT (supplementary material Fig. S8C).Taken together, these data suggest that WRN and ATR form a

complex that is enhanced and/or stabilized in CPT-treated cells.

DiscussionBy using CPT, HU and PUVA, several reports show that WRN

could be involved in the resolution of recombinationalintermediates that arise from replication arrest or collapse(Pichierri et al., 2001; Dhillon et al., 2006; Sidorva et al.,

2008). We have recently shown that WRN is involved infacilitating an ATM-mediated checkpoint in response to PUVAinduced collapsed forks (Cheng et al., 2008). These previously

published reports strongly document a role for WRN,downstream to DSBs, in the repair of collapsed forks. Here, wepresent data directly implicating a further upstream role of WRNin the S-phase-specific checkpoint to prevent CPT-induced fork

collapse. We find that both CPT-induced S-phase checkpoint andATR signaling are severely compromised in WRN-depleted cells(Figs 2, 5). However, the CPT-induced G2 checkpoint is

fully functional in both WT and WRN-KD cells (Fig. 6A,B).Interestingly, a defective S-phase checkpoint and ATR–CHK1signaling in WRN-KD cells seems to be a CPT-specific response

because both wild-type and WRN-KD cells show robust CHK1phosphorylation and S-phase arrest in the presence of HU(Fig. 3A–C and Fig. 5C).

CPT-induced DNA damage appears to communicate withATR–CHK1 through WRN by at least three mechanisms. First,stabilized TOPI–DNA complexes could be recognized by WRN.Second, CPT-induced DSBs might elicit a checkpoint response.

Third, replication-derived complex DNA structures or unresolvedpositive supercoils, as suggested by Koster and co-workers(Koster et al., 2007), could also be sensed by WRN. WRN is

known to stimulate the TOPI-mediated relaxation cycle (Laineet al., 2003) and collaborate with proteins (RAD51, RAD52,MRN complex, ku70, DNA-PKcs) involved in DSB repair by

homologus recombination and non-homologus end joining(Sakamoto et al., 2001; Baynton et al., 2003; Cheng et al.,2004; Otsuki et al., 2007), suggesting a putative role of WRN inthe first two mechanisms mentioned above. In this study,

however, our results showed that a significantly higher fractionof WRN colocalizes with RPA32 (replication forks sites)compared with 53BP1 (DSBs site) (,73 vs 31%) in response

to CPT (Fig. 4A,C). Of note, WRN is found at most of the CPTinduced DSB sites (53BP1) (Fig. 4A), supporting its role in DSBrepair through homologous recombination and non-homologous

end joining. However, we also observed a fraction of WRN focithat did not colocalize with DSB sites, and these are likely torepresent WRN molecules engaged in other functions, possibly

related to CPT-induced stalled replication forks. A recent studydocumented the role of WRN in preventing fork collapse andDSBs upon replication arrest (Ammazzalorso et al., 2010).

Intriguingly, Ammazzalorso and co-workers demonstrated that

ATR-mediated phosphorylation of WRN facilitates properaccumulation of WRN in nuclear foci, colocalization with RPAand prevention of fork collapse upon replication stress

(Ammazzalorso et al., 2010). Thus, the role of WRN in CPT-induced toxicity is not just confined to DSB related repair andresolution of recombinational intermediates but it also plays acrucial role in preventing fork collapse. The presence of WRN at

replication forks (Fig. 4C), and its role in checkpoint activation(Fig. 5) and prevention of DSB formation (Fig. 1B) raise acompelling argument for an intriguing, previously unconsidered,

checkpoint-regulating role of WRN at stalled replication forks.

We further addressed how WRN mediates the slow down ofDNA synthesis in response to CPT (Fig. 2). Recent work usingDNA fiber labeling showed that the replication initiation

was normal in WS primary fibroblast cells whereas themajority of fork elongations were asymmetrical (Rodriguez-Lopez et al., 2002). Our analysis, using alkaline sucrose gradient

centrifugation, led to the interesting observation that thefrequency of origin firing is reduced to similar extents inWRN-KD and WRN-WT cells, whereas fork elongation is less

affected in WRN-KD cells compared with WRN-WT cells in thepresence of CPT (Fig. 7B). Implicit in this result is the ability ofthe WRN-induced checkpoint to impede fork progression

selectively in the presence of CPT. However, our results do notoverrule the idea that inhibition of the origin firing near the forksalso inhibits fork restart in WRN-WT cells in response to CPT,which warrants further study.

Importantly, Kumar and colleagues recently showed thepersistence of replication intermediates in MMS-treated fissionyeast by using 2D-gel analysis of replication intermediates

(Kumar et al., 2009). This suggests the inhibition of fork progressin response to MMS. The effects were dependent on Cds1 (acheckpoint kinase in fission yeast) and were abolished in cellsoverexpressing cdc25 phosphatase, showing evidence for

checkpoint-induced regulation of fork elongation in response toDNA damage. Similarly, unchecked fork progression in WRN-depleted cells might lead to frequent encounters of replication

forks with accumulated positive supercoils or TOPI–DNAcomplexes resulting in enhanced fork collapse (supplementarymaterial Fig. S5; Fig. 1A). Here, we propose that a WRN-

dependent checkpoint regulates fork progression by a globalmechanism in which the activated checkpoint acts in trans toaffect all fork stalling in response to CPT. Few individual pauses

at DNA–TOPI complexes would not significantly delayreplication, but the S-phase-checkpoint-mediated cumulativeeffect of forks pausing at many sites would lead to an overallreduction in replication rate.

Mechanistic insight into CPT-induced activation of theATR–CHK1 pathway by WRN

The ATR–CHK1 pathway is the primary pathway regulating the

S-phase checkpoint in response to stalled replication forksinduced by UVC, HU, aphidicolin etc. (Zhou and Elledge,2000; Cortez et al., 2001; Melo and Toczyski, 2002). These

agents specifically cause independent recruitment of ATR and anRFC-related protein, Rad17, to RPA-coated single-strandedDNA. Subsequently, the Rad9–Rad1–Hus1 (9-1-1) complex is

recruited to chromatin by the Rad17–RFC complex, and ATRphosphorylates Rad17 in a Hus1-dependent manner (Zou et al.,2002; Zou et al., 2003). Together, these events regulate

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phosphorylation of chromatin-bound CHK1, which in turn causes

rapid dissociation of phosphorylated CHK1 from the chromatin

(Smits et al., 2006; Niida et al., 2007). Although the ATR–CHK1pathway is required for S-phase arrest in response to CPT

treatment, it is not known how this pathway is activated. Several

lines of evidence in this study suggest a role of WRN in CPT-

induced checkpoint activation. Thus, we have demonstrated a

functional interaction between WRN and ATR by co-

immunoprecipitation and colocalization experiments, and shownthat WRN regulates CHK1 phosphorylation and subsequent

release from chromatin (Fig. 5B, Fig. 8; supplementary material

Figs S3, S8). We further show evidence here that WRN depletion

intriguingly caused no significant effect on ssDNA levels at DSBs

generated by resection, but the ssDNA level was drastically

reduced at the replication sites (Fig. 7A–C). These results suggestthat the WRN-dependent checkpoint signal does not emanate from

the (resected) DSBs per se. Moreover our results are consistent

with the hypothesis that WRN has an upstream role to facilitate

ssDNA formation for ATR-mediated checkpoint activation upon

CPT stress. Specifically, in response to UVC, aphidicolin and

cisplatin, the MCM helicase is thought to become functionallyuncoupled from DNA polymerase to generate a common ssDNA

intermediate. This is in turn coated with RPA to elicit checkpoint

signaling (Byun et al., 2005). In the case of CPT-induced lesions

(accumulated TOPI–DNA covalent complexes and associated

positive supercoilings), it is not possible for the MCM helicase to

bypass these lesions to uncouple from DNA polymerase. Thus, it ispossible that WRN senses CPT-induced lesions in order to

stimulate MCM helicase activity or induce transient uncoupling

between leading and lagging strand synthesis to generate ssDNA.

Alternatively, WRN might directly generate ssDNA through

its intrinsic helicase activity. Although WRN alone shows

preference for recessed 39 ends (Huang et al., 2000), it mightalso unwind fork substrates without a 39 single-strand tail in the

presence of RPA (Ahn et al., 2009). A third possibility is that WRN

helicase promotes reverse branch migration of the nascent

templates away from a stalled fork. Any or all of these processes

could account for the presence of ssDNA at forks. It is of future

interest to determine in detail how WRN causes generation ofssDNA in response to stabilized TOPI-DNA complex.

In summary, we have demonstrated that WRN is involved in

the induction of an intra S-phase checkpoint in response to CPT.

Our finding that WRN is involved in the generation of ssDNA atreplication forks, but not at sites of DSB end resection, shows

that WRN plays distinct roles in inducing checkpoint signaling

that results from TOPI–DNA complexes encountering forks. This

raises the possibility that the CPT sensitivity of WS cells reflects

loss of an S-phase surveillance mechanism, in addition to the

known function of WRN during resolution of intermediates inrecombinational DNA repair and the NHEJ pathway (Otsuki

et al., 2007; Kawabe et al., 2006; Pichierri et al., 2001). Our

findings have important implications for understanding resistance

mechanisms provided by WRN to a clinically important group of

cancer therapeutic drugs, specifically TOPI and CHK1 inhibitors

in combination with WRN silencing (Futami et al., 2008).

Materials and MethodsCell lines and culture conditions

U-2 OS osteosarcoma cells were routinely grown in DMEM supplemented with10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 C in 5% CO2. The generation andmaintenance of U-2 OS based WRN-WT (expressing control shRNA) and

WRN-KD (expressing WRN shRNA) cells have been described previously (Cheng

et al., 2008). WS-KO-375 (WRN mutated) and ATR-defective Seckel cells

(GM18367) were grown in RPMI and MEM medium supplemented with 10–15%

FBS, glutamine and antibiotics, as above. All cell culture media were purchased

from GIBCO. Nocodazole, CPT, HU, protease inhibitors (leupeptin, pepstatin andaprotinin) and phosphatase inhibitors cocktails were purchased from Sigma (St.

Louis, MO). The ATM inhibitor, KU55933 was purchased from Calbiochem.

(EMD Bioscience, La Jolla, CA).

Cell extracts, chromatin fractionation, immunoblotting and antibodies

Whole-cell lysates and immunoblot analysis were described previously (Cheng

et al., 2004). To analyze histone H3 Ser10 phosphorylation, cells were scraped,

pooled with nonattached cells, washed, and lysed with buffer containing 50 mM

Tris-HCl, 0.5% NP-40, 150 mM NaCl, protease and phosphatase inhibitors. After

incubation for 30 minutes on ice, the extracts were centrifuged (14,000 r.p.m.,

20 minutes) and supernatant was collected for blotting. Chromatin fractionation of

mammalian cells was performed essentially as described previously (Mandez and

Stillman, 2000). In brief, PBS washed (26106) cells were resuspended in 200 mlof solution A [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M

sucrose, 10% glycerol, 1 mM DTT, 0.1% Triton X-100, protease and phosphatase

inhibitors]. After incubation on ice for 5 minutes, the cytoplasmic (S1) and nuclear

fractions (P1) were harvested by centrifugation at 1300 g for 4 minutes. Next, the

P1 fraction was washed twice in solution A, lysed in 150 ml solution B (3 mM

EDTA, 0.2 mM EGTA, 1 mM DTT, protease and phosphatase inhibitors). After

incubating on ice for 10 minutes, the soluble nuclear (S2) and chromatin fractions

(P2) were harvested by centrifugation at 1700 g for 4 minutes. Isolated chromatin(P2) was then washed in solution B, spun down at 10,000 g and resuspended in

150 ml Laemmli sample buffer. The S1 and S2 fractions were pooled for

analyzing proteins in non-chromatin fraction. Primary antibodies were used in

immunoblotting against the following proteins: WRN, Lamin B, CHK1, TOPI

(Santa Cruz Biotechnology, Santa Cruz, CA), WRN, FANCD2, phosphor-

histone3-ser10 (Abcam, Cambridge, UK), p345CHK1 (Epitomics, Burlingame,

CA and Cell Signaling Technology, Beverly, MA), ATR (Oncogene, Darmstadt,

Germany), RPA32 (Bethyl, Montgomery, TX), 53BP1 (Novus, Littleton, CO),

ORC2 (BD Biosciences, San Jose, CA) and cH2AX (Upstate Biotechnology, LakePlacid, NY).

Immunoprecipitation

All steps in immunoprecipitations were performed at 4 C. After treating with CPT

(1 mM), cells were resuspended in 750 ml of TNN lysis buffer [50 mM Tris-HCl,

pH 7.6, 120 mM NaCl and 0.5 % (v/v) NP-40 and protease inhibitors]. Cell lysateswere kept on ice for 30 minutes, treated with 50 U of RNase-free DNase I (Roche,

BM) at 25 C for 30 minutes and centrifuged at 12,800 g for 10 minutes.

Approximately 1 mg/ml protein cell lysates were incubated with antibody against

ATR or an IgG control antibody overnight in the presence of ethidium bromide

(25 mg/ml). Subsequently, Protein-G–agarose beads (Invitrogen) were added and

incubated for an additional 3 hours under rotation. Antibody–protein complexes

were harvested by centrifugation, and the pellet was washed four times in 500 ml

of TNN buffer. Pellets were boiled in SDS-PAGE loading buffer and subjected to

electrophoresis and western blotting by standard procedures. Control experimentsshowed that the positive control IgG did not interact with ATR and WRN in cells

untreated treated with CPT (supplementary material Fig. S8B).

Immunofluorescence

Cells washed in PBS were fixed in ice-cold methanol and permeabilized in Triton

X-100 (0.5% in PBS, 10 minutes). Binding with anti-WRN (Santacruz and

Abcam), anti-53BP1 (Novus) and anti-ATR (Oncogene) antibodies was

performed in 5% BSA in PBS [1 hour, room temperature (RT)]. Primary

antibody detection was achieved using donkey anti-mouse Alexa Fluor 488 and

donkey anti-rabbit Alexa Fluor 555 secondary antibodies (Invitrogen) (1 hour,

RT). Slides were mounted in Antifade Gold containing DAPI (Invitrogen). Nuclei

were examined at a magnification of 636 using a Carl Zeiss fluorescence

microscope. The ssDNA patch assay was performed as reported previously(Syljuasen et al., 2005; Lee et al., 2006). Briefly, BrdU-prelabeled (10 mg/ml,

30 hours) cells were treated with CPT. Then cells washed in PBS were fixed

(chilled in methanol, 30 minutes), permeabilized [chilled in methanol:acetone,

7:3 (v/v), 30 minutes] and blocked in 5% BSA. Cells were then incubated

overnight with mouse monoclonal anti-BrdU antibody (which recognizes BrdU in

ssDNA only; BD Biosciences, 1:500) and 53BP1 (Novus, 1:4000) or RPA32

(Bethyl, 1:5000). Secondary antibodies, mounting and fluorescence microscopy

was performed as described above. Larger BrdU foci were quantified manuallyby carefully monitoring the foci that colocalized with 53BP1. The remainder of

the prominent and bright smaller foci were quantified as smaller ssDNA foci. At

least 100–200 nuclei were analyzed in each untreated and treated samples in two

to four independent experiments.

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Flow cytometry analysis

Single cell suspensions were fixed with 80% ethanol in PBS (24 hours, 220 C).Fixed cells were stained in solution containing 20 mg/ml propidium iodide (Sigma)and 50 mg/ml RNAse (Roche). Data were collected with a Becton DickinsonFACS machine (Mountain View, CA) and analyzed using FlowJo software.

Quantification of inhibition of DNA replication

This assay was performed as previously described (Heffernan et al, 2002) withminor modifications. Briefly, WRN-WT and WRN-KD (16105) cells were grownin the presence of [14C]thymidine (specific activity, 10 nCi/ml; 50 Ci/mol) for48 hours, and then in non-radioactive medium overnight to chase and label DNAuniformly. Cells were mock treated or exposed to CPT (0.5 mM), incubated at37 C for 1 hour 45 minutes, and then [3H]thymidine (10–50 mCi/ml; specificactivity, 56 Ci/mmol) was added to the same medium for 15 minutes. Cells wereharvested in lysis buffer (1 M NaOH and 0.02 M EDTA) and cell lysate waslayered on top of a 36 ml 5–20% alkaline sucrose gradient containing 0.4 MNaOH, 2.0 M NaCl, and 0.01 M EDTA. Gradients were placed under laboratoryfluorescent lights for l hour for cell lysis, centrifuged in an SW28 rotor (BeckmanInstruments) at 25,000 r.p.m. at 20 C for 12 hours. Gradients were fractionatedinto 24 equal fractions through the bottom of the centrifuge tube. Acid-insoluble3H/14C radioactivity was determined for each fraction by liquid scintillationspectrometry. After correction for spillover of 14C into the 3H channel, net 3Hc.p.m. in each gradient fraction were divided by the total l4C cpm in the gradient tonormalize DNA synthesis to cell number.

Neutral comet assay

DSB formation in WRN-WT and WRN-KD cells in response to CPT weremeasured as reported earlier (Lin et al., 2008).

AcknowledgementsWe thank W. H. Cheng for critically reading the manuscript and forhis valuable suggestions.

FundingThis research was supported by grants from the EuropeanCommission [grant number LSHM-CT-2004-512020]; LundbeckFoundation [grant number 4-55951-95094019]; The Danish ResearchCouncil [grant number 271-08-0697]; The Carlsberg Foundation[grant number 2008_01_0533]; The Danish Cancer Society [grantnumber DP05024]; and The Danish Aging Research Center (VeluxFoundation). This research was also partly supported by funds fromthe intramural program of the National Institute on Aging, NationalInstitutes of Health [grant number Z01 AG000721-01]. Deposited inPMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.081372/-/DC1

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