Physical and Functional Interaction between p53 and the Werner’s Syndrome Protein*

Werner’s syndrome is a human autosomal recessive disorder leading to premature aging. The mutations responsible for this disorder have recently been localized to a gene (WRN) encoding a protein that possesses DNA helicase and exonuclease activities. Patients carrying WRN gene mutations exhibit an elevated rate of cancer, accompanied by increased genomic instability. The latter features are also characteristic of the loss of function of p53, a tumor suppressor that is very frequently inactivated in human cancer. Moreover, changes in the activity of p53 have been implicated in the onset of cellular replicative senescence. We report here that the WRN protein can form a specific physical interaction with p53. This interaction involves the carboxyl-terminal part of WRN and the extreme carboxyl terminus of p53, a region that plays an important role in regulating the functional state of p53. A small fraction of WRN can be found in complex with endogenous p53 in nontransfected cells. Overexpression of WRN leads to augmented p53-dependent transcriptional activity and induction of p21Waf1 protein expression. These findings support the existence of a cross-talk between WRN and p53, which may be important for maintaining genomic integrity and for preventing the accumulation of aberrations that can give rise to premature senescence and cancer.

Werner's syndrome is a human autosomal recessive disorder leading to premature aging. The mutations responsible for this disorder have recently been localized to a gene (WRN) encoding a protein that possesses DNA helicase and exonuclease activities. Patients carrying WRN gene mutations exhibit an elevated rate of cancer, accompanied by increased genomic instability. The latter features are also characteristic of the loss of function of p53, a tumor suppressor that is very frequently inactivated in human cancer. Moreover, changes in the activity of p53 have been implicated in the onset of cellular replicative senescence. We report here that the WRN protein can form a specific physical interaction with p53. This interaction involves the carboxyl-terminal part of WRN and the extreme carboxyl terminus of p53, a region that plays an important role in regulating the functional state of p53. A small fraction of WRN can be found in complex with endogenous p53 in nontransfected cells. Overexpression of WRN leads to augmented p53-dependent transcriptional activity and induction of p21 Waf1 protein expression. These findings support the existence of a cross-talk between WRN and p53, which may be important for maintaining genomic integrity and for preventing the accumulation of aberrations that can give rise to premature senescence and cancer.
Mutational inactivation of the p53 tumor suppressor gene is a very common event in human cancer (1,2). Loss of wild-type (WT) 1 p53 function results in a failure to respond properly to a variety of stress signals, leading to increased genomic instability and eventually cancer (reviewed in Refs. 3 and 4). On the other hand, induction of WT p53 activity can lead to a variety of cellular outcomes, most notably cell cycle arrest and apoptosis.
Biochemically, the most prominent feature of p53 is its ability to serve as a sequence-specific transcriptional activator (for general reviews on p53, see Refs. 4 -7). This requires the specific binding of p53 to defined recognition elements within the DNA, triggering the subsequent activation of genes carrying such elements. The products of these genes mediate, to a great extent, the various biological effects of WT p53.
One of the features often associated with cancer cells is acquisition of the potential to undergo an indefinite number of cell divisions. This feature, commonly described as cellular immortalization, requires the loss of molecular mechanisms that normally mediate the induction of cellular replicative senescence (reviewed in Refs. 8 and 9). Numerous studies suggest that p53 plays an important role in the orchestration of replicative senescence and may actually be required for this process to occur effectively. The specific DNA binding activity and transcriptional potency of p53 increase markedly, with or without a concomitant increase in overall p53 protein levels, when cells approach the end of their replicative life span (10 -13). Replicative senescence can be substantially delayed by abrogation of endogenous p53 function through antisense or dominant-negative mutants (14,15). Furthermore, loss of p53, usually in combination with additional genetic alterations, is conducive to immortalization of cells in culture (16 -18). Finally, excessive WT p53 activity can impose an irreversible senescent phenotype upon cancer cells, providing an alternative mechanism for p53-mediated tumor suppression (19,20).
Unlike the recent rapid progress in understanding cellular replicative senescence, relatively little remains known about the molecular basis of aging in higher eukaryotes. A possible clue may be offered by human genetic disorders that result in premature aging. Werner's syndrome, a rare autosomal recessive disorder (21)(22)(23), is one such example. Werner's syndrome patients exhibit a wide array of features characteristic of premature aging, including arteriosclerosis, osteoporosis, hair loss, cataracts, and many more. Of particular note, they are also highly predisposed to the emergence of benign and malignant neoplasms (24). A possible link between cellular replicative senescence and human aging is suggested by the fact that whereas normal human fibroblasts approach senescence in culture after Ͼ60 population doublings, this happens in their Werner's syndrome counterparts already after as little as 20 population doublings (25).
The gene affected in Werner's syndrome, designated WRN, was recently cloned (26,27). The human WRN gene encodes a protein of 1432 amino acids with an N-terminal 3Ј-5Ј exonuclease domain (28,29) and a central domain that contains seven helicase motifs and exhibits a 3Ј-5Ј DNA helicase activity (30 -32). The fact that mutations in WRN predispose to accelerated aging implies that the WRN protein plays a critical role in preventing premature aging. This conjecture gains further support from the observation that expression of WRN decreases in normal fibroblasts undergoing senescence in culture (33).
A number of observations raise the interesting possibility that p53 and WRN may be functionally linked. As discussed above, defects in both genes strongly predispose to cancer. Moreover, like loss of p53, defects in WRN function also result in enhanced genomic instability (34,35). Finally, physical and functional interactions exist between p53 and several DNA helicases, including the TFIIH subunits XPB, XPD, and CSB (36,37). We therefore investigated the possibility of a cross-talk between the p53 and WRN proteins. We report here that p53 and WRN can form specific protein-protein interactions through their respective C-terminal domains. Overexpression of WRN results in elevated p53-dependent transcriptional activity. This suggests that some of the cellular activities of WRN may involve a cross-talk with p53, perhaps as a means for maintaining genomic stability and preventing the accumulation of irreversible genetic damage that may eventually lead to loss of replicative capacity.

EXPERIMENTAL PROCEDURES
Plasmids-Expression plasmids were constructed using pcDNA3FLAG as a backbone; pcDNA3FLAG is a derivative of pcDNA3 (Invitrogen) modified to include a FLAG epitope (38). To construct a p62 expression plasmid, HeLa cell mRNA was subjected to reverse transcription followed by PCR amplification with appropriate p62 primers. The PCR products and plasmid vector DNA were cleaved with the restriction enzymes KpnI and XbaI and ligated in frame to the FLAG epitope. pcDNA3FLAG Werner's syndrome protein (WRN) expression plasmids (WRN, WRN-M, WRN-N, and WRN-C) were similarly constructed by a reverse transcription-PCR-based approach using a basophilic leukemia cell line, KU812 (39), as the RNA source. The PCR products and the pcDNA3FLAG plasmid DNA were cleaved with KpnI and ApaI, followed by ligation in frame to the FLAG epitope. In some of the experiments, expression of full-length WRN protein was directed by plasmid pBRCMVPAWRNwt. This plasmid was constructed as follows. A DNA cassette containing the cytomegalovirus early enhancer/promoter, multiple cloning sites, and the SV40 polyadenylation signal was first integrated into the pBR322 vector. Wild-type WRN cDNA, containing the entire open reading frame, was reverse transcription-PCR-amplified from a normal lymphoblast cell line and cloned into this modified vector, giving rise to pBRCMV-PAWRNwt. Additional plasmids used in this study encode the following proteins: mouse WT p53 (40); human WT p53 (41); pRB (42); luciferase (Promega); and the mouse p53-derived miniproteins p53DD, p53Dt360, and p53D⌬SS (43). Plasmids encoding various GST-p53 fusion proteins (pGThp53, pGThp53C-(160-393), pGThp53C1-(160-318), pGThp53C2-(318-393), and pGThp53N-(1-160)) were kindly provided by Dr. T. Shenk (44). p21 Waf1 -luciferase was constructed by excising the whole mouse p21 Waf1 genomic DNA fragment from the WWP-Luc plasmid (45) and inserting it into pGL3-basic (Promega).
Cell Lines and Transfections-Human p53-null H1299 (46) nonsmall cell lung carcinoma cells were maintained in RPMI 1640 medium containing 10% fetal calf serum. H1299Val135 (Clone 3) cells were derived by stable transfection of H1299 cells with DNA encoding the temperature-sensitive mouse p53 mutant p53Val135 2 ; the temperature-sensitive p53 in these cells regains WT p53 activity when the cells are cultured at 32°C. The adenovirus type 5-transformed human epithelial kidney 293 cell line and the RKO colorectal carcinoma cell line were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were incubated at 38°C in a 5-6% CO 2 atmosphere. Transfection was by the calcium phosphate method (40).
Antibodies-PAb419 is a monoclonal antibody directed against the SV40 large T antigen (47). PAb421 (47), DO-1 (48), and PAb1801 (49) are p53-specific monoclonal antibodies; DO-1 and PAb1801 are specific for human p53, whereas PAb421 reacts with both human and mouse p53. The monoclonal antibodies PAb246 and PAb248 are specific for mouse p53. Purified murine monoclonal antibody directed against the FLAG epitope was purchased from Eastman Kodak Co. The anti-p21 polyclonal antibody C-19 was from Santa Cruz Biotechnology. The polyclonal serum Ab3 was raised against a recombinant fusion protein encompassing GST followed in frame by residues 1223-1432 of human WRN.
Protein Extraction-Cell extracts were prepared by resuspending phosphate-buffered saline-washed cell pellets in 0.5-1 ml of Nonidet P-40 extraction buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% Nonidet P-40) supplemented with 1% aprotinin and 300 g/ml phenylmethylsulfonyl fluoride. Following incubation on ice for 20 min, nonextractable material was removed by centrifugation at 17,000 ϫ g for 15 min at 4°C, and the cleared supernatants were employed for further analysis.
Immunoprecipitation-Cell extracts for immunoprecipitation were prepared as described above. In vitro translated proteins were synthesized in a TNT coupled reticulocyte lysate system (Promega). Samples containing the desired proteins were incubated in Nonidet P-40 extraction buffer together with antibody and protein A beads for 1 h at 4°C. Following centrifugation, bead pellets were washed two times with Nonidet P-40 extraction buffer or three times with buffer containing 5% sucrose, 50 mM Tris-HCl (pH 7.4), 500 mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40, followed by one wash with buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 5 mM EDTA. Bound proteins were released by resuspending the beads in 30 l of protein sample buffer, followed by vigorous vortexing, boiling for 5 min, and centrifugation at 17,000 ϫ g for 1 min. The cleared supernatants were taken for SDS-PAGE analysis. For [ 35 S]methionine-labeled proteins, polyacrylamide gels were fixed for 1 h in 10% acetic acid and fluorographed in 1 M sodium salicylate for 30 min and then dried and exposed to x-ray film.
In Vitro Binding Assays-To determine the binding of radioactive polypeptides to nonradioactive recombinant GST fusion proteins, the tested polypeptides were produced by in vitro transcription and translation in a rabbit reticulocyte lysate (TNT). The translation products were then incubated with the indicated GST fusion proteins, prebound to glutathione-agarose beads. Incubation was in 54K buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, and 2 mg/ml bovine serum albumin) for 2 h at 4°C. Bead pellets were washed as described above. Coprecipitated proteins were analyzed by SDS-PAGE followed by autoradiography as described above. To maintain an identical input of radioactivity in each binding reaction, the in vitro translation products were first analyzed by SDS-PAGE, and inputs were adjusted accordingly.
Alternatively, in vitro translated polypeptides were mixed with the products of parallel in vitro translation reactions carried out in the presence of nonradioactive amino acids only. The mixture was incubated in 54K buffer for 2 h at 4°C and then subjected to immunoprecipitation as described above, employing antibodies against the nonradioactive protein. Coprecipitated radioactive proteins were analyzed by SDS-PAGE followed by autoradiography as described above.
Luciferase Assays-H1299 cells were transfected with reporter plasmid DNA together with the indicated expression plasmid combinations. 26 h later, cells were rinsed with cold phosphate-buffered saline, resuspended in cell lysis buffer (Promega), and incubated for 10 min at room temperature. Insoluble material was spun down, and luciferase activity in the cleared supernatant was quantitated in the presence of luciferin (Promega) and ATP using a Turner Design Model 20 luminometer.

RESULTS
The WRN Protein Binds p53 in Vitro-The p53 protein can engage in a multitude of protein-protein interactions, some of which are known to modulate its activity in a variety of ways (reviewed in Refs. 4 -6 and 50). To explore the possibility of a cross-talk between p53 and the WRN protein, we tested whether these two proteins can interact directly. Full-length WRN protein was translated in vitro in the presence of [ 35 S]methionine and incubated with recombinant GST-p53, consisting of a fusion between GST and human WT p53. As shown in Fig.  1, WRN bound much more efficiently to GST-p53 (lane 1) than to GST alone (lane 2). The preferential binding of WRN to p53 was even more prominent than that of p62 (lanes 5 and 6), a TFIIH subunit that binds p53 in vitro (51,52). Luciferase, serving as a negative control, bound to neither GST nor GST-p53 (lanes 3 and 4). Thus, WRN and p53 can engage in specific protein-protein interactions in vitro.
To map the p53-binding domain(s) within WRN, human WT p53 was produced by in vitro translation in the presence of nonradioactive amino acids. This p53 was then incubated with an array of 35 S-labeled, in vitro translated polypeptides, including the pRB tumor suppressor protein, the p62 TFIIH subunit, luciferase, and various portions of the WRN open reading frame as indicated in Fig. 2A. Radioactive polypeptides associated with the unlabeled p53 protein were co-immunoprecipi-tated with a mixture of the p53-specific monoclonal antibodies DO-1 and PAb421 and resolved by SDS-PAGE. As expected, p62 was efficiently coprecipitated with p53 (Fig. 2B, lane 2 with a series of recombinant GST fusion proteins containing different segments of human WT p53 (Fig. 3A). Although the N-terminal part of p53 (residues 1-160) did not interact detectably with WRN (Fig. 3B, lane 4), prominent binding was seen with a fragment encompassing the C-terminal part of p53 (residues 160 -393; lane 1). Binding was further mapped to residues 318 -393 (lane 3), whereas residues 160 -318 were negative (lane 2). Hence, the WRN interaction domain is fully contained within the last 76 residues of p53. This region encompasses several important functional elements (Fig. 3A), including the nuclear localization sequence (NLS), the oligomerization/tetramerization domain (OD), and a negative regulatory domain (NR) that down-regulates sequence-specific DNA binding by p53 (53). To further narrow down the WRN interaction domain of p53, the WRN-C segment ( Fig. 2A), which binds p53 (Fig. 2B), was cloned in frame downstream of a FLAG epitope and translated in vitro in the presence of nonradioactive amino acids. Several miniproteins, derived from mouse WT p53 (43), were each translated in vitro in the presence of [ 35 S]methionine and tested for association with nonradioactive FLAG-WRN-C. In agreement with Fig. 3B, the p53DD miniprotein comprising the last 89 residues of mouse p53 (Fig. 3C) bound efficiently to WRN-C (Fig. 3D, lane 1). However, binding was completely abolished by removal of the last 30 residues of p53 (p53Dt360; lane 2). Hence, the interaction between p53 and WRN requires the extreme C-terminal part of p53. Interestingly, a derivative of p53DD carrying an internal deletion within the oligomerization domain (p53D⌬SS; Fig. 3C) was markedly impaired in WRN-C binding (Fig. 3D,  lane 3). The region deleted in p53D⌬SS may be part of the actual WRN interaction domain; alternatively, WRN may associate preferentially with p53 oligomers rather than monomers. Of note, preferential binding to p53 oligomers has been documented for Mdm2, a cellular protein acting as a negative regulator of p53 (54). In conclusion, WRN and p53 can undergo a specific interaction in vitro, and this interaction requires the C-terminal portions of both proteins.
In Vivo Association between WRN and p53-We next wished to determine whether p53 and WRN interact also in vivo. To this end, human 293 cells were transiently transfected with mouse WT p53 either alone or in combination with a WRN expression plasmid. Cell extracts were subjected to immunoprecipitation with mouse p53-specific monoclonal antibodies, followed by immunoblotting with anti-WRN serum. As shown in Fig. 4A (upper panel), the WRN protein was brought down from extracts of (p53 ϩ WRN)-cotransfected cells with p53specific antibodies (lane 4), but not with control antibodies (lane 5). Lanes 1 and 2 show aliquots of the corresponding total cell extracts, applied directly to the gel; the faint band in lane 1 represents the endogenous WRN protein of the 293 cells.
The existence of complexes between p53 and endogenous WRN was explored through the use of human p53-null H1299 cells and their derivative cells, H1299Val135, stably expressing a temperature-sensitive mouse p53 mutant. This mutant, p53Val135, encodes a protein that is largely inactive at 37°C, but that regains WT p53 activity at 32°C (55). As shown in Fig.  4B (upper panel), WRN was coprecipitated with p53 from extracts of H1299Val135 cells (lane 4), whereas no WRN was brought down from such extracts by an irrelevant control antibody (lane 5). The p53-specific antibodies did not bring down a significant amount of WRN from extracts of parental p53-null H1299 cells (lane 3), despite the fact that they contained somewhat higher total amounts of WRN than their H1299Val135 derivatives (compare lanes 1 and 2); the latter observation is in line with the report that the WRN gene promoter is repressed by excess WT p53 (56).  1. In vitro association between p53 and WRN. Glutathioneagarose beads loaded with GST-p53 (lanes 1, 3, and 5) or GST only (lanes 2, 4, and 6) were incubated with in vitro translated WRN protein (lanes 1 and 2), luciferase (Luc; lanes 3 and 4), or the p62 subunit of TFIIH (lanes 5 and 6). Bound proteins were eluted, resolved by SDS-PAGE, and visualized by autoradiography. See "Experimental Procedures" for details.

p53-Werner's Syndrome Protein Interactions
WRN Overexpression Elevates the Transcriptional Activity of p53-The most notable biochemical property of p53 is the sequence-specific transcriptional activation of target genes. To find out whether WRN can influence p53 function, human p53-null H1299 cells were transiently transfected with various combinations of expression plasmids together with a luciferase reporter gene driven by the p53-responsive p21 Waf1 promoter, a major target for sequence-specific transcriptional activation by p53 (45). We deliberately employed a limiting amount of p53 expression plasmid, which elicited only a very modest increase in luciferase activity (Fig. 5, bars 1 and 6). Whereas WRN alone had only a very mild effect on basal transcription from the p21 Waf1 promoter (bar 2), its cotransfection with p53 led to a significantly higher activity relative to p53 alone (bar 7). A similar picture was revealed with several other p53-responsive promoters, including those of the bax, mdm2, and PIG3 genes, as well as a synthetic promoter containing 17 tandem repeats of a p53-binding motif from the ribosomal gene cluster (data not shown). These observations suggest that WRN overexpression elevates the overall transcriptional activity of p53.
The ability of various WRN segments to modulate p53-dependent transcription was also investigated. Unlike the fulllength protein, none of the three WRN segments tested could enhance the activation of the p21 Waf1 promoter by p53 (Fig. 5,  compare bars 8 -10 and bar 6). This was true also for WRN-C, which binds WRN very efficiently in vitro (see Fig. 2B). Hence, although binding to p53 may be necessary, additional function(s) of WRN also appear to be required for its ability to potentiate the transcriptional activity of p53.
To determine whether WRN can also enhance the activation of endogenous target genes by p53, we monitored the levels of p21 Waf1 protein in cells overexpressing WRN. As shown in Fig.  6, transfection of WRN caused a substantial accumulation of p21 Waf1 protein in H1299 cells cotransfected with p53 (compare lanes 1 and 3), whereas neither WRN-N nor WRN-C had any measurable effect on p21 Waf1 (data not shown). Thus, WRN can interact with p53 not only physically, but also functionally. DISCUSSION The data presented in this study demonstrate that p53 and WRN can engage in a direct physical association, mediated through the C-terminal portion of WRN and the extreme Cterminal domain of p53. The fact that only a relatively small fraction of WRN was coprecipitated with p53 from cell extracts might imply that only a particular subpopulation of WRN can bind p53 (e.g. as a result of particular post-translational modifications). Alternatively, the interaction in vivo may be transient or may occur effectively only under very special conditions such as particular stress signals.
High WRN levels elicit increased p53-mediated transcription. This may be due to the ability of WRN to bind the extreme C terminus of p53, a negative regulatory domain (53). In fact, a variety of macromolecules that bind to this domain relieve its inhibitory effect and activate p53 for DNA binding (53,(57)(58)(59). However, the failure of WRN-C to potentiate the transcriptional activity of p53 (Fig. 5) despite binding p53 very efficiently in vitro (Fig. 2) argues that the mere binding to p53 is not sufficient. Rather, an additional function of WRN, residing outside WRN-C, may also contribute to the enhanced p53 response. An attractive hypothesis is that p53 may recruit WRN to particular p53 target genes, where the helicase activity of WRN may facilitate transcription by opening up the DNA template.
Our findings are reminiscent of the interactions between p53 and the TFIIH subunits XPB and XPD. Like WRN, both are DNA helicases, and both bind p53 very efficiently in vitro. Moreover, both XPB and XPD bind the C terminus of p53 (36), very much like WRN. Of particular note, XPB and XPD may be required for effective induction of p53-mediated apoptosis (37).
What is the physiological relevance of the association be- tween p53 and WRN? Two apparently opposing scenarios come to mind. On the one hand, loss of function of either p53 or WRN results in genomic instability and increased predisposition to cancer. This would suggest that the two proteins may possess similar roles and perhaps even act synergistically to prevent accumulation of genomic damage. On the other hand, p53 and WRN seem to act very differently when it comes to senescence. Thus, loss of p53 function delays the onset of senescence and facilitates cellular immortalization, whereas loss of proper WRN function, as in cells of Werner's syndrome patients, accelerates senescence in culture and promotes premature aging in vivo.
The data presented here are more consistent with the first scenario, namely that p53 and WRN serve a common interest under conditions of imminent genomic instability. The analogy with XPD and XPD, molecules involved in DNA repair and in the response to DNA damage, also supports such a working hypothesis. In the case of XPB and XPD, which are components of a general transcription factor (TFIIH) (60), this association may recruit p53 to sites of DNA damage within transcribed genes. This could perhaps serve to halt the transcription of the damaged genes or to facilitate DNA repair through interactions of p53 with other proteins such as RPA and Rad51. Moreover, it might "alert" p53 to the presence of DNA damage, perhaps through covalent modifications of p53 that occur preferentially at arrested transcription complexes. The picture is less obvious in the case of WRN, mainly because the exact roles of this helicase remain to be established. An appealing clue is provided by the finding that WRN is highly homologous to FFA-1, a protein associated with the origin recognition complex described in Xenopus laevis (61). FFA-1 has been proposed to be the helicase that unwinds the DNA at the origin of replication, allowing initiation of DNA replication (61). WRN might play a similar role in the unwinding of mammalian DNA replication origins. Such a notion is consistent with the defective S phase transit in Werner's syndrome patients' cells (62) and with the fact that the wild-type, but not the mutant, WRN protein is physically associated with a multiprotein DNA replication complex (63). It is tempting to speculate that the interaction of p53 with WRN may recruit p53 to such replication origins in response to DNA damage and somehow help prevent initiation at  1 and 3). Cell extracts were prepared 24 h later. Aliquots containing 3 mg of total protein were subjected to immunoprecipitation (IP) with a combination of the mouse p53-specific monoclonal antibodies PAb246 and PAb248 (p53; lanes 3 and 4) or an equal amount of the control monoclonal antibody PAb419, directed against the SV40 large T antigen (C; lane 5). Immunoprecipitated proteins were resolved by SDS-PAGE, followed by immunoblotting (IB) with anti-WRN polyclonal serum Ab3. Lanes 1 and 2 contain aliquots of unprocessed extracts from cells transfected with p53 plus pcFLAG or p53 plus WRN, respectively (100 g/lane), applied directly to the gel. Lanes 1 and 2 were reprobed with PAb248 to visualize the transfected p53 in the unprocessed cell extracts (lower panel). B, p53null H1299 cells (upper panel, lanes 1 and 3) and H1299Val135 cells, carrying a temperature-sensitive mouse p53 mutant (lanes 2, 4, and 5), were maintained at 32°C for 16 h and then extracted and subjected to immunoprecipitation analysis exactly as described for A. Lanes 3-5 represent immunoprecipitates corresponding to 3 mg of total cell protein. Lanes 1 and 2 contain aliquots of unprocessed extracts from the indicated cell lines (100 g/lane), applied directly to the gel. Lanes 1 and 2 were reprobed with PAb248 to visualize p53 in the unprocessed cell extracts (lower panel). such origins. p53 might thereby ensure that only intact DNA is replicated. Such a mechanism would operate in addition to the well studied ability of p53 to utilize its checkpoint function to prevent the entry of cells with damaged DNA into S phase (4). Thus, the increased genomic instability in the cells of Werner's syndrome patients may be due, at least in part, to a failure of the defective WRN to mobilize p53 in an attempt to prevent the propagation of damaged DNA. The fact that the p53-binding domain of WRN resides near its C terminus, a region eliminated by practically all the mutations found in Werner's syndrome patients, would appear consistent with this possibility.
This notion is reminiscent of recent findings relating to the Bloom's syndrome gene (BLM). Like WRN, the BLM protein is also a member of the RecQ family of DNA helicases and has been implicated in DNA replication (64). Interestingly, fibroblasts derived from Bloom's syndrome patients exhibit a delayed induction of p53 in response to UV exposure (65).
While this paper was under revision, Harris and co-workers (66) also reported that p53 and WRN interact in vitro and in vivo. Furthermore, this interaction appears to be important for p53-induced apoptosis (66); our functional analysis provides a possible biochemical explanation for this finding.
Recently, it has been reported that transcription of the WRN gene is negatively regulated by p53 in Saos-2 cells (56). This may delineate a negative feedback loop, where a transient increase in WRN protein augments the cellular activity of p53, resulting, in turn, in the down-regulation of WRN expression. It could also explain the decreased WRN expression in senescent fibroblasts (33), where the transcriptional activity of p53 is greatly enhanced (11,12).
As discussed earlier, there exists also an alternative scenario in which p53 and WRN play opposing roles in the regulation of senescence. Although our data would appear inconsistent with such a scenario, studies with Sgs1p, a WRN-related protein from the yeast Saccharomyces cerevisiae (67), suggest that the phenotypic effects of Sgs1p overexpression may be very similar to those of an Sgs1p null mutant. It thus remains possible that vast WRN overexpression also somehow mimics the biological consequences of WRN inactivation. One should therefore not rule out the possibility that the aim of the p53-WRN interaction is actually to silence p53 rather than to activate it. In this manner, WRN might bypass the prosenescent effect of p53, whereas its inactivation will allow p53 to become fully active and to drive the cells into senescence.
Finally, although this study addresses only the effects of WRN on p53, it is conceivable that the molecular interaction described here is mainly designed to modulate WRN function, rather than p53 function. Alternatively, the p53-WRN complex might serve as a signaling molecule whose roles are distinct from those of each component alone. This latter possibility is compatible with the observation that only a small fraction of each protein appears to be in the complex (Fig. 4). Future experiments should resolve this interesting question.