P53 Modulates RPA-Dependent and RPA-Independent WRN Helicase Activity

Research Article p53 Modulates RPA-Dependent and RPA-Independent WRN Helicase Activity

Joshua A. Sommers,1 Sudha Sharma,1 Kevin M. Doherty,1 Parimal Karmakar,1 Qin Yang,2 Mark K. Kenny,3 Curtis C. Harris,2 and Robert M. Brosh, Jr.1

1Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland; 2Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland; and 3Montefiore Medical Center, Department of Radiation Oncology, and Albert Einstein Cancer Center, Bronx, New York

Abstract metabolic pathways that influence genome integrity as suggested Werner syndrome is a hereditary disorder characterized by by the catalytic activities of the WRN protein and its interactions the early onset of age-related symptoms, including cancer. The with nuclear proteins implicated in DNA replication, repair, and absence of a p53-WRN helicase interaction may disrupt the recombination (8). signal to direct S-phase cells into apoptosis for programmed The tumor suppressor gene p53 plays a critical role in the DNA cell death and contribute to the pronounced genomic damage response pathway to activate checkpoint control in instability and cancer predisposition in Werner syndrome mammalian cells. Loss of p53 function results in genomic instability, cells. Results from coimmunoprecipitation studies indicate a key feature of carcinogenesis (reviewed in refs. 9–11). Key that WRN is associated with replication protein A (RPA) and components of checkpoint control mediated by p53 include arrest p53 in vivo before and after treatment with the replication of cell cycle progression, inhibition of DNA replication, and inhibitor hydroxyurea or ;-irradiation that introduces DNA activation of DNA repair. A DNA damage signaling pathway leads strand breaks. Analysis of the protein interactions among to elevated p53, which up-regulates transcription of several genes, purified recombinant WRN, RPA, and p53 proteins indicate including the cyclin-dependent kinase inhibitor p21 (12, 13). In that all three protein pairs bind with similar affinity in the low addition to growth arrest, p53 mediates damage-induced apoptosis nanomolar range. In vitro studies show that p53 inhibits RPA- in certain cell types (14). Induction of apoptosis by p53 is thought to stimulated WRN helicase activity on an 849-bp M13 partial be important for the removal of cells from the population that are duplex substrate. p53 also inhibited WRN unwinding of a short unable to repair the damage. (19-bp) forked duplex substrate in the absence of RPA. WRN Several recent findings suggest that p53 and WRN proteins unwinding of the forked duplex substrate was specific, function together to maintain genomic stability: (a) p53-mediated because helicase inhibition mediated by p53 was retained in apoptosis is attenuated in Werner syndrome cells (15); (b) the presence of excess competitor DNA and was significantly overexpression of WRN results in enhanced p53-dependent reduced or absent in helicase reactions catalyzed by a WRN transcriptional activity (16); (c) Sp1-mediated transcription of the helicase domain fragment lacking the p53 binding site or the WRN gene is modulated by p53 (17); and (d) WRNÀ/À knockout human RECQ1 DNA helicase, respectively. p53 effectively mice display accelerated mortality in a p53-null background (18). In inhibited WRN helicase activity on model DNA substrate support of a molecular interaction between WRN and p53, a intermediates of replication/repair, a 5V ssDNA flap structure physical interaction between the proteins has been reported (15, and a synthetic replication fork. Regulation of WRN helicase 16). Further work has shown p53 can modulate WRN exonuclease activity by p53 is likely to play an important role in genomic activity (19) and Holliday junction unwinding (20). integrity surveillance, a vital function in the prevention of Recently, several physical and functional interactions between tumor progression. (Cancer Res 2005; 65(4): 1223-33) helicase domain-containing proteins and p53 have been discov- ered. p53 binds to the human transcription factor IIH subunits XPB Introduction and XPD and inhibits their helicase activities (21, 22). p53 also interacts with BLM (23, 24), the Cockayne syndrome group B Werner syndrome is an autosomal recessive disorder that protein (22), and SV40 large T antigen (25, 26). p53 is also able to displays symptoms of premature aging, including an elevated inhibit the helicase activity catalyzed by the large T antigen (27, 28) incidence of neoplastic cancers, particularly sarcomas (1–3). as well as its replication function (29). In addition to helicases, Werner syndrome cells grown in culture display marked p53 physically and functionally interacts with the ssDNA binding chromosomal instability characterized by elevated rates of protein replication protein A (RPA; refs. 30, 31). p53 inhibits ssDNA chromosome translocation, rearrangements, and deletions. Muta- binding by RPA and blocks SV40 viral replication via its interaction tions in the Werner syndrome (WRN) gene are found in patients with RPA (30). exhibiting the clinical symptoms of Werner syndrome (4). The Our recent work showed that RPA physically interacts with WRN WRN gene encodes a protein that harbors both DNA helicase (5) (32), BLM (33), and RECQ1 (34) helicases and stimulates their and exonuclease (6, 7) activities. The precise molecular role(s) of respective helicase activities. A specific interaction between human the WRN protein is not known but presumably relates to DNA RecQ helicases and RPA is further supported by the absolute requirement for RPA in the WRN-, BLM-, or RECQ1-catalyzed unwinding of long DNA duplexes (32–34). Because RPA has been Requests for reprints: Robert M. Brosh, Jr., Laboratory of Molecular Gerontology, shown to have roles in DNA replication, recombination, and repair, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224. Phone: 410-558-8578; Fax: 410-558-8157; E-mail: [email protected]. human RecQ helicases are likely to function with the ssDNA I2005 American Association for Cancer Research. binding protein during one or more of these processes. www.aacrjournals.org 1223 Cancer Res 2005; 65: (4). February 15, 2005

The p53-RPA, RPA-WRN, and p53-WRN connections suggest that polynucleotide kinase was obtained from New England Biolabs (Beverly, the phenotypes of cancer and genomic instability in Werner MA). T7 Sequenase version 2.0 was purchased from U.S. Biochemical syndrome may possibly involve p53 modulation of WRN catalytic (Cleveland, OH). functions affected by RPA. To investigate this possibility, we have Nucleotides and DNA. M13mp18 single-stranded circular DNA was from New England Biolabs. The two oligonucleotides used for the 34-bp telomeric directly examined the effect of p53 on WRN helicase activity in the forked duplex DNA substrate were 5V-TTTTTTTTTTTTTTTTTAGGGT- presence or absence of RPA. Our results show that p53 can TAGGGTTAGGGTTAGGGCATGCACTAC-3V and 5V-GTAGTGCATGCCC- modulate RPA-dependent WRN helicase activity on a long 849-bp TAACCCTAACCCTAACCCTAACCCTAATTTTTTTTTTTTTTT-3V.Yeast DNA substrate. On forked DNA structures with a short (19-bp) tRNA was from Boehringer Mannheim (Indianapolis, IN). [g-32P]ATP was duplex region, p53 directly inhibited WRN helicase activity in a from Perkin Elmer (Wellesley, MA). specific manner. Regulation of DNA unwinding activity by p53 is Duplex DNA Helicase Substrates. The 849-bp M13mp18 partial duplex relevant to the important roles of RecQ helicases in the substrate was constructed as described previously (32). Briefly, the gel- maintenance of genome stability. purified 849-bp duplex DNA fragment from the HaeIII digest of M13mp18 RF was treated with calf intestinal phosphatase to remove 5V phosphates and subsequently labeled at their 5V ends using T4 polynucleotide kinase and 32 Materials and Methods [g- P]ATP. The complementary fragments were then annealed to M13mp18 ssDNA circle. M13 partial duplex DNA substrates were purified by gel Cells and Treatments. The normal human diploid fibroblasts (MRC-5) filtration column chromatography using A-5M resin (Bio-Rad, Hercules, CA). or Werner syndrome fibroblasts (AG11395) were grown in DMEM The 34-bp forked duplex DNA substrate was prepared as described previously j supplemented with 10% fetal bovine serum at 37 Cin5%CO2. Hydroxyurea (37). The 19-bp forked duplex, 26-nucleotide 5Vflap with either the upstream (Sigma, St. Louis, MO) was added to the 50% to 80% confluent cultures from or the downstream primer 5V32P labeled, and synthetic replication fork DNA a 200 mmol/L stock solution to a final concentration of 2 mmol/L for 15 substrates were constructed as described previously (38). hours until harvesting. For treatment with ionizing radiation, cells were Helicase Assays. Helicase assay reaction mixtures (20 AL) for assays 137 washed with PBS and then irradiated at 6 Gy using Gammacell 40, a Cs using the 849-bp M13 partial duplex substrate (0.125 nmol/L) contained source emitting at a fixed dose rate of 0.82 Gy/min (Nordion International, 40 mmol/L Tris (pH 7.4), 4 mmol/L MgCl2, 5 mmol/L DTT, 2 mmol/L ATP, Inc., Ottawa, Ontario, Canada). After ionizing radiation treatment, cells were 0.5 mg/mL tRNA, and the indicated concentration of WRN protein. For cultured in DMEM for 2 hours before harvesting. WRN or WRN500-946 helicase assays using the oligonucleotide-based duplex Coimmunoprecipitation Experiments. MRC-5 and AG111395 cells substrates, reaction mixtures (20 AL) contained 30 mmol/L HEPES Â 7 (1 10 ) were collected by low-speed centrifugation, washed in cold PBS, (pH 7.5), 40 mmol/L KCl, 8 mmol/L MgCl2, 2 mmol/L ATP, 100 ng/AL and lysed in whole cell lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L bovine serum albumin (BSA), 5% glycerol, and DNA substrate concen- NaCl, 5 mmol/L EDTA, 1% NP40, 2 mmol/L Na3VO4, 10 mmol/L NaF] trations indicated in the figure legends. For those WRN helicase reactions containing protease inhibitors (Roche, Indianapolis, IN). For coimmunopre- containing RPA or wild-type p53, the concentrations are indicated in the cipitation experiments, whole cell lysate (1 mg protein) was incubated with figure legends. Reaction mixtures were preincubated with RPA and the either rabbit polyclonal anti-WRN antibody (H-300, 1:40 dilution, Santa indicated concentration of p53 protein on ice for 4 minutes and Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG (Santa Cruz subsequently initiated by the addition of WRN protein. WRN helicase Biotechnology) either in the presence or in the absence of ethidium reactions were incubated at either 24jC for 60 minutes for the assays bromide (10 Ag/mL) for 4 to 6 hours at 4jC. The mixture was subsequently using the M13 partial duplex substrates or 37jC for 15 minutes using the tumbled with 40 AL protein G agarose (Roche) at 4jC overnight. Beads oligonucleotide-based duplex substrates. For RECQ1 helicase assays, were washed thrice with the whole cell lysis buffer supplemented with 0.1% reaction mixtures (20 AL) contained 20 mmol/L Tris-HCl (pH 7.4),

Tween 20. Protein complexes were eluted by boiling in SDS sample buffer 8 mmol/L DTT, 5 mmol/L MgCl2, 5 mmol/L ATP, 10 mmol/L KCl, 4% and resolved on 10% polyacrylamide Tris-glycine SDS gels followed by (w/v) sucrose, 80 Ag/mL BSA, and 0.5 nmol/L forked duplex DNA transfer to polyvinylidene difluoride membranes (Amersham Biosciences, substrate. RECQ1 helicase reactions were incubated at 37jC for 15 Piscataway, NJ). The membranes were blocked with 5% nonfat dry milk in minutes. Reactions were terminated by the addition of 10 ALof50 PBS containing 0.1% Tween 20 and probed for WRN, RPA, and p53 using mmol/L EDTA-40% glycerol-0.9% SDS-0.1% bromophenol blue-0.1% xylene antibodies against WRN (mouse monoclonal at 1:250, BD Transduction, cyanol. The products of helicase reactions were resolved on 6 or 12% San Diego, CA), 70- and 32-kDa subunits of RPA (Ab-1 and Ab-3 at 1:20, nondenaturing polyacrylamide gels. Radiolabeled DNA species in poly- Oncogene Research, San Diego, CA), and p53 (Ab-6 at 1:1,000, Oncogene acrylamide gels were visualized using a PhosphorImager and quantitated Research), respectively, followed by horse anti-mouse IgG conjugated to using the ImageQuant software (Molecular Dynamics, Piscataway, NJ). The horseradish peroxidase (Amersham Biosciences). Proteins on immunoblot percentage helicase substrate unwound was calculated as described were detected using Enhanced Chemiluminescence Plus (Amersham previously (38). Biosciences). For ssDNA competitor experiments, p53 (42 nmol/L) was preincubated Proteins. Baculovirus constructs for recombinant hexa-histidine-tagged, with the indicated concentrations (0-50 nmol/L) of ‘‘unlabeled’’ ssDNA full-length wild-type or exonuclease-defective WRN protein (WRN-E84A, (25-mer oligonucleotide) in standard helicase reaction buffer for unwinding designated X-WRN) were kindly provided by Dr. Matthew Gray (University assays with oligonucleotide-based substrates (see above) containing of Washington, Seattle, WA) and Dr. Judith Campisi (Lawrence Berkeley 2 mmol/L ATP for 3 minutes at 24jC. Forked duplex substrate (10 fmol National Laboratory, Berkeley, CA), respectively. Amplified baculovirus was radiolabeled 19 bp, final concentration 0.5 nmol/L) and WRN (final used to infect Sf9 insect cells for WRN protein overexpression, and WRN concentration 3 nmol/L) was simultaneously added to the reaction mixture was purified to apparent homogeneity as described elsewhere (35). after the 3-minute preincubation and incubated subsequently for 7 minutes Recombinant glutathione S-transferase-WRN500-946 fusion protein was at 37jC. Reactions were then quenched and resolved on native overexpressed in Escherichia coli and purified using glutathione beads polyacrylamide gels as described above. Typically, 70% to 85% of the forked (Amersham Biosciences) as described previously (35). Recombinant human duplex substrate were unwound in reactions lacking the DNA competitor RECQ1 helicase was overexpressed in insect cells using a baculovirus molecule. Unwinding (% control) is expressed relative to the control encoding recombinant RECQ1 kindly provided by Dr. Alessandro Vindigni reactions lacking the competitor DNA. (International Center for Genetic Engineering and Biotechnology, Trieste, Strand Displacement Synthesis Reactions. WRN and p53 were Italy) and purified as described elsewhere (34). Human RPA containing all preincubated on ice for 3 minutes in the presence of 10 fmol of a 26- three subunits (RPA70, RPA32, and RPA14) was purified as described nucleotide 5V flap substrate (upstream primer 5V 32P labeled) in 30 previously (36). p53 was purified as described previously (22). T4 mmol/L HEPES (pH 7.5), 40 mmol/L KCl, 8 mmol/L MgCl2, 2 mmol/L

Cancer Res 2005; 65: (4). February 15, 2005 1224 www.aacrjournals.org

ATP, 100 Ag/mL BSA, 0.5 Amol/L deoxynucleotide triphosphates, and 5% Effect of p53 on WRN Helicase Activity on M13 Partial glycerol. T7 Sequenase (U.S. Biochemical, 25 mU) was added and the 20 AL Duplex Substrates. To determine if p53 exerts a direct effect on reactions were incubated at 37jC for 5 minutes and terminated with the WRN unwinding of standard B-form duplex DNA, we preincubated addition of 10 AL formamide loading buffer. Reactions were resolved on 20% WRN helicase (92 nmol/L monomer) with increasing amounts of denaturing gels containing 7 mol/L urea. Radiolabeled DNA species in p53 (0, 47, 94, and 184 nmol/L monomer) before incubation with a polyacrylamide gels were visualized using a PhosphorImager. M13 28-bp partial duplex DNA substrate and found that p53 did Electrophoretic Mobility Shift Assays. Electrophoretic mobility shift not stimulate or inhibit WRN-catalyzed unwinding of the M13 assays were similar to those described previously (20). Briefly, reaction mixtures (20 AL) contained 30 mmol/L HEPES (pH 7.5), 40 mmol/L KCl, partial duplex (19). Moreover, p53 (47-184 nmol/L) did not stimulate WRN helicase activity on the 28-bp partial duplex 8 mmol/L MgCl2, 100 ng/AL BSA, 5% glycerol, 0.5 nmol/L of the 19-bp forked duplex DNA substrate, and the indicated p53 concentrations. substrate with an amount of WRN protein (26 nmol/L monomer) Reactions were incubated at 24jC for 20 minutes followed by fixation for 10 that unwinds only 10% of the duplex DNA substrate (19). minutes at 37jC in the presence of 0.25% glutaraldehyde. Products were We reported previously a physical and functional interaction resolved by 5% nondenaturing PAGE at 4jC for 2.5 hours and visualized between WRN and RPA (32). The presence of RPA stimulates using a PhosphorImager. WRN helicase to unwind DNA duplexes as long as 849 bp (32). ELISA Detection of Protein Interaction among WRN, RPA, and p53. p53 has been shown to associate with RPA both in vivo and in Purified WRN, p53 or RPA proteins were diluted to a concentration of 1 ng/ vitro (30, 31, 39). In addition, human p53 can inhibit SV40 viral A L in carbonate buffer [0.016 mol/L Na2CO3, 0.034 mol/L NaHCO3 (pH 9.6)], DNA replication by its association with RPA (30). The nature of A coated to appropriate wells of a 96-well microtiter plate (50 L/well), and the replication function of RPA that is inhibited by p53 is not allowed to incubate overnight at 4jC. Control wells were incubated with known. We hypothesized that p53 may inhibit RPA-dependent carbonate buffer and incubated overnight at 4jC. Wells were aspirated and blocked for 2 hours at 30jC with blocking buffer (3% BSA in PBS with 0.5% WRN helicase activity. To address this issue, an 849-bp M13 Tween 20). Wells were aspirated and washed again; all washing steps were partial duplex DNA substrate was tested for WRN helicase activity done with the blocking buffer. Wells were then coated with serial dilutions of in the presence of RPA and increasing concentrations of p53 (0, 47, WRN, RPA, or p53 in 30 mmol/L HEPES (pH 7.5), 40 mmol/L KCl, 8 mmol/L 94, and 188 nmol/L p53 monomer). Inhibition of WRN helicase

MgCl2,100ng/AL BSA, and 5% glycerol starting with 36 nmol/L protein. For activity was detected at a p53 concentration of 47 nmol/L (Fig. 1, the ethidium bromide treatment, 10 Ag/mL ethidium bromide were included lane 3) and more evident at p53 concentrations of 94 and 188 nmol/ in the incubation with WRN, RPA, or p53 during the binding step. Following L (Fig. 1, lanes 4 and 5). At 188 nmol/L p53, WRN unwinding of the incubation for 1 hour at 30jC, wells were aspirated, washed five times, and 849-bp substrate was inhibited f70% compared with WRN helicase j allowed to incubate for 1 hour at 30 C with anti-WRN (1:500, rabbit reactions lacking p53. Addition of up to 4-fold more RPA (final polyclonal, Novus Biologicals, Littleton, CO), anti-RPA (1:100 mouse concentration 372 nmol/L) in the WRN helicase reaction on the 849- monoclonal, Oncogene Research), or anti-p53 antibody (1:1,000, Oncogene bp duplex substrate did not alleviate the p53 inhibition (data not Research) diluted in blocking buffer. Following three washings, horseradish peroxidase–conjugated anti-rabbit (1:5,000, Santa Cruz Biotechnology) or shown), suggesting that the mechanism of p53 inhibition involves anti-mouse (1:5,000, Amersham Biosciences) antibody was added to the wells more than simply p53 sequestration of RPA. Inhibition of WRN and incubated for 30 minutes at 30jC. After washing five times, WRN and helicase activity was dependent on the duplex length of the M13 p53 were detected using OPD substrate (Sigma). The reaction was terminated substrate, because less inhibition was observed for 341- or 100-bp after 3 minutes with 3 N H2SO4 and absorbance readings were taken at 490 partial duplex substrates, and p53 failed to inhibit WRN helicase nm. The absorbance was corrected for the background signal in the presence activity on a 69-bp M13 partial duplex substrate in the presence of of BSA. RPA (data not shown). ELISA Data Analysis. The fraction of the immobilized WRN, RPA, or p53 Effect of p53 on Telomere Duplex Fork Unwinding by WRN. bound to the microtiter well that was specifically bound by WRN or p53 The p53 inhibition of RPA-dependent WRN helicase activity on protein was determined from the ELISA assays. A Hill plot was used to long M13 partial duplex substrates characterized by vast regions of analyze the data as described previously (33). ssDNA (f7,000 nucleotides) raised the question if p53 might retain its ability to modulate WRN-catalyzed DNA unwinding on a more Results It was shown previously that the COOH terminus of p53 (15, 16) physically interacts with the COOH terminus of WRN. In support of the direct physical interaction, it was shown that p53 modulates the exonuclease activity of WRN protein (19). No direct effect of p53 on WRN ATPase or helicase activity on a 28-bp M13 partial duplex substrate was observed (19). However, p53 has been shown to inhibit both WRN and BLM helicase activities on a Holliday junction (20). The physical interaction between p53 and WRN or RPA (30, 31, 39) suggested to us that WRN helicase activity on longer duplex DNA substrates might be affected by the presence of p53 in the reaction, because RPA is an essential ancillary factor for WRN unwinding of DNA duplexes at least 257 bp (32). In addition, we have examined the effect of p53 on WRN helicase activity on biologically relevant DNA structures that are proposed intermedi- Figure 1. p53 inhibits RPA-dependent WRN helicase activity on an 849-bp M13 ates during cellular DNA transactions. The ability of p53 to directly partial duplex substrate. WRN protein (55 nmol/L monomer) and RPA modulate the unwinding reaction of a human helicase would alter (93 nmol/L heterotrimer) were incubated with the 849-bp M13 partial duplex the ability of the enzyme to function in a pathway of DNA substrate (0.125 nmol/L) in the presence of the indicated concentrations of p53 monomer under the standard helicase reaction conditions as described metabolism and may have potential consequences for genome in Materials and Methods. Products were resolved on native 6% polyacrylamide integrity. gels. Heat-denatured DNA substrate control (E). www.aacrjournals.org 1225 Cancer Res 2005; 65: (4). February 15, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research physiologic substrate with reduced ssDNA character. Biochemical characterization of the DNA substrate specificity of the WRN helicase showed that a forked DNA duplex with noncomplemen- tary 3V and 5V ssDNA tails is a preferred structure for DNA unwinding by WRN (38). Forked DNA substrates are important DNA structural intermediates in a variety of DNA metabolic pathways, including replication, repair, and recombination. To investigate if p53 modulates WRN helicase activity on B-form DNA of a more physiologically relevant substrate, we tested the effect of p53 on RPA-dependent WRN helicase activity on a 34-bp duplex with 15-nucleotide 3V and 5V ssDNA tails. Because WRN may have a direct role in telomere metabolism, we chose a forked duplex of human telomeric repeat sequences (TTAGGG). In order for this telomeric duplex substrate to be unwound by WRN, RPA is required in the helicase reaction (37). We elected to test the effect of p53 on RPA-stimulated helicase activity of a mutant WRN protein (X-WRN) devoid of exonuclease activity due to a missense mutation in the active site of the exonuclease domain. The use of purified X-WRN recombinant protein enabled us to examine the effect of p53 on RPA-stimulated WRN helicase on the 34-bp forked duplex substrate without having to take into account the inhibitory effect of p53 on WRN 3V to 5V exonuclease activity that can degrade from the 3V termini of blunt ends of forked duplex molecules that the helicase does not efficiently unwind (37). As shown in Fig. 2, lane 3, the 34-bp forked duplex was not detectably unwound by X-WRN (1.5 nmol/L monomer). However, in the presence of RPA (9.8 nmol/L), WRN helicase unwound 62% of the 34-bp duplex (Fig. 2, lane 4). The presence of p53 in the WRN-RPA helicase reaction resulted in the inhibition of WRN helicase activity as evidenced by the disappearance of the released strands with the concomitant appearance of intact duplex DNA substrate resolved on the native polyacrylamide gel (Fig. 2, lanes Figure 3. p53 inhibits WRN helicase activity on a short (19-bp) forked duplex 5-9). Inhibition of WRN helicase activity on the 34-bp forked substrate. A, WRN protein (3 nmol/L) was incubated with the 19-bp forked duplex substrate was p53 concentration dependent. At the highest duplex (0.5 nmol/L) in the presence of the indicated concentration of p53 for 15 minutes at 37jC as described in Materials and Methods. Products were p53 concentration tested (123 nmol/L), WRN helicase activity was resolved on native 12% polyacrylamide gels. Heat-denatured DNA substrate inhibited by f50% of the control (no p53) unwinding reaction. control (E). B, quantitation of % control helicase activity from experiments as These results show that p53 effectively inhibits WRN helicase conducted in A. activity that requires RPA to unwind forked DNA duplexes as short as 34 bp. These results also show that the inhibition of RPA- dependent WRN helicase activity exerted by p53 does not require long ssDNA regions on the helicase substrate because the forked duplex with 15-nucleotide 3V and 5V ssDNA tails is effectively inhibited by p53. We conclude that RPA-dependent WRN- catalyzed unwinding of the telomeric duplex fork is significantly reduced by p53. p53 Inhibition of RPA-Independent WRN Helicase Activity. To determine if p53 had the ability to inhibit RPA-independent WRN helicase activity, we tested a forked DNA substrate with a short (19-bp) duplex region. WRN (3 nmol/L) was able to unwind 78% of the forked duplex substrate in the absence of RPA (Fig. 3A, lane 2). In the presence of 11 nmol/L p53, WRN helicase activity on the forked duplex decreased by 30% compared with control reactions lacking p53 (Fig. 3A, lane 3, and B). When the p53 concentration was increased to 21 nmol/ L, there was a 70% decrease in WRN helicase activity on the forked duplex (Fig. 3A, lane 4,andB). At the highest Figure 2. p53 inhibits RPA-dependent WRN helicase activity on a forked DNA concentration of p53 (42 nmol/L), there was a 90% decrease in duplex of human telomeric sequence. An exonuclease defective WRN mutant protein, X-WRN (1.5 nmol/L), was incubated with the 34-bp forked WRN helicase activity (Fig. 3A, lane 5, and B). These results duplex (0.9 nmol/L) in the absence or presence of RPA (9.8 nmol/L heterotrimer) show that p53 inhibits WRN helicase activity on a short (19-bp) and the indicated concentrations of p53 monomer for 15 minutes at 37jC as described in Materials and Methods. Products were resolved on native 12% forked duplex that WRN unwinds efficiently in the absence of its polyacrylamide gels. Lane 1, heat-denatured substrate control. auxiliary factor RPA.

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glutathione S-transferase-WRN helicase domain fragment (glutathione S-transferase-WRN500-946) that lacks the COOH-terminal p53 interaction domain (WRN residues 1,013-1,432). p53 (21 nmol/L) inhibited the unwinding catalyzed by the WRN helicase domain fragment to some extent (29% inhibition) but not as greatly as that observed for full-length WRN (55% inhibition; Fig. 6A and B), suggesting that the p53 protein interaction with WRN significantly contributes to the helicase inhibition. To evaluate the specificity of WRN helicase inhibition by p53, we tested its effect on another human RecQ DNA helicase, RECQ1. As shown in Fig. 6C and D, RECQ1 DNA helicase activity was completely resistant to a concentration of p53 (42 nmol/L) that under the same reaction conditions nearly eliminated WRN unwinding of the 19-bp forked duplex substrate. These results indicate that p53 inhibition of WRN helicase activity is specific and not a general effect exerted on any human RecQ helicase. p53 Inhibition of WRN Helicase Activity on DNA Replication and Repair Intermediates. Our previous studies showed that WRN helicase efficiently unwinds two important DNA intermediates of replication/repair, a 5V ssDNA flap and a synthetic replication fork (38). WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of Figure 4. p53 binds specifically to the forked DNA duplex substrate. The 19-bp the fork. For the 5V flap structure, WRN specifically displaced the forked duplex (0.5 nmol/L) was incubated in the presence of wild-type p53 (1-21 nmol/L) as described in Materials and Methods. Binding reaction mixtures 5V flap oligonucleotide, suggesting a role of WRN in Okazaki were resolved on native 5% polyacrylamide gels. Phosphoimage of gels that fragment processing or DNA repair. To address a potential role of is representative of at least three independent experiments. p53 in modulation of WRN helicase activity on these replication/ repair intermediates, we tested for its effect on WRN-catalyzed An interaction between p53 and the forked duplex DNA unwinding of the 5V flap (Fig. 7A) or synthetic replication fork substrate might contribute to the inhibition of WRN helicase structures (Fig. 7C). The results, shown quantitatively in Fig. 7B activity on this substrate because p53 is known to bind to DNA and D, show that p53 effectively inhibited WRN helicase activity on structures, including Holliday junctions (40). Using a gel mobility both DNA substrates. The ability of p53 to block WRN unwinding shift assay to measure DNA binding, we observed that indeed of the replication fork structure indicates that the p53 inhibition p53 was able to bind the forked duplex substrate used for the does not require any preexisting ssDNA in the helicase substrate to helicase studies in a specific manner dependent on the p53 prevent WRN from unwinding the DNA structure. concentration (Fig. 4). Because p53 inhibited WRN helicase activity on the 5V flap The ability of p53 to bind the forked 19-bp duplex raised the structure, we were interested in the possibility that it would possibility that the p53 inhibition of WRN helicase activity may in modulate the extent of strand displacement by DNA polymerase part be mediated by a p53-DNA interaction as opposed to a p53- synthesis that is stimulated by WRN helicase activity. To address WRN protein interaction. To address this issue, we examined the this experimentally, we did primer extension assays using a 5V effect of p53 on WRN unwinding of the same forked duplex flap substrate in which the upstream primer was 5V 32P-labeled. substrate but with the additional presence of a 25-nucleotide Strand displacement DNA synthesis was stimulated by WRN ssDNA competitor, because p53 binds to ssDNA and the forked helicase activity as evidenced by the increased production of duplex substrate contains 25- and 26-nucleotide ssDNA arms. As longer 32P-labeled single strands resolved on denaturing shown in Fig. 5, p53 retained its ability to inhibit WRN unwinding polyacrylamide gels (compare lane 2 with lane 3 in Fig. 7E). of the forked duplex substrate (0.5 nmol/L) in the presence of Enhancement of strand displacement DNA synthesis was competitor ssDNA (12.5-50 nmol/L), suggesting that p53 is dependent on WRN helicase activity, because a helicase-dead inhibiting WRN helicase activity by a mechanism other than p53 ATPase mutant of WRN did not stimulate DNA synthesis under simply binding to the ssDNA arms of the forked duplex substrate conditions in which the wild-type WRN enzyme did as observed and preventing WRN from either binding or efficiently initiating previously for the effect of WRN on strand displacement DNA unwinding of the forked duplex substrate. Similar compe- synthesis by polymerase (42). When p53 was included in the 4 tition experiments with cold forked duplex DNA (41) or blunt DNA synthesis reaction mixture with WRN (Fig. 7E, lane 4), the 5 duplex DNA inhibited WRN helicase activity in the absence of synthesized DNA products were shorter and more closely p53, which prevented us from assessing the ability of p53 to resembled those from reactions lacking WRN (Fig. 7E, lane 2). inhibit WRN helicase activity in the presence of these DNA In control reactions, p53 did not have an effect on DNA competitor molecules. synthesis catalyzed in the absence of WRN (Fig. 7E, lane 5). Effect of p53 on Unwinding Catalyzed by WRN Helicase Under the reaction conditions for strand displacement DNA Domain Fragment or Human RECQ1 Helicase. To better understand the nature of the p53 inhibition of WRN-catalyzed unwinding of the forked duplex, we compared the effect of p53 on 4 Unpublished data. DNA unwinding catalyzed by full-length WRN with a recombinant 5 Unpublished data. www.aacrjournals.org 1227 Cancer Res 2005; 65: (4). February 15, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research synthesis, p53 inhibited WRN unwinding of the 5V flap specificity of the WRN antibody was shown by the result that RPA oligonucleotide.6 These results indicate that p53 modulates or p53 was not precipitated by the anti-WRN antibody from the strand displacement DNA synthesis by inhibiting WRN helicase WRNÀ/À cell extracts in which WRN was absent (Fig. 9B). In activity on the 5V flap structure. addition, both p53 and RPA were coimmunoprecipitated with Physical Analysis of the Protein Interactions among WRN, WRN by the anti-WRN antibody in the presence of ethidium p53, and RPA. Collectively, the results from the p53-WRN helicase bromide from the normal cell extracts (Fig. 9B), suggesting that a studies suggested that direct protein interactions among WRN, RPA, DNA bridge was not responsible for their coimmunoprecipitation. and p53 are likely to be important in the inhibition of WRN helicase These results show that endogenous WRN-p53 and WRN-RPA activity on the various DNA substrates. To characterize the physical complexes exist in human cells during normal DNA metabolism protein interactions, we did ELISA studies to determine the relative and persist during replication arrest or DNA processing in binding affinities of the protein pairs (WRN-RPA, WRN-p53, and response to DNA damage. RPA-p53). Purified recombinant p53 protein (0-36 nmol/L) was incubated in the presence of 3% BSA with WRN or RPA that had Discussion been immobilized on microtiter wells. Bound p53 was detected In this report, we have shown that p53 modulates both RPA- using anti-p53 antibodies. The specificity of this interaction was dependent and RPA-independent WRN helicase activity on B-form duplex DNA substrates. To gain some insight to the nature and shown by very low absorbance values (0.089 F 0.012 A490) for wells that had been precoated with BSA compared with the intense signal specificity of WRN helicase inhibition by p53, we have focused on obtained with WRN or RPA (Fig. 8A). The colorimetric signal from further characterizing the effect of p53 on short oligonucleotide- the WRN-p53 or RPA-p53 interaction was both dose dependent and based substrates that have only minimal ssDNA character. From saturable. The data analyzed by Scatchard binding theory using a these analyses, we conclude that p53 modulates WRN helicase Hill plot were linear, indicating a single site on p53 for binding to activity in a specific manner because it does not impair the DNA unwinding reaction on a forked duplex catalyzed by a related either WRN or RPA. The apparent dissociation constants (Kd) for p53-WRN and p53-RPA were 1.81 and 2.22 nmol/L, respectively (Table 1), indicating that p53 binds to WRN with similar affinity to that observed with the p53-RPA interaction. To validate the results, we modified the ELISA procedure by coating either p53 or RPA on the microtiter wells and incubating with WRN in the presence of 3% BSA in the helicase reaction buffer. Bound WRN was detected using anti-WRN antibodies. As observed in the previous set of ELISA assays, the colorimetric signal was both dose dependent and saturable (Fig. 8B). The apparent dissociation constants for WRN-RPA and WRN-p53 were 1.40 and 1.23 nmol/L, respectively (Table 1), indicating that WRN bound to RPA with similar affinity to that observed with the WRN-p53 interaction. The colorimetric signals from the WRN-p53 (Fig. 8C), WRN-RPA (Fig. 8C), and RPA-p53 (Fig. 8D) interactions were resistant to the presence of ethidium bromide during binding, indicating that a contaminating DNA bridge was not responsible for the positive signal. In vivo Interaction of WRN with RPA and p53 in Untreated andDNADamagedCells.To explore the possibility that endogenous WRN, RPA, and p53 are associated with each other in vivo, we did coimmunoprecipitation experiments using whole cell lysates prepared from human diploid fibroblasts that were either untreated or treated with the replication inhibitor hydroxyurea or ionizing radiation that introduces DNA strand breaks. Polyclonal antibody against WRN protein was used to precipitate endogenous WRN and associated p53 or RPA. As shown in Fig. 9A, both p53 and RPA were coimmunoprecipitated with WRN in untreated cells (lane 4). The association of WRN with p53 and RPA was also evident in cells that had been exposed to 2 mmol/L hydroxyurea (lane 5) or 6 Gy ionizing radiation (lane 6). Input for the various lysates represented 10% of the total protein Figure 5. p53 retains its ability to inhibit WRN helicase activity in the used for the coimmunoprecipitation (lanes 1-3). In control presence of excess competitor ssDNA. A, reaction mixtures (20 AL) containing 42 nmol/L p53 (lanes 3-6) and the indicated concentrations of experiments, no detectable signal was obtained for WRN, RPA, ssDNA (25-mer) were incubated at 24jC for 3 minutes under standard or p53 when antibody was omitted or normal rabbit IgG was used conditions. After 3 minutes, radiolabeled 19-bp forked duplex DNA substrate (10 fmol, final concentration 0.5 nmol/L) and WRN (final concentration 3 nmol/L) in the immunoprecipitation reactions (data not shown). The were added to each reaction and incubated for an additional 7 minutes at 37jC. Helicase reaction products were resolved on native 12% polyacrylamide gels. Lane 1, no enzyme control; lane 8, heat-denatured DNA substrate control (E). B, quantitative analysis of WRN helicase data. Points, average 6 Unpublished data. of at least three independent experiments; bars, SD.

Cancer Res 2005; 65: (4). February 15, 2005 1228 www.aacrjournals.org

Figure 6. Effect of p53 on unwinding catalyzed by WRN helicase domain fragment or human RECQ1 helicase. A, glutathione S-transferase-WRN500-946 (9.4 nmol/L) or WRN (1.5 nmol/L) was incubated with the 19-bp forked duplex (0.5 nmol/L) in the presence of p53 (21 nmol/L) for 15 minutes under standard reaction conditions at 37jC as described in Materials and Methods. C, RECQ1 (10 nmol/L) or WRN (0.8 nmol/L) was incubated with the 19-bp forked duplex (0.5 nmol/L) in the presence of p53 (42 nmol/L) for 15 minutes under RECQ1 helicase reaction conditions at 37jC as described in Materials and Methods. Products were resolved on native 12% polyacrylamide gels. Heat-denatured DNA substrate control (E). B and D, quantitation of percent helicase activity from experiments as conducted in A and C, respectively. Columns, average of at least three independent experiments; bars, SD.

human RecQ helicase, RECQ1. A significant component of the The ability of p53 to deter WRN unwinding of the 5V flap and WRN helicase inhibition by p53 is likely to be mediated by a direct synthetic replication fork structures suggests that p53 may modu- protein interaction because the presence of exogenous DNA late WRN helicase activity on DNA intermediates that arise during competitor did not alleviate the inhibitory effect of p53 on DNA replication and/or repair. A role of WRN in processing 5VDNA WRN unwinding. In support of this, a WRN helicase domain flap structures is suggested by our biochemical and genetic results fragment lacking the p53 binding site was not inhibited to as great that show a specific interaction between WRN and FEN-1 an extent as the full-length WRN protein, suggesting that p53 (35, 43, 44), a structure-specific nuclease implicated in trimming binding to the DNA substrate was not the major contributor to of 5V flaps created during strand displacement synthesis in either WRN helicase inhibition by p53. In addition to these functional lagging strand replication or DNA repair processes, such as base assays, physical analyses of the WRN-p53 interaction show that excision repair (reviewed in ref. 45). The 5V flap substrate is the two proteins interact with a high affinity, and this interaction efficiently unwound by WRN (38), and modulation of this activity is not mediated by DNA. by p53 may be important to prevent the creation of long 5V flaps www.aacrjournals.org 1229 Cancer Res 2005; 65: (4). February 15, 2005

Figure 7. p53 modulates WRN helicase activity on synthetic DNA intermediates of replication and repair. WRN (3 or 6.8 nmol/L, respectively) was incubated with the 5Vflap DNA substrate (0.5 nmol/L; A) or the synthetic replication fork substrate (0.5 nmol/L; C) in the presence of p53 (42 nmol/L) for 15 minutes under standard reaction conditions at 37jC as described in Materials and Methods. Products were resolved on native 12% polyacrylamide gels. Heat-denatured DNA substrate control (E). B and D, quantitation of percent helicase activity from experiments as conducted in A and C, respectively. Columns, average of at least three independent experiments; bars, SD. E, effect of p53 on strand displacement DNA synthesis stimulated by WRN helicase activity. As described in Materials and Methods, T7 Sequenase was incubated with 26-nucleotide 5V flap substrate (10 fmol, upstream primer 5V 32P labeled) for 5 minutes at 37jC in the absence of WRN and p53 (lane 2), in the presence of 4.8 nmol/L WRN (lane 3), in the presence of 4.8 nmol/L WRN and 70 nmol/L p53 (lane 4), or in the presence of 70 nmol/L p53 only (lane 5). Products were resolved on denaturing polyacrylamide gels. Representative phosphoimage of a gel.

Cancer Res 2005; 65: (4). February 15, 2005 1230 www.aacrjournals.org

Figure 8. Detection of WRN-RPA, WRN-p53, and p53-RPA complexes by ELISA. A, WRN (.) or RPA (o) was coated onto ELISA plates. Following blocking with 3% BSA, the wells were incubated with increasing concentrations of purified recombinant p53 (0-36 nmol/L) for 1 hour at 30jC, and bound p53 was detected by a mouse monoclonal antibody against p53. B, p53 (o), or RPA (.) was coated onto ELISA plates. Following blocking with 3% BSA, the wells were incubated with increasing concentrations of purified recombinant WRN protein (0-18 nmol/L) for 1 hour at 30jC, and bound WRN was detected by ELISA using a rabbit polyclonal antibody against WRN. A and B, Points, mean of three independent experiments in duplicate; bars, SD. C and D, interaction among protein pairs of WRN, RPA, and p53 is not mediated by DNA. Either BSA (control) or purified recombinant p53 (18 nmol/L) or RPA (9 nmol/L) were coated onto ELISA plates. After a blocking step with BSA, the wells were incubated with either recombinant WRN (C) or recombinant RPA (D) either alone (white columns) or in the presence of ethidium bromide (10 Ag/mL; gray columns). Wells were aspirated and washed thrice, and bound WRN was detected using anti-WRN antibodies (C) and bound RPA was detected using anti-RPA antibodies (D), each followed by detection with secondary horseradish peroxidase (HRP)–labeled antibodies.

resistant to subsequent processing by FEN-1. By its ability to Information pertaining to the physical interaction sites among regulate WRN helicase activity, p53 would serve to insure proper WRN, RPA, and p53 should prove useful to understanding the maturation of the newly synthesized lagging strand. In this context, mechanism of p53 inhibition of WRN-catalyzed unwinding. WRN we have shown that p53 modulates the ability of WRN helicase to protein can bind to p53 via its COOH-terminal 419 residues, and facilitate strand displacement by DNA polymerase synthesis. p53 an interaction domain for WRN has been mapped to the last 100 may be involved in replication- or repair-related processes by its amino acids of p53 (full-length 393 amino acids; refs. 15, 16). ability to modulate WRN helicase activity on key DNA metabolic Both an amino region [residues 1-73 (31) and 2-117 (30)] and a intermediates. COOH region [residues 289-393 (30)] of p53 interact with the 70- The inhibition of RPA-dependent WRN helicase activity on long kDa subunit of the RPA heterotrimer. Residues 1 to 221 and 411 DNA duplex substrates is of interest because p53 did not have an to 492 of RPA70 interact with p53 (39). Thus, it is possible that effect on WRN helicase activity on shorter partial duplex p53 can bind to both WRN and RPA simultaneously; therefore, substrates characterized by the same M13 ssDNA backbone. we cannot rule out that a dual protein interaction may be The fact that p53, WRN, and RPA all bind ssDNA and each other responsible for p53 inhibition of RPA-stimulated WRN helicase makes it difficult to elucidate the mechanism of inhibition. activity. WRN binds to the 70-kDa subunit of RPA, and the WRN Nonetheless, the results presented also show that WRN can block binding motif is located within amino acids 100 to 300 and RPA-dependent unwinding of a more physiologically relevant overlaps with the ssDNA binding domain (amino acids 150-450) substrate, the telomeric forked duplex, suggesting that the of RPA70 (46). Because p53 interacts with the NH -terminal half involvement of p53 in DNA transactions catalyzed by WRN may 2 be biologically significant. (residues 1-221) of the 70-kDa RPA subunit (39), it is possible that binding of p53 to the 70-kDa subunit prevents the interaction of RPA with WRN during the unwinding reaction. Table 1. Physical analysis of WRN, RPA, and p53 protein Alternatively, it was shown previously that p53 associates with interactions RPA and prevents RPA from binding ssDNA (30). A more detailed understanding of the mechanism for RPA stimulation of WRN helicase activity will be helpful to delineate the precise Protein interaction Kd (nmol/L) (protein titrated-protein immobilized) mechanism of p53 inhibition of RPA-dependent WRN helicase activity. WRN-RPA 1.395 The inhibition of WRN helicase activity by p53 is likely to WRN-p53 1.233 have biological consequences. p53 is able to inhibit SV40 viral p53-WRN 1.808 replication (29) and nuclear DNA replication in a transcription- p53-RPA 2.219 free DNA replication extract from Xenopus eggs (47), suggesting that p53 may bind directly to proteins of the replication complex NOTE: Kd determinations were based on the average of three and interfere with DNA replication. RPA is required for independent ELISA experiments as shown in Fig. 8. replication of chromosomal DNA and displays specific functional and physical interactions with the WRN and BLM DNA helicases www.aacrjournals.org 1231 Cancer Res 2005; 65: (4). February 15, 2005

Figure 9. WRN forms protein complexes with RPA and p53 in vivo. A, WRN antibody coimmunoprecipitates WRN, RPA, and p53 from whole cell extracts of human diploid MRC-5 fibroblasts that were either untreated or exposed to 2 mmol/L hydroxyurea (HU) or ionizing radiation (IR; 6 Gy). Proteins from immunoprecipitates were resolved on SDS denaturing gels and transferred to membranes, and blots were probed with antibodies against WRN, RPA 70-kDa subunit, p53, or RPA 32-kDa subunit as described in Materials and Methods. Lanes 1-3, whole cell extracts (10% of input) from untreated, hydroxyurea-treated, or ionizing radiation–treated cells, respectively. Lanes 4-6, anti-WRN immunoprecipitates from untreated, hydroxyurea-treated, or ionizing radiation–treated cells, respectively. B, coimmunoprecipitation of WRN, RPA, and p53 is independent of DNA. Coimmunoprecipitation using anti-WRN antibody was done in the presence of 10 Ag/mL ethidium bromide (EtBr) from whole cell extracts of MRC-5 cells or the AG11395 (WS) cells. Proteins from immunoprecipitates were resolved and detected as above. Lanes 1 and 2, whole cell extract (10% of input); lanes 3 and 4, anti-WRN immunoprecipitates from AG11395 cells and MRC-5 cells.

(32, 33). WRN may function during replication as suggested by unwinds the duplex region ahead of a synthetic replication fork the extended S phase (48) and a reduced frequency of initiation (38) and p53 may serve to control this activity. Alternatively, p53 sites in Werner syndrome cells (49, 50). Genomic instability in may have a role to inhibit strand exchange or replication fork Werner syndrome cells, characterized by extensive deletions and regression that is promoted by Rad51 (53) and possibly facilitated chromosomal rearrangements, may arise due to basic defects in by WRN. a replication-associated process that involves both RPA and p53. p53 modulation of WRN catalytic function may be an important The interaction of p53 with WRN protein and/or the WRN-RPA feature of p53-mediated apoptosis. Defects in this pathway may complex may be critical to deter entry into S phase or to direct S- contribute to the prevalent cancer disposition in Werner syndrome phase cells into apoptosis. Recent studies suggest that RPA patients. Spillare et al. (15) showed that p53-mediated apoptosis in binding by p53 is less important for growth suppression than the Werner syndrome fibroblasts could be rescued by expression of transactivation and transrepression functions of p53 (51). Thus, wild-type WRN protein. It would be insightful to determine if the p53-RPA interaction may be critical for other cellular expression of the COOH-terminal domain of WRN responsible for processes, such as DNA repair or apoptosis. The attenuation of physical interaction with p53 is sufficient for rescue of the p53-mediated apoptosis in Werner syndrome cells (15) may attenuated p53-mediated apoptosis in WSÀ/À cells. Perhaps, possibly be explained by the absence of a p53-WRN direct other domains of WRN are also involved in p53-mediated interaction that could serve as a signal for programmed cell apoptosis as they are required for enhancement of p53-dependent death. p53-mediated arrest of WRN-catalyzed unwinding during transcriptional activity (16). p53 may exert its effect on WRN via its the initiation or elongation phases of DNA replication may be interaction with RPA. The genomic instability and cancer responsible for the apoptotic signal. Protein synthesis is not predisposition observed in Werner syndrome may be related to a required for p53-induced apoptosis, suggesting that p53 directly defect in p53-mediated apoptosis that is dependent on the helicase targets downstream members, such as WRN, in the apoptotic function of WRN. Precisely defining the cellular DNA metabolic pathway (52). Our findings suggest that the WRN as well as BLM pathways on which p53 regulates WRN function in vivo is the next enzymes (24) may be downstream targets of p53 in a pathway of challenge. DNA metabolism regulated by p53. p53 has been proposed to serve as a ‘‘molecular governor’’ of homologous recombination by functionally interacting with BLM and Rad51 during resolution of Acknowledgments stalled replication forks, supporting a S-phase-specific and Received 1/31/2003; revised 9/23/2004; accepted 11/5/2004. transcription-independent function of p53 (24). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance In cells arrested in S phase with hydroxyurea, WRN exits the with 18 U.S.C. Section 1734 solely to indicate this fact. nucleolus and colocalizes with p53 in the nucleoplasm (19). It has We thank Mark Sawyer, a summer student in the Laboratory of Molecular been proposed that RecQ helicases may play a role in the Gerontology, National Institute on Aging, NIH, for technical assistance in helicase assays on oligonucleotide-based substrates and Dr. Vilhelm Bohr and the members of processing of certain DNA structures at a stalled replication fork, the Laboratory of Molecular Gerontology (National Institute on Aging) and Laboratory such as the Holliday junction. p53 may function to inhibit WRN of Human Carcinogenesis (National Cancer Institute) for helpful discussions. We helicase activity at a replication fork or recombination interme- thank Drs. Alessandro Vindigni (ICGEB) and Cayetano von Kobbe (Laboratory of Molecular Gerontology, NIA, NIH) for RECQ1 baculovirus and GST-WRN500-946, diate by directly interacting with the DNA molecule. WRN respectively.

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References 21. Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, exonuclease of Werner syndrome protein. J Biol Chem Harris CC. Hepatitis B virus X protein inhibits p53 2001;276:7693–700. 1. Epstein CJ, Martin GM, Schultz AL, Motulsky AG. sequence-specific DNA binding, transcriptional activ- 38. Brosh RM, Waheed J, Sommers JA. Biochemical Werner’s syndrome a review of its symptomatology, ity, and association with transcription factor ERCC3. characterization of the DNA substrate specificity of natural history, pathologic features, genetics and Proc Natl Acad Sci U S A 1994;91:2230–4. Werner syndrome helicase. J Biol Chem 2002;277: relationship to the natural aging process. Medicine 22. Wang XW, Yeh H, Schaeffer L, et al. p53 modulation 23236–45. (Baltimore) 1966;45:177–221. of TFIIH-associated nucleotide excision repair activity. 39. Lin YL, Chen C, Keshav KF, Winchester E, Dutta A. 2. Salk D. Werner’s syndrome: a review of recent Nat Genet 1995;10:188–95. Dissection of functional domains of the human DNA research with an analysis of connective tissue 23. Garkavtsev IV, Kley N, Grigorian IA, Gudkov AV. The replication protein complex replication protein A. metabolism, growth control of cultured cells, and Bloom syndrome protein interacts and cooperates J Biol Chem 1996;271:17190–8. chromosomal aberrations. Hum Genet 1982;62:1–5. with p53 in regulation of transcription and cell growth 40. Lee S, Cavallo L, Griffith J. Human p53 binds 3. Goto M, Miller RW, Ishikawa Y, Sugano H. Excess of control. Oncogene 2001;20:8276–80. Holliday junctions strongly and facilitates their rare cancers in Werner syndrome (adult progeria). 24. Sengupta S, Linke SP, Pedeux R, et al. BLM helicase- cleavage. J Biol Chem 1997;272:7532–9. Cancer Epidemiol Biomarkers Prev 1996;5:239–46. dependent transport of p53 to sites of stalled DNA 41. Driscoll HC, Matson SW, Sayer JM, Kroth H, Jerina 4. Yu CE, Oshima J, Fu YH, et al. Positional cloning of replication forks modulates homologous recombina- DM, Brosh RM Jr. Inhibition of Werner syndrome the Werner’s syndrome gene. Science 1996;272:258–62. tion. EMBO J 2003;22:1210–22. helicase activity by benzo[c]phenanthrene diol epox- 5. Gray MD, Shen JC, Kamath-Loeb AS, et al. The 25. Braithwaite AW, Sturzbecher HW, Addison C, ide dA adducts in DNA is both strand- and Werner syndrome protein is a DNA helicase. Nat Palmer C, Rudge K, Jenkins JR. Mouse p53 inhibits stereoisomer-dependent. J Biol Chem 2003;278: Genet 1997;17:100–3. SV40 origin-dependent DNA replication. Nature 41126–35. 6. Huang S, Li B, Gray MD, Oshima J, Mian IS, Campisi J. 1987;329:458–60. 42. Harrigan JA, Opresko PL, von Kobbe C, et al. The The premature ageing syndrome protein, WRN, is a 26. Wang EH, Friedman PN, Prives C. The murine p53 Werner syndrome protein stimulates DNA polymerase 3V5V exonuclease. Nat Genet 1998;20:114–6. protein blocks replication of SV40 DNA in vitro by h strand displacement synthesis via its helicase 7. Shen JC, Gray MD, Oshima J, Kamath-Loeb AS, Fry M, inhibiting the initiation functions of SV40 large T activity. J Biol Chem 2003;278:22686–95. Loeb LA. Werner syndrome protein. I. DNA helicase antigen. Cell 1989;57:379–92. 43. Sharma S, Otterlei M, Sommers JA, et al. WRN and DNA exonuclease reside on the same polypeptide. 27. Sturzbecher HW, Brain R, Maimets T, Addison C, helicase and FEN-1 form a complex upon replication J Biol Chem 1998;273:34139–44. Rudge K, Jenkins JR. Mouse p53 blocks SV40 DNA arrest and together process branch-migrating DNA 8. Brosh RM, Bohr VA. Roles of the Werner syndrome replication in vitro and downregulates T antigen DNA structures associated with the replication fork. Mol protein in pathways required for maintenance of helicase activity. Oncogene 1988;3:405–13. Biol Cell 2004;15:734–50. genome stability. Exp Gerontol 2002;37:491–506. 28. Sakurai T, Suzuki M, Sawazaki T, Ishii S, Yoshida S. 44. Sharma S, Sommers JA, Brosh RM Jr. In vivo 9. Lane DP. Cancer. p53, guardian of the genome. Anti-oncogene product p53 binds DNA helicase. Exp function of the conserved non-catalytic domain of Nature 1992;358:15–6. Cell Res 1994;215:57–62. Werner syndrome helicase in DNA replication. Hum 10. Bennett MR. Mechanisms of p53-induced apoptosis. 29. Friedman PN, Kern SE, Vogelstein B, Prives C. Wild- Mol Genet In Press. Biochem Pharmacol 1999;58:1089–95. type, but not mutant, human p53 proteins inhibit the 45. Liu Y, Kao HI, Bambara RA. Flap endonuclease 1: 11. el-Deiry WS. p21/p53, cellular growth control and replication activities of simian virus 40 large tumor a central component of DNA metabolism. Annu Rev genomic integrity. Curr Top Microbiol Immunol antigen. Proc Natl Acad Sci U S A 1990;87:9275–9. Biochem 2004;73:589–615. 1998;227:121–37. 30. Dutta A, Ruppert JM, Aster JC, Winchester E. 46. Shen JC, Lao Y, Kamath-Loeb A, Wold MS, Loeb LA. 12. KastanMB,ZhanQ,el-DeiryWS,etal.A Inhibition of DNA replication factor RPA by p53. The N-terminal domain of the large subunit of human mammalian cell cycle checkpoint pathway utilizing Nature 1993;365:79–82. replication protein A binds to Werner syndrome p53 and GADD45 is defective in ataxia-telangiectasia. 31. Abramova NA, Russell J, Botchan M, Li R. Interac- protein and stimulates helicase activity. Mech Ageing Cell 1992;71:587–97. tion between replication protein A and p53 is Dev 2003;124:921–30. 13. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a disrupted after UV damage in a DNA repair-dependent 47. Cox LS, Hupp T, Midgley CA, Lane DP. A direct potential mediator of p53 tumor suppression. Cell manner. Proc Natl Acad Sci U S A 1997;94:7186–91. effect of activated human p53 on nuclear DNA 1993;75:817–25. 32. Brosh RM, Orren DK, Nehlin JO, et al. Functional replication. EMBO J 1995;14:2099–105. 14. Levine AJ. p53, the cellular gatekeeper for growth and physical interaction between WRN helicase and 48. Poot M, Hoehn H, Runger TM, Martin GM. and division. Cell 1997;88:323–31. human replication protein A. J Biol Chem 1999;274: Impaired S-phase transit of Werner syndrome cells 15. Spillare EA, Robles AI, Wang XW, et al. p53- 18341–50. expressed in lymphoblastoid cells. Exp Cell Res 1992; mediated apoptosis is attenuated in Werner syndrome 33. Brosh RM, Li JL, Kenny MK, et al. Replication 202:267–73. cells. Genes Dev 1999;13:1355–60. protein A physically interacts with the Bloom’s 49. Takeuchi F, Hanaoka F, Goto M, et al. Altered 16. Blander G, Kipnis J, Leal JF, Yu CE, Schellenberg GD, syndrome protein and stimulates its helicase activity. frequency of initiation sites of DNA replication in Oren M. Physical and functional interaction between J Biol Chem 2000;275:23500–8. Werner’s syndrome cells. Hum Genet 1982;60:365–8. p53 and the Werner’s syndrome protein. J Biol Chem 34. Cui S, Arosio D, Doherty KM, Brosh RM, Falaschi 50. HanaokaF,YamadaM,TakeuchiF,GotoM, 1999;274:29463–9. A, Vindigni A. Analysis of the unwinding activity of Miyamoto T, Hori T. Autoradiographic studies of 17. Yamabe Y, Shimamoto A, Goto M, Yokota J, the dimeric RECQ1 helicase in the presence of DNA replication in Werner’s syndrome cells. Adv Exp Sugawara M, Furuichi Y. Sp1-mediated transcription human replication protein A. Nucleic Acids Res Med Biol 1985;190:439–457. of the Werner helicase gene is modulated by Rb and 2004;32:2158–70. 51. Lieter LM, Chen J, Marathe T, Tanaka M, Dutta A. p53. Mol Cell Biol 1998;18:6191–200. 35. Brosh RM, von Kobbe C, Sommers JA, et al. Werner Loss of transactivation and transrepression function, 18. Lombard DB, Beard C, Johnson B, et al. Mutations syndrome protein interacts with human flap endonu- and not RPA binding, alters growth suppression by in the WRN gene in mice accelerate mortality in a p53- clease 1 and stimulates its cleavage activity. EMBO J p53. Oncogene 1996;12:2661–8. null background. Mol Cell Biol 2000;20:3286–91. 2001;20:5791–01. 52. Caelles C, Helmberg A, Karin M. p53-dependent 19. Brosh RM, Karmakar P, Sommers JA, et al. A. p53 36. Kenny MK, Schlegel U, Furneaux H, Hurwitz J. The apoptosis in the absence of transcriptional activation Modulates the exonuclease activity of Werner syn- role of human single-stranded DNA binding protein of p53-target genes. Nature 1994;370:220–3. drome protein. J Biol Chem 2001;276:35093–102. and its individual subunits in simian virus 40 DNA 53. Yoon D, Wang Y, Stapleford K, Wiesmuller L, Chen J. 20. Yang Q, Zhang R, Wang XW, et al. The processing of replication. J Biol Chem 1990;265:7693–700. p53 inhibits strand exchange and replication fork Holliday junctions by BLM and WRN helicases is 37. Opresko PL, Laine JP, Brosh RM, Seidman MM, regression promoted by human Rad51. J Mol Biol regulated by p53. J Biol Chem 2002 2002;277:31980–7. Bohr VA. Coordinate action of the helicase and 3Vto 5V 2004;336:639–54.

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MEETING OF THE RADIATION RESEARCH SOCIETY

The annual meeting of the Radiation Research Socie S. Harris. (2) On Monday night, June 22, a lecture by ty will be held at the State University of Iowa, Iowa Dr. L. W. Alvarez on meson physics has been tentative City, on June 22â€”24,1953. The Society will be the guest ly scheduled. On Tuesday night, June 23, Dr. L. H. of the University, and all meetings will be held on the Gray of the Hammersmith Hospital, London, will speak campus. The program will consist of: (1) Two symposia, on a topic to be announced. Dr. Gray's lecture is spon one on â€œTheEffects of Rwliation on Aqueous Solu sored by the Iowa Branch of the American Cancer Soci tions,â€• which includes the following speakers: E. S. G. ety. Those desiring to report original research in radia Barren, Edwin J. Hart, Warren Garrison, J. L. Magee, tion effects, or interested in attending or desiring addi and A. 0. Allen. The second is â€œPhysicalMeasurements tional information, please contact the Secretary of the for Radiobiologyâ€•and companion talks by Ugo Fano, Society, Dr. A. Edelmann, Biology Department, Brook Burton J. Moyer, G. Failla, L. D. Marinelli, and Payne haven National Laboratory, Upton, L.I., New York.

ERRATUM The following correction should be made in the arti by the glucose utilized by 16 per cent in CLL. If the as cle by Beck and Valentine, â€œTheAerobic Carbohydrate sumption is made that, in this respect, the myeloid and Metabolism of Leukocytes in Health and Leukemia. I. lymphoid celLsof leukemia are similar to those of nor Glycolysis and Respiration,â€• November, 1952, page 821; ma! blood, it may be that the computed normal figure substitute for the last paragraph: represents a summation of the myeloid (M) and The data in Table 3 permit several interesting calcu lymphoid (L) cells that make up the normal leukocyte lations. If one compares the amount of glucose actually population. Thus, if M = +0.27 and L = â€”0.16 and disappearing with the sum of the amount equivalent to the normal differential is 65 per cent M and So per cent lactic acid produced plus that equivalent to 02 con L, then sumption, it is seen that the amount of glucose â€œcleav 0.65 (+0.27) + 0.35 (â€”0.16) = +0.12 age productsâ€•exceedsthe amount of glucose utilized b a figure identical to the observed +0.12 for normal 12 per cent in N and 27 per cent in CML and is exceeded leukocytes.

308 p53 Modulates RPA-Dependent and RPA-Independent WRN Helicase Activity

Joshua A. Sommers, Sudha Sharma, Kevin M. Doherty, et al.

Cancer Res 2005;65:1223-1233.

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