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Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland 21224 [J-P. L., P. L. O., F. E. I., J. A. H., C. v. K., V. A. B.], and Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France [J-P. L.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent data indicate cooperation between helicases and topoisomerases in DNA metabolism during progression of the replication fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, recombination, and DNA repair (15 , 16) . DNA topoisomerases solve the topological problems associated with DNA manipulation by introducing a transient break in the DNA and resolving the DNA structure (16) . Type I topoisomerases are monomeric, cleave one strand of duplex DNA, and require no energy cofactor. In contrast, the type II enzymes function as dimers, cleave both strands of the DNA, and pass an intact DNA duplex through the transient double-stranded break in an ATP-dependent manner (17) .
There is increasing evidence to suggest that RecQ helicases may act in concert with topoisomerases to preserve genomic integrity. Previous studies in bacteria and yeast have identified an interaction between topoisomerases and members of the RecQ helicase family. In Escherichia coli, RecQ and topo III together catalyze the linking and unlinking of covalently closed circular DNA molecules (18) . The yeast homologue of RecQ helicases, SGS1, interacts both physically and genetically with topo III, a type I topo. Mutations in SGS1 partially suppress the pleiotropic effects of TOP3 mutations, suggesting that Sgs1p and Top3p act in the same pathway (19 , 20) . Wu et al. (21) have shown that the Bloom protein (BLM) also interacts with human topo III and have reported recently that BLM stimulates the ability of topo III to relax supercoiled DNA (22) . Furthermore, a previous study found that topo I was coimmunoprecipitated with an antibody against WRN from human colon carcinoma SW480 cell extracts (23) . In addition, the inhibition of topo I activity by CPT results in a higher induction of chromosomal damage and apoptosis in WS cells, compared with normal cell lines, in the S and G2 phases of the cell cycle (13 , 24 , 25) . Topo I is a molecular target of CPT, which interferes with the topo I mediated DNA breakage-reunion reaction by stabilizing the enzyme bound to the DNA single-strand break intermediate and consequently inhibiting the DNA rejoining activity of topo I (26) . Furthermore, recent work in yeast showed that expression of an sgs1 mutant lacking DNA helicase activity was sufficient to rescue the hypersensitivity of the sgs1 mutant strain to topo inhibitors but failed to rescue the premature aging phenotype (27) .
In this study, we report that there is a physical and two-way functional interaction between WRN and topo I. Topo I inhibits the WRN ATPase and helicase activities, whereas WRN stimulates the topo I relaxation activity and stimulates the religation step of the topo I relaxation cycle. WS fibroblast cells are sensitive to CPT, and WS cell extracts show a decrease in DNA relaxation activity, compared with controls. These data suggest that WRN and topo I function together in a common pathway in the maintenance of genomic stability.
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MATERIALS AND METHODS |
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Characterization of WRN-Topo I Interaction by ELISA.
ELISA was performed as described previously (30)
, but with some modifications. The blocking and binding steps were performed in the same buffer (2% BSA, 0.1% Tween 20 in PBS). Wells were coated with 75 ng of purified topo I diluted in carbonate buffer (50 µl) or with BSA as a background control. After blocking, various amounts of purified WRN (0, 75, or 150 ng) were added in the binding step (50 µl), and ethidium bromide (10 µg/ml) was included in a control reaction. As a positive control and to estimate the amount of WRN protein bound to topo I, wells were coated with various amounts of WRN (0–300 ng) and were incubated with blocking buffer during the binding step. Following washes, primary antibody (1:1,500, anti-rabbit IgG against WRN; Novus, Littleton, CO) was added and incubated for 1 h at 37°C. Wells were washed, and secondary antibody (1:10,000, anti-rabbit IgG-horseradish peroxidase; Vector, Burlingame, CA) was added and incubated for 1 h at 37°C. Bound WRN was detected with o-phenylenediamine dihydrochloride followed by termination with 3 M H2SO4. Absorbance was read at 490 nm, and values were corrected for background observed in BSA only wells. To estimate the amount of WRN protein bound to topo I, the absorbance values for wells coated with WRN only were plotted against WRN protein amounts. Linear regression analysis was performed on the linear portion of the curve and used to calculate the amount of WRN protein bound to topo I-coated wells. Only those absorbance readings in the linear range were used. Values represent the means of four experiments, and error bars represent the SD.
Cell Lines and Culture.
h-TERT GM01604 (normal) and h-TERT AG03141 (WS) cell lines were a generous gift from Jerry W. Shay. The h-TERT cell lines, SV40-transformed VA13 (normal) and AG11395 (WS), were cultured in DMEM (Life Technologies, Inc.) supplemented with penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum. Normal (WI38) and WS (AG03141C) primary fibroblasts were cultured in MEM (Life Technologies) with 15% fetal bovine serum, essential and non-essential amino acids, vitamins, 2 mM L-glutamine, and penicillin/streptomycin. Cultures were incubated at 37°C in a 5% CO2 atmosphere.
Immunofluorescence.
Exponentially growing normal cells (h-TERT GMO1604) on chamber slides (Nalge-Nunc, Naperville, IL) were fixed in methanol at -20°C for 10 min and then permeabilized in 0.15% Triton X-100 for 10 min. Fixed cells were processed for immunofluorescence as described (31)
, except that TBS (20 mM Tris, pH 7.0, 150 mM NaCl) was used instead of PBS. Cells were stained with rabbit anti-WRN (H300; Santa Cruz Biotechnology Labs, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-topo I mAb (Novo Castra Lab, Newcastle upon Tyne, United Kingdom) and then with Cy3- conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA), and Alexa 488-conjugated goat anti-mouse secondary mAb (Molecular Probes, Eugene, OR). Prior to mountings, cells were treated with 20 µg/ml RNase A for 20 min (Worthington, Lakewood, NJ) and with the DNA stain DAPI (Molecular Probes, Eugene, OR). The position of nucleoli in the cells was monitored using Nomarski optics and DAPI staining. Approximately 100 cells from several randomly selected fields were evaluated for the presence of WRN/topo I colocalizing foci, both in nontreated and CPT-treated cells. Images were captured with a Zeiss Axioplan 2 microscope equipped with a Hamamatsu Orca 2 CCD camera. Thin optical sections of 100 nm were collected along the Z-axis of the cell in each of the three wavelengths examined. These sections were deconvolved, which is a mathematical process that assigns in-focus light to the proper section and removes out-of-focus light from all sections (Openlab software; Improvision, Lexington, MA). Some deconvolved sections were used for the volumetric reconstruction (Volocity software; Improvision) that provided the data for Table 1
. Figures were prepared with Adobe Photoshop 6.0 (San Jose, CA).
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Electrophoretic Mobility Shift Assay.
Binding (20 µl) was conducted in standard reaction buffer. Protein and DNA substrate concentrations were as indicated in figure legends. Reactions were incubated for 20 min at 4°C and stopped by the addition of 3x dye (0.125% bromphenol blue and 40% glycerol) to a 1x final concentration. Samples were loaded on 4% polyacrylamide (29:1) gels and electrophoresed overnight at 30 V, 4°C in 1x TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8). Products were visualized using a PhosphorImager.
ATPase Assay.
Reactions (10 µl) contained 150 ng of DNA cofactor, 1 µCi of [
-32P]ATP (3,000 Ci/mmol) in standard reaction buffer, with protein amounts indicated in the figure legend. Reactions were conducted at 37°C for 30 min and then stopped by adding 5 µl of 0.5 M EDTA. Samples (2 µl) were analyzed on a polyethylenimine-cellulose TLC and developed in 0.75 M KH2PO4. ATP hydrolysis was visualized by PhosphorImager and quantitated by ImageQuant. Results from at least three independent experiments are reported as mean ± SD.
Topo I DNA Relaxation Assay.
Topo I activity was measured by the relaxation of supercoiled plasmid pBluescript SK II DNA. The 20-µl assay mixture contained 40 mM Tris-HCl (pH 8), 4 mM MgCl2, 5 mM DTT, 7.5 µg/ml pBluescript SK II, and 0.1 mg/ml BSA. 2 mM ATP was added only where indicated. The reaction was initiated by the addition of 1 unit of topo I and allowed to proceed at 37°C for 30 min. Full-length WRN or WRN fragments were added prior to the addition of topo I at concentrations described in the figure legends. Products were run on a 1% agarose gel at 75 V for 30 min in TBE buffer. The gels were stained with ethidium bromide (0.5 µg/ml) for 30 min. The bands were visualized by illumination from below with short-wave UV light and photographed. Experiments using WCEs were conducted with the same buffer and under the same conditions. Amounts of total proteins are specified in the figure legends. Agarose gels were scanned using FluorImager SI software and quantitated using ImageQuant software (Molecular Dynamics, Inc.). The percentage of relaxation activity was calculated using the following formula: relaxed/(relaxed + supercoiled) x 100. The values for the percentage of relaxation activity were corrected for background in the no-enzyme control experiment. Data were analyzed by a simple t test.
WCE Preparation.
Cell pellets (100-µl packed cell volume) were resuspended in an equal volume of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP40, 2 mM Na3VO4, 10 mM NaF, and protease inhibitors (1 µg/ml chymotrypsin, pepstatin, aprotinin, leupeptin, and 1 mM phenylmethylsulfonyl fluoride) for 30 min on ice. Cell lysates were centrifuged at 4°C, 15,000 x g for 20 min, and the supernatants were aliquoted. WCE concentrations were determined by the Bio-Rad assay using BSA as a standard (Bio-Rad Laboratories, Hercules, CA).
Western Blot Analysis.
Equal amounts of WCE were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated in blocking buffer (5% nonfat milk, 0.5% Tween 20 in 1x PBS) for 1.5 h at room temp. The membranes were then incubated with rabbit anti-topo I (1:2,500; Topogen) and goat anti-lamin B (1:1,000; Santa Cruz Biotechnology; NM marker) polyclonal antibodies overnight at 4°C. After washing twice (10 min each) with blocking buffer, the membranes were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (1:10,000; Vector Laboratories) for 1 h at room temperature. After three washes (15 min each), enhanced chemiluminescence was performed (Amersham Biosciences), following the manufacturer’s instructions.
Detection of Cell Death.
Cells were grown on coverslips and exposed for 1 h to 15 µM CPT (stocks were dissolved in DMSO; Sigma; Ref. 13
), washed twice, and recovered in drug-free medium for 14 h. Samples were subsequently analyzed for cell death. The final DMSO concentration did not exceed 1% in CPT-treated cultures. PI (0.5 µg/ml; Sigma) dye was used to distinguish the dead cells from viable cells. After treatment, the coverslips were washed once in 1x PBS, followed by incubation with PI for 20 min at room temperature. After three washes in 1x PBS, the coverslips were mounted on Vectashield containing DAPI stain and viewed under a fluorescence microscopy (Zeiss). Reported values represent the means of two independent experiments in which at least 1,000 cells were scored for each.
Topo I DNA Strand Transfer/Religation Assay.
Reactions were conducted as described previously (33)
. Briefly, an 18-mer DNA oligonucleotide (5'-CGTGTCGCCCTTATTCCC) was 5' end-labeled in the presence of [-32P]ATP and T4 polynucleotide kinase and then hybridized to a complementary 30-mer strand (5'-TGTAGTCACTATCGGAATAAGGGCGCAACG) to form the 18-mer/30-mer cleavage substrate. Covalent topo I-DNA complexes were formed in the identical buffer as in the relaxation assay, containing (per 20 µl) 10 fmol of 18-mer/30-mer DNA and 150 fmol of topo I. The mixture was incubated for 5 min at 37°C. The strand transfer reaction was initiated by the addition of 0.5 pmol of an 18-mer acceptor strand, 5'-ATTCCCATAGTGACTACA (a 50-fold molar excess over the input DNA substrate), while simultaneously adjusting the reactions to 0.3 M NaCl. Addition of NaCl during the reaction promotes dissociation of the topo I after strand ligation and prevents recleavage of the strand transfer product. The reactions were terminated by the addition of SDS and formamide to 0.2 and 50%, respectively. The samples were heat-denatured and then electrophoresed through a 14% polyacrylamide gel containing 7 M urea in TBE. The extent of strand transfer was visualized by PhosphorImager and quantitated by ImageQuant. The percentage of 30-mer strand transferred product was calculated as the amount of 30-mer divided by total input labeled DNA (free 18-mer, 30-mer final product and the cleaved 12-mer complexed with topo I). The migration of the cleaved 12-mer/topo I complex was retarded in the gel (data not shown).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, columns 3 and 4). This interaction is specific, because BSA fails to show an interaction with WRN under the same conditions (Fig. 1A
, column 1). The WRN/topo I interaction was not mediated by DNA because the binding was unaffected by the presence of ethidium bromide (data not shown). To estimate the percent of WRN that bound to topo I, we coated wells with WRN protein alone, performed ELISA, and measured the absorbance for each WRN amount. Absorbance values were plotted against WRN protein amounts (Fig. 1B)
, and the linear portion of the curve was analyzed by linear regression (see "Materials and Methods"). On the basis of this analysis, for the binding reaction in which 75 ng of WRN was added to the topo I-coated wells, 
26% of the total WRN remained bound to topo I (Fig. 1A
, column 4). The absorbance values for the binding reaction containing 150 ng of WRN were outside the linear range. These experiments indicate that WRN and topo I bind to each other in vitro.
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, yellow). After cells were treated with CPT, both WRN and topo I were no longer present in the nucleolus (Fig. 2B)
. The distribution of topo I was similar in the WS cell line AGO3141 (data not shown). In GM01604 cells stained for WRN (green) and topo I (red), 60% of the cells examined showed colocalization of both proteins in the nucleolus (Fig. 2A
, yellow merge). In contrast, after treatment with CPT, only 6% of the cells showed nucleolar colocalization, and both WRN and topo I were mostly absent from the nucleolus (Fig. 2B)
. However, in 13% of the cells treated with CPT, a discrete non-nucleolar WRN/topo I colocalization can be discerned, indicating the presence of WRN/topo I protein complexes in the nucleoplasm and peripheral to the nucleolus (Fig. 2
B, yellow stain in enlarged panels). We further examined the colocalization of these proteins in a volumetric reconstruction of a CPT-treated cell (Table 1)
. We found that 58% of the WRN spots localized with 5% of the topo I spots. Another abundant nuclear protein that is associated with WRN in the nucleolus, the AAA ATPase VCP,4
did not localize with topo I (data not shown), thus supporting the specificity of the WRN/topo I colocalization.
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, Lane 2). The incubation of WRN (12 fmol), together with increasing amounts of topo I (12–96 fmol), resulted in an inhibition of WRN helicase activity (Fig. 3A
, Lanes 5–8). The addition of the highest amount of topo I tested (96 fmol) inhibited the WRN unwinding activity 3.6-fold (Fig. 3A
, compare Lanes 2 to 8). This inhibition was not attributable to components in the topo I storage buffer (Fig. 3A
, compare Lanes 2 to 3). The ability of topo I to inhibit WRN helicase may be attributable, in part, to topo I binding to the DNA substrate. Standard gel-shift analysis indicates that incubation of topo I with the helicase substrate led to a shift in the migration of the substrate corresponding to a topo I-DNA complex (Fig. 3B
, compare Lanes 1 and 3). Moreover, WRN did not stably bind the substrate (Lane 2), which is in accordance with our previous observations (28)
. Next, we investigated the effect of topo I on the ATPase activity of WRN. WRN (75 fmol) was incubated alone or with topo I (300 fmol) in the presence of either the M13 ssDNA or supercoiled DNA as effectors. The addition of topo I to the reaction led to a substantial inhibition (75%) of the WRN ATPase activity with the M13 ssDNA, from 10.2 ± 0.2% (WRN alone) to 2.4 ± 0.4% (WRN and topo I; Fig. 3C
, compare Lanes 1 and 2). Topo I does not bind ssDNA with high efficiency compared to dsDNA, suggesting that the inhibition observed using the M13 ssDNA as DNA effector may be attributable to the direct interaction between topo I and WRN. A similar inhibition of the WRN ATPase activity (78%), from 6.3 ± 0.6% (WRN alone) to 1.4 ± 0.5% (WRN and topo I), was observed using the supercoiled DNA as the DNA effector, a substrate that topo I can act upon (Fig. 3C
, compare columns 3 and 4). These results indicate that topo I inhibits the WRN ATPase activity. Because WRN is an ATP-dependent helicase, topo I inhibition of the WRN helicase may be attributable, at least partially, to its ability to inhibit the WRN ATPase.
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, Lane 2). As observed in Fig. 4A
, topo I relaxation activity was stimulated by the addition of an increasing amount of WRN (compare Lanes 2 to Lanes 3–6). Titration of WRN (0.3, 0.6, 1.2, to 2.4 pmol) led to an increase in topo I activity up to 64 ± 6%, whereas the highest amount of WRN alone did not affect the substrate (data not shown). Because these reactions were conducted in the absence of ATP, the stimulation of topo I by WRN does not require the catalytic activity of the WRN ATPase-dependent helicase. Addition of ATP did not alter the level of stimulation (data not shown).
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. N-WRN, corresponding to the exonuclease-proficient NH2 terminus of WRN (Fig. 4C
, Lanes 4 and 5), and C-WRN, corresponding to the COOH terminus (Fig. 4C
, Lanes 8 and 9), stimulated topo I activity similarly to the full-length WRN. The topo I stimulation by C-WRN indicates that neither the catalytic helicase nor exonuclease activities of WRN are required for stimulation. No stimulation was observed with the truncated WRN protein corresponding to the helicase domain of WRN (Hel-WRN; Fig. 4C
, Lanes 6 and 7). These data suggest the existence of two separate topo I-interactive sites in the WRN protein, one present in the NH2 terminus and another in the COOH terminus. In an effort to further map the topo I-interactive sites, we tested a battery of smaller GST-WRN protein fragments (Fig. 4D)
for their ability to stimulate the topo I relaxation activity. Each fragment (0.6 pmol) was incubated with 0.15 pmol of topo I. Only the GST-WRN fragment 2 (corresponding to the first 51 amino acids) was able to stimulate topo I, narrowing the first topo I-interactive site in the NH2 terminus to the first 51 amino acids of WRN (Fig. 4E
, Lane 4). GST alone and GST-WRN fragments 3 (WRN949–1092), 4 (WRN949–1236), and 5 (WRN1072–1236) corresponding to regions in the C-WRN (amino acids 949-1432) all failed to stimulate topo I activity (Fig. 4C
, Lane 10, and Fig. 4E
, Lanes 5–7). Incubation of either GST (Fig. 4C
, Lane 11) alone or the different GST-WRN fragments (data not shown) with the DNA substrate alone did not affect the substrate. These data suggest that the WRN region corresponding to amino acids 1236–1432 (because that region was not present in the GST subfragments) is necessary for the stimulation of topo I observed with C-WRN. This region represents part of the second topo I-interactive site but is not necessarily sufficient for the stimulation of topo I by the COOH terminus of WRN. Regions encompassed in the COOH-terminal GST-WRN fragments (3, 4, 5)
were not sufficient for topo I stimulation but could still contribute to the stimulation observed with full-length C-WRN. Taken together, these data indicate that the WRN N-terminal amino acids 1–51 and COOH-terminal amino acids 949-1432 functionally interact with topo I (Fig. 4G)
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To test the specificity of the functional interaction between WRN and topo I, i.e., the ability of WRN to stimulate the topo I relaxation activity, we assayed various proteins known to be implicated in DNA replication, repair, and recombination processes and that bind a variety of DNA structures. APE-1, Pol ß, and FEN-1 were incubated (Fig. 4F
, Lanes 3 and 4; Lanes 5 and 6; and 7, respectively) with topo I. No stimulation was detected upon addition of these proteins, supporting the specificity of the WRN protein to stimulate topo I activity.
WS Cell Extracts Exhibit a Decrease in DNA Relaxation Activity.
The observation that WRN stimulates topo I DNA relaxation activity in vitro predicts that WS cells should exhibit a decreased relaxation activity of negatively supercoiled DNA, compared with WRN-proficient cell lines. To test this hypothesis, we prepared WCEs from various WS and normal cell lines and compared levels of DNA relaxation activity using a negatively supercoiled plasmid. The assay conditions and substrate were chosen to specifically favor the detection of DNA relaxation activity attributable to topo I (see "Discussion"). DNA relaxation assays with equal amounts of WCE were carried out under the same conditions as the experiments with purified protein. We first tested fibroblast cell lines that were immortalized through the expression of exogenous h-TERT. The h-TERT WS (AG03141) cell line exhibited significantly less relaxation activity compared with the h-TERT normal (GM01604) cell line (46 ± 6% and 75 ± 5% activity, respectively; P < 0.001; Fig. 5A
). A similar deficiency was observed when comparing SV40-transformed WS (AG11395) and normal (VA13) cell lines, 60 ± 3% and 80 ± 11% respectively (P < 0.001; Fig. 5B
), and WS (AG03141C) and normal (WI38) primary fibroblasts, 27 ± 5% and 44 ± 6%, respectively (P < 0.001; Fig. 5C
). To determine whether the decrease in relaxation activity exhibited by the WS cell lines was attributable to decreased levels of topo I, Western blot analyses were carried out. Equal amounts of WCE were separated by PAGE, transferred, and blotted with an antibody against human topo I, along with an antibody against lamin B to control for loading. WCE, from h-TERT (Fig. 5D)
, SV40 transformed (Fig. 5E)
, and primary fibroblast (Fig. 5F)
cells displayed a similar level of topo I, suggesting that the decrease in the topo relaxation activity observed in the WS cell lines was not attributable to decreased expression levels of topo I.
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, treated normal cells (Fig. 6A, g)
, and untreated WS cells (Fig. 6B, c)
, approximately 2, 5.5, and 5%, respectively. In contrast, 17% of treated WS cell lines exhibited a staining pattern consistent with cell death (Fig. 6B, g)
. These results are consistent with previous reports using lymphoblast cell lines and demonstrate that the WS fibroblast cell line used in this study also exhibits an increase of cell death after treatment with CPT.
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. Site-specific cleavage of the DNA substrate by topo I triggered the release of a six-nucleotide oligomer from the 3' extremity of the 18-mer top strand. The resulting DNA cleavable complex consisted of a 12-mer strand annealed to a 30-mer top strand, covalently bound to topo I. The strand transfer reaction was initiated by adding 50-fold molar excess of an 18-mer acceptor strand, complementary to the 5' single-stranded tail of the DNA cleavable complex, together with increasing amounts of WRN (0, 0.15, and 0.3 pmol). These reactions were simultaneously adjusted to 0.3 M NaCl to promote topo I dissociation after ligation and to prevent recleavage of the strand transfer product (see "Materials and Methods"). Ligation of the 18-mer strand to the 12/30-mer complex leads to the formation of a 30-mer top strand product (Fig. 7A)
. After 1-h incubation at 37°C, 8.2 ± 3.8% of the substrates were converted to the expected 30-mer strand by topo I alone (Fig. 7B
, Lane 3 and 7C
, column 1), whereas 9.4 ± 1.5% and 18.2 ± 4.2% were converted upon addition of the WRN protein (0.15 and 0.3 pmol, respectively; Fig. 7B
, Lanes 4 and 5, and 7C
, columns 2 and 3). This 2.2-fold increase in strand transfer upon addition of the WRN protein indicates that the WRN protein stimulates the religation step of the topo I catalytic cycle.
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In the reciprocal interaction, we showed that WRN stimulated the ability of topo I to relax negatively supercoiled DNA and that this stimulation was not dependent on WRN catalytic activities. Moreover, we found that the topo I functionally interactive sites of WRN are located in the NH2 terminus (1–51 amino acids) and in the COOH terminus (949–1432) of WRN. The WRN extreme COOH terminus (1236–1432 amino acids) is necessary for topo I stimulation via interaction with the WRN COOH terminus. Our results are consistent with previous studies that found yeast Top3p interacts with the NH2 terminus and COOH terminus of Sgs1p (20) , and human topo III interacts with the extreme NH2 terminus (1–212 amino acids) and the COOH terminus (1266–1417) of the BLM protein (21) . A study in Saccharomyces cerevisiae demonstrated that a helicase-deficient mutant of the yeast RecQ homologue, sgs1, failed to rescue the premature aging phenotype of the SGS1 mutant strain but was still able to rescue the hypersensitivity of the SGS1 mutant to topo inhibitors (27) . Another study suggests a bipartite structure of sgs1p and concluded that the NH2 terminus of Sgs1p has an essential role, distinct from its DNA helicase activity (34) . Furthermore, BLM stimulates the DNA relaxation activity of human topo III (22) , as reported here for WRN and topo I. However, a truncated BLM mutant that retains helicase activity, but lacks the NH2 and COOH termini, failed to stimulate topo III (22 , 35) . Similarly, we found that a WRN truncated mutant that also retains helicase activity but lacks the NH2 and COOH termini failed to stimulate topo I. Together these data suggest that modes of interaction between RecQ helicases and their topo partners may be conserved. However, it is important to note that topo I and topo III belong to different type I subfamilies, differ mechanistically and structurally, and have distinct cellular roles (17) . Although WRN and BLM belong to the same RecQ helicase family, defects in these proteins give rise to distinctly different syndromes. Differences in protein partners, including associated topoisomerases, may reflect differences in WRN and BLM involvement in DNA metabolic pathways.
GFP-topo I has been shown to shuttle dynamically between the nucleoplasm and nucleolus (36) . After treatment with the CPT derivative, topotecan, GFP-topo I is rapidly sumoylated (37) . Sumoylation of the NH2-terminal region of topo I can prevent topo I binding to nucleolar sites, and CPT treatment increases sumoylation (38) . Our finding that WRN domains interact with topo I can provide the means by which this rapidly cycling protein is recruited to WRN-associated repair complexes. CPT treatment relocates both WRN and topo I to the nucleoplasm, where we observed several foci containing both WRN and topo I, indicating that a subset of both proteins are present in the same protein complex. However, there may be additional proteins in the WRN/topo I complex that may be essential for topo I function. Topo I is present in the nucleoli of WRN-null cells such as AGO3141, arguing that WRN does not have a direct role in sequestering topo I to the nucleolus or retaining it there. Further research is required to understand the dynamics of these multifunctional proteins.
Studies have shown a hypersensitivity of WS cell lines to CPT (13) , and one characteristic of this sensitivity is an increase in apoptotic induction after treatment with a sufficient dose of CPT. Here, we report that h-TERT WS fibroblast cell lines are also sensitive to CPT. Interestingly, cell extracts from h-TERT, SV40, and primary WS fibroblast cell lines exhibited a decrease in the overall relaxation activity on a supercoiled DNA plasmid, compared with the control cell lines. The reaction conditions were chosen to enrich specifically for topo I activity. ATP was omitted from the relaxation buffer to prevent potential relaxation activity from topo II, which is ATP dependent (39) . Moreover, topo III-catalyzed relaxation of negatively supercoiled DNA is more efficient at a higher temperature (52°C; Ref. 40 ). Furthermore, E. coli and eukaryotic topo III characterized to date require a hypernegatively supercoiled plasmid DNA substrate for efficient relaxation (41, 42, 43) . The above evidence suggests that the relaxation activity observed with the various cell extracts was primarily attributable to topo I. Our evidence is consistent with the decrease in the relaxation activity exhibited by the WS cell extracts as a likely consequence of the lack of WRN protein, and in turn, a lack of stimulation of topo I activity.
In this study, we show that WS cells display sensitivity to CPT without a significant increase in the level of topo I (Fig. 6)
. CPT interferes with the DNA breakage-ligation reaction by stabilizing the intermediate DNA-topo I complex, consequently inhibiting the religation activity of topo I. We suggest that WRN might shorten the duration of this cleavable complex in the cell by stimulating topo I relaxation activity and, more specifically, the religation step of the topo I relaxation cycle (Fig. 7)
. Fig. 8
shows a model for how WRN and topo I might act together in vivo. An intermediate step in the relaxation cycle of topo I is the formation of the cleavable complex. If the relaxation cycle goes to completion, the DNA is relaxed. Control of the superhelical state of the DNA and its relaxation are important during replication, transcription, and the segregation of newly replicated chromosomes. Accumulation of negatively (-) or positively (+) supercoiled regions behind or ahead of the replication and transcription machineries can lead to genomic instability by blocking these DNA metabolic processes and by inducing illegitimate recombination events (44
, 45)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These authors contributed equally to this work. 
2 To whom requests for reprints should be addressed, at Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224. Phone: (410) 558-8162; Fax: (410) 558-8157; E-mail: vbohr{at}nih.gov
3 The abbreviations used are: WS, Werner syndrome; topo, topoisomerase; CPT, camptothecin; h-TERT, human telomerase reverse transcriptase; mAb, monoclonal antibody; DAPI, 4',6-diamidino-2-phenylindole; WCE, whole cell extract; PI, propidium iodide; WCE, whole cell extract; GST, glutathione S-transferase.
4 Fred Indig, personal communication.
Received 5/ 5/03. Revised 8/ 8/03. Accepted 8/18/03.
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, wild-type cell lines; 
, WS cell lines. Values represent the means from six independent experiments; bars, SD. D–F, detection of the amount of topo I in each cell extract. WCEs were tested by Western blot analysis using a rabbit polyclonal antibody against human topo I. WCEs from cell lines used in A–C were loaded. The control for equal loading is indicated by blotting with an antibody against lamin B.
, topo I and 0.3 pmol WRN. The values represent the averages from at least three independent experiments; bars, SD.