A Novel Protein Interacts with the Werner's Syndrome Gene Product Physically and Functionally*

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Wernerhelicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells andsgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1disruptants, respectively. In contrast, disruption of theyWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.

Werner's syndrome (WS) 1 is a rare autosomal recessive disorder characterized by premature aging and an early onset of age-related diseases including arteriosclerosis, malignant neoplasms, melituria, and cataract (1). Somatic cells derived from WS patients show chromosome instability, a shorter life span in in vitro culture, and accelerated telomere shortening (2,3). WS cells have subtle defects in DNA replication, resulting in a reduced frequency of firing of replication origins (4). In addition, a large number of reports have shown that many cellular events including DNA repair, transcription, and apoptosis are affected in WS cells (5)(6)(7). The gene responsible for WS encodes a protein (WRN) that is a member of the RecQ family of DNA helicases (8). Most of the WS mutations that have been identified are nonsense or frameshift mutations, resulting in the truncation of WRN (9,10). The clinical features and cellular phenotypes of most WS patients seem to be due to an absolute lack of WRN in the nucleus because the nuclear localization signal of WRN resides in its C-terminal end (11).
WRN has been shown to have DNA helicase and exonuclease activity (26 -29). Recent studies (30 -32) have revealed that WRN interacts with replication protein A, PCNA, DNA topoisomerase I, and DNA polymerase ␦, indicating the involvement of WRN in some aspects of DNA replication. WRN also interacts with the p53 and Ku 70/86 heterodimer, suggesting that WRN is involved in apoptosis and the repair of DNA double strand breaks (7,(33)(34)(35). Despite these observations, it is not clear how the dysfunction of WRN is related to the observed phenotypes of WS cells. To obtain further insight into the process in which WRN is involved, we performed a two-hybrid screening using mouse WRN (mWRN) as bait and identified three interacting proteins: Ubc9, SUMO-1 (small ubiquitinrelated modifier-1), and a novel protein, WHIP (Werner Helicase Interacting Protein), which is conserved from E. coli to human (36). Here we report that mWRN physically interacts with mWHIP, and the yeast homologue of WRN, Sgs1, genetically interacts with yWHIP. mWHIP cDNA lacking the 5Ј region by two-hybrid screening. To obtain the 5Ј region of mWHIP, we performed nested PCR using a Cap-site cDNA library of mouse testis (Nippon Gene) as a template, appropriate primers, and the Advantage GC2-PCR kit (CLONTECH). Based on the 5Ј sequence of mWHIP obtained by nested PCR, full-length mWHIP cDNA was amplified by a reverse transcriptase-mediated polymerase chain reaction (RT-PCR) using the Advantage GC2-PCR kit and appropriate primers containing BglII and BamHI sites on total RNA from testes of C57BL/6 mice and cloned into the pGEM-T-Easy vector (Promega). hWHIP cDNA was cloned by RT-PCR using primers synthesized based on the hWHIP sequence as revealed by Expressed Sequence Tag and the Cap-site hunting method.
Northern Blot Analysis-The expression of hWHIP and WRN mRNA was studied using multiple tissue Northern blots (CLONTECH). The filters were hybridized with [␣-32 P]dCTP-labeled hWHIP and WRN cDNA fragments, respectively, at 42°C overnight in a 5ϫ SSPE buffer containing 50% formamide, 2% SDS, 10ϫ Denhardt's solution, and 100 g/ml depurinated salmon sperm DNA. The washing was done under highly stringent conditions: three washes with 2ϫ SSC, 0.1% SDS at room temperature and one with 0.2ϫ SSC, 0.1% SDS for 30 min at 65°C. The filters were analyzed using a BAS 1500 system (Fuji Film).
Expression of WRN and WHIP-Human 293 EBNA cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were grown to 70% confluence in 10-cm dishes, transfected with plasmid DNA using LipofectAMINE (Life Technologies, Inc.), and incubated for 48 h.
Immunoprecipitation and Western Blot Analysis-Transfected and nontransfected 293 EBNA cells were used to detect the interaction of exogenous and endogenous proteins, respectively. The cells were washed once with phosphate-buffered saline, lysed with 0.5% Triton buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM dithiothreitol) containing Complete protease inhibitor mixture (Roche Molecular Biochemicals), and left standing for 20 min on ice. The cell lysates were centrifuged, and the supernatants were incubated with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) for 1 h at 4°C and centrifuged. The resultant supernatants were incubated for 1.5 h at 4°C with anti-FLAG M2 antibody (Sigma)-coated protein A-Sepharose and WHIP antiserum-coated protein A-Sepharose to immunoprecipitate exogenous and endogenous proteins, respectively. The protein-bound beads were precipitated by centrifugation, washed three times with lysis buffer, and then suspended in SDS-sample buffer. Samples were fractionated in 7% SDS-polyacrylamide gels. The gels were transferred to polyvinylidene difluoride membranes (Millipore). For the detection of interaction between exogenous proteins the membrane was immunoblotted with anti-FLAG M2 (Sigma) and anti-HA (MBL Laboratories) antibody, followed by the secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (Dako). Bands were visualized using ECL detection reagents (Amersham Pharmacia Biotech). Interaction between exogenous proteins was detected by immunoblotting with a WHIP antiserum and hWRN monoclonal antibody (Santa Cruz Biotechnology) followed by secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (New England Biolabs), or anti-goat IgG (Dako).
In Vitro Binding Assay-The MBP-mWHIP and MBP were expressed in E. coli BL21(DE3) and purified using amylose resin (New Englands Biolabs). mWRN was synthesized in the presence of [ 35 S]methionine using the TNT SP6-coupled reticulocyte lysate system (Promega). The lysate containing [ 35 S]methionine-labeled mWRN was incubated with amylose resin bound with MBP or MBP-mWHIP in binding buffer (40 mM Tris-HCl, pH 7.6, 100 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.1 mM EDTA, 1 mM MgCl 2 , 1 mM dithiothreitol) at 4°C for 2 h. The beads were washed once with binding buffer and twice with binding buffer devoid of glycerol and Triton X-100 and then were suspended in SDS-sample buffer. Samples were fractionated in 7% SDS-polyacrylamide gel. The gel was fixed, soaked with Amplify (Amersham Phar-macia Biotech), dried, and subjected to autoradiography.
Generation of Anti-WHIP Sera-Rabbit polyclonal antisera against mouse WHIP was generated by immunizing rabbits with purified MBP-mWHIP expressed in E. coli. The antisera were confirmed to cross-react with human WHIP and to be able to immunoprecipitate both mWHIP and hWHIP (data not shown).
Immunofluorescence-The 293 EBNA cells were grown on poly-Llysine coated 8-chamber culture slides and transfected with plasmid DNA by lipofection. Cells cultured for 24 h after transfection were rinsed three times with PBS, fixed with 4% paraformaldehyde in PBS containing 2% sucrose for 10 min, and then permeabilized with Triton buffer (20 mM HEPES, pH 7.4, 0.5% Triton X-100, 50 mM NaCl, 3 mM MgCl 2 , 300 mM sucrose) for 5 min. After being rinsed three times with PBS, the cells in each well were overlaid with blocking buffer (0.1 mM citrate buffer, pH 6.0, skim milk, 0.05% NaN 3 ) for 3 h at 37°C, and the blocking buffer was removed. The cells were incubated with anti-FLAG M2 monoclonal antibody (Eastman Kodak Co.) in PBS containing 1% bovine serum albumin for 12 h at 4°C. After three washes with PBS, cells were treated for 3 h at 37°C with Texas red-conjugated goat anti-mouse IgG (Vector Laboratories, Inc.) and washed five times with PBS. Samples were mounted in Permafluor (Lipshaw-Immunon, Inc.) and analyzed with a Bio-Rad MRC-1024 confocal microscope.
Life Span Analysis-The life span of yeast strains was measured using a micromanipulator as described by Kennedy et al. (38).
Analysis of MMS Sensitivity--Stationary phase YPAD grown cells were diluted with distilled water and spotted at 10-fold dilutions (10 5 -10 2 cells) onto YPAD plates containing MMS, incubated at 30°C for 3 days, and photographed.

RESULTS AND DISCUSSION
To gain an insight into the cellular processes in which WRN is involved, we tried to identify proteins that interact with WRN by a yeast two-hybrid screening using cDNA encoding the mouse WRN as bait. We identified three proteins: a novel protein, which we designated as WHIP, and Ubc9 and SUMO-1 (36). Recently, it has been established that Ubc9 is the enzyme conjugating SUMO-1 to other proteins (39). We have recently obtained results showing that mWRN is covalently attached to SUMO-1 (36). The mWHIP consists of 660-amino acid polypeptides and shows partial homology to replication factor C family proteins, which are required for loading PCNA to the end of elongating DNA (40). A search of DNA data bases indicated that WHIP is conserved from E. coli to human (Fig. 1A), and in the N-terminal region (20 -39 aa), eukaryotic WHIPs have a conserved zinc finger motif (CX 2 CX 11 H 3 C), which is homologous to that in the post-replicational repair protein, RAD18 (Fig. 1B). Fig. 1C shows expression of WHIP and WRN mRNAs in various human tissues. Although WRN was transcribed in a relatively tissue-specific manner, WHIP was transcribed ubiquitously.
To confirm the binding of WHIP to WRN, FLAG-tagged mWRN (FLAG-mWRN) and HA-tagged mWHIP (HA-mWHIP) were expressed in human 293 EBNA cells and immunoprecipi-tated with an anti-FLAG antibody. The immunoprecipitants were analyzed by Western blotting using the anti-FLAG antibody and an anti-HA antibody, revealing co-precipitation of mWRN and mWHIP ( Fig. 2A). In addition, the direct association between mWRN and mWHIP was confirmed by a pulldown assay for in vitro translated mWRN using MBP-mWHIP (Fig. 2B). To address the interaction between endogenous WRN and WHIP in the cell, we generated anti-WHIP antisera and performed immunoprecipitation using the antisera. As shown in Fig. 2C, endogenous WRN was co-immunoprecipitated with WHIP. We next determined the region of WRN where WHIP binds by using the two-hybrid system. Deletion mutants of mWRN were transfected into yeast cells, and ␤-galactosidase activity was assayed. Positive results were obtained with constructs encoding polypeptides containing the N-terminal portion of mWRN (1-271 aa) including the exonuclease domain (78 -219 aa) but not with the construct encoding the polypeptide corresponding to the region where Ubc9 binds (272-514 aa) (36) (Fig. 2D).
In addition to WRN, there are four recQ homologues in human cells; RECQL1, BLM, RECQL4, and RECQL5. We have previously shown that mUbc9 interacts with both mWRN and mBLM but not with mRECQL1 isozymes (36). Thus, we examined whether mWHIP interacts with RecQ family proteins  c., YNL218w), and E. coli (E.c., BAA35624) WHIPs. Alignment was performed using the CLUSTALW program. The identity of amino acids is 94, 38, 32, and 35%, for human, fission yeast, budding yeast, and E. coli, respectively, compared with mouse WHIP. The zinc-finger motif, Walker A and B motifs, and Sensor 1 and 2 motifs are underlined. B, alignment of the conserved zinc-finger motifs of hWHIP and hRAD18. C, expression of WHIP and WRN mRNAs in various human tissues. Multiple tissue Northern blots containing 2 g of poly(A)ϩ RNA/lane were hybridized with cDNA fragments of human WHIP and WRN genes as described under "Experimental Procedures." The arrows show the major species of WHIP and WRN transcripts. pbl, peripheral blood lymphocyte; kb, kilobase pair(s). other than WRN. As shown in Fig. 3A, mWHIP did not interact with either isomer of mRECQL1 or mBLM. In this context, it is interesting that exogenously expressed mWHIP co-localized with exogenously expressed mWRN in granular structures in the nucleus (Fig. 3B).
Numerous studies have shown that many cellular events including telomere maintenance and DNA replication, DNA repair, transcription, and apoptosis are affected in WS cells. Recently, the focus-forming activity 1, which was found as a factor for recruiting RPA to the pre-replicative foci in a cell-free system, was identified as the Xenopus leavis homologue for WRN (41). In addition, it has been reported that WRN interacts with PCNA, DNA topoisomerase I, and DNA polymerase ␦, suggesting that WRN plays some role in DNA replication (30 -32). Thus, it is quite interesting that WHIP has motifs similar to those in replication factor C. To examine the functional relationship between WHIP and WRN, we took advantage of yeast genetics, because WHIP is conserved from yeast to human.
In budding yeast, a sole recQ homologue was identified as SGS1 (13). An original mutant allele of SGS1 was identified as a suppressor of the slow growth phenotype of top3 mutants. Deletion mutants of the SGS1 gene showed pleiotropic phenotypes including premature aging of mother cells, poor sporulation, a reduction in the fidelity of chromosome segregation during mitosis and meiosis, and a mitotic hyper-recombination phenotype (13, 20 -22, 37, 42, 43). In addition, the sgs1 mutants were shown to be hypersensitive to hydroxyurea and MMS (22)(23)(24)(25)37). Cells derived from WS patients show chromosome fected with pGAD-mWHIP or control plasmid into the Y190 yeast strain. ␤-Galactosidase activity was assayed as described previously (36). A, B, C, D, and E indicate the exonuclease domain, repeat region, acidic region, helicase domain, and nuclear localization signal, respectively. a.a., amino acid position.  (2). In Bloom's syndrome cells, the interchanges between homologous chromosomes are increased, and an abnormally large number of sister chromatid exchanges are present (44). Because similar phenotypes were observed between sgs1 disruptants and WS and Bloom's syndrome cells, sgs1 disruptants seem to be a good model for both WS and Bloom's syndrome cells.
The sgs1 disruptants showed an accelerated aging phenotype (Fig. 4) and high sensitivity to MMS (Fig. 5) as described previously (20,37). In contrast, ywhip disruptants showed a slightly accelerated aging phenotype and no apparent sensitivity to MMS. We constructed whip/sgs1 double gene disruptants. The ywhip/sgs1 showed a slow growth phenotype (Fig. 5). Deletion of the yWHIP gene intensified the accelerated aging phenotype of the sgs1 disruptants (Fig. 4). In contrast, disruption of the yWHIP gene partially alleviated the MMS sensitivity of sgs1 disruptants (Fig. 5). Although these results suggest that under normal growth conditions, yWHIP functions in a pathway not involving Sgs1, whereas under DNA damage-induced conditions, it acts upstream of Sgs1 in a pathway involving Sgs1, these issues must be clarified in a future study. In this context, the levels of WRN mRNA and WHIP mRNA in various human tissues were not necessarily correlated (Fig. 1B). Recently, we and others (45,46) found that Sgs1 plays dual functions, that is, to suppress recombination under normal growth conditions and to induce recombination under DNA damage-induced conditions. Thus, it seems likely that yWHIP functions as a modulator for Sgs1 when DNA is damaged. The yWHIP gene was identified independently as encoding a protein possessing Walker A and B motifs, which are substantially homologous to those of the E. coli RuvB protein.
The protein encoded by this gene, MGS1 (yWHIP), was shown to possess DNA-dependent ATPase and single-stranded DNA annealing activity (47). Thus, Sgs1 and yWHIP catalyze opposite reactions, the unwinding of double-stranded DNA and annealing of single-stranded DNA, respectively. This fact may help to explain the alleviation of the MMS sensitivity of sgs1 disruptants by disruption of the yWHIP gene.
In conclusion, the physical interaction in mammalian cells and the genetic interaction in budding yeast between WHIP and WRN (Sgs1) indicate a functional link between WHIP and WRN that might be conserved from yeast to human.