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

• DOI: 10.1074/jbc.c100035200
• Publication Date: 2002-07-26T15:09:24Z
• Authors: Yoh-ichi Kawabe; Dana Branzei; Tomoko Hayashi; Hirobumi Suzuki; Takashi Masuko; Fumitoshi Onoda; Seok-Jin Heo; Hideo Ikeda; Akira Shimamoto; Yasuhiro Furuichi; Masayuki Seki; Takemi Enomoto
• Keywords: 0301 basic medicine; 0303 health sciences; Base Sequence; Sequence Homology, Amino Acid; Molecular Sequence Data; 3. Good health; DNA-Binding Proteins; 03 medical and health sciences; ATPases Associated with Diverse Cellular Activities; Humans; Amino Acid Sequence; Werner Syndrome; Carrier Proteins; DNA Primers; Protein Binding

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes protein homologous to Escherichia coli RecQ. Here we describe novel Wernerhelicase interacting (WHIP), which interacts with the N-terminal portion of Werner (WRN), containing exonuclease domain. WHIP, shows homology replication factor C family proteins, conserved from E. human. Ectopically expressed WHIP and WRN co-localized in granular structures nucleus. functional relationship between was indicated genetic analysis yeast cells. Disruptants SGS1 Saccharomyces cerevisiae, homologue yeast, show an accelerated aging phenotype high sensitivity methyl methanesulfonate as compared wild-type Disruption (yWHIP) cells andsgs1 disruptants resulted slightly enhancement sgs1disruptants, respectively. In contrast, disruption theyWHIP partially alleviated sgs1 disruptants. slow growth suppressor 1 proliferating cell nuclear antigen helicase-interacting polymerase chain reaction reverse transcriptase-mediated PCR maltose-binding hemagglutinin green fluorescent mouse human calcium- magnesium-free phosphate-buffered saline amino acid extract-peptone-adenine-dextrose. (WS)1is early onset age-related diseases including arteriosclerosis, malignant neoplasms, melituria, cataract (1Epstein C.J. Martin G.M. Schultz A.L. Motulsky A.G. Medicine. 1966; 45: 177-221Crossref PubMed Scopus (745) Google Scholar). Somatic derived patients chromosome instability, shorter life span inin vitro culture, telomere shortening (2Martin Am. J. Pathol. 1977; 89: 484-511PubMed Scholar,3Schulz V.P. Zakian V.A. Ogburn C.E. McKay Jarzebowicz A.A. Edland S.D. Hum. Genet. 1996; 97: 750-754Crossref (209) have subtle defects DNA replication, resulting reduced frequency firing origins (4Fujiwara Y. Higashikawa T. 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Smithson W.A. Lindor N.M. 82-84Crossref (574) 19Kitao Ohsugi I. Ichikawa Genomics. 1998; 54: 443-452Crossref (238) also some phenotype, predisposition various neoplasms. caused hyper-recombination (20Sinclair D.A. Guarente 91: 1033-1042Abstract (1176) 21Watt P.M. Hickson I.D. Borts R.H. Louis E.J. Genetics. 144: 935-945Crossref sgs1mutants showed higher MMS hydroxyurea (22Yamagata Kato Ikeda Proc. Natl. Acad. Sci. U. 95: 8733-8738Crossref (268) 23Kusano Berres M.E. Engels W.R. 151: 1027-1039PubMed 24Onoda Seki Miyajima Mutat. 2000; 459: 203-209Crossref (78) 25Frei Gasser S.M. 81-96Crossref 37Miyajima Onoda Shiratori Odagiri Ohta Kikuchi Ohno 20: 6399-6409Crossref (44) Thus, mutants exhibit WS. has activity (26Gray J.C. Kamath-Loeb Blank Sopher B.L. Loeb L.A. 17: 100-103Crossref (519) 27Suzuki Kitao 25: 2973-2978Crossref (195) 28Huang Li Mian I.S. Campisi 114-116Crossref (377) 29Shen Fry 273: 34139-34144Abstract (221) Recent studies (30Brosh Jr., R.M. Ravn P.H. Kenny M.K. 274: 18341-18350Abstract (256) 31Lebel Spillar Leder P. 37795-37799Abstract 32Kamath-Loeb Johansson Burgers 4603-4608Crossref (157) Scholar) revealed A, PCNA, topoisomerase I, δ, indicating involvement aspects replication. p53 Ku 70/86 heterodimer, suggesting involved repair double strand breaks (7Spillare 33Blander G. Kipnis Leal J.F., Oren 29463-29469Abstract 34Cooper M.P. Ramsden 907-912PubMed 35Li Comai 275: 28349-28352Abstract (176) Despite these observations, it not clear how dysfunction related observed To obtain further insight into process involved, performed two-hybrid screening using (mWRN) bait three proteins: Ubc9, SUMO-1 (small ubiquitin-relatedmodifier-1), protein, (WernerHelicase Interacting Protein), (36Kawabe W.S. Saitoh 20963-20966Abstract (87) report mWRN physically mWHIP, genetically yWHIP. strains plasmids were described previously We cloned partialmWHIP cDNA lacking 5′ region screening. nested Cap-site library testis (Nippon Gene) template, appropriate primers, Advantage GC2-PCR kit (CLONTECH). Based on sequence ofmWHIP obtained PCR, full-length mWHIPcDNA amplified (RT-PCR) primers BglII BamHI sites total RNA testes C57BL/6 mice pGEM-T-Easy vector (Promega). hWHIP RT-PCR synthesized based Expressed Sequence Tag hunting method. plasmid pGBT9-mWRN, variously truncated cDNA, pGBT9-mouse BLM, RECQL1α RECQL1β, mammalian expression pFLAG-mWRN prepared For translation, full-lengthWRN SalI site subcloned aSalI-digested pSPUTK (Stratagene). bacterial encoding MBP-fused mWHIP (pMAL-mWHIP) vectors HA-tagged (pHA-mWHIP) GFP-tagged (pEGFP-mWHIP) constructed inserting in-frame at theBamHI pMAL-c2 (New Englands Biolabs), pHA (pCMV5-HA), pEGFP-C1 (CLONTECH), hWHIPand mRNA studied multiple tissue Northern blots filters hybridized [α-32P]dCTP-labeled WRNcDNA fragments, respectively, 42 °C overnight 5× SSPE buffer 50% formamide, 2% SDS, 10× Denhardt's solution, 100 μg/ml depurinated salmon sperm DNA. washing done under highly stringent conditions: washes 2× SSC, 0.1% SDS room temperature one 0.2× 30 min 65 °C. analyzed BAS 1500 system (Fuji Film). Human 293 EBNA cultured Dulbecco's modified Eagle's medium supplemented 10% fetal calf serum. Cells grown 70% confluence 10-cm dishes, transfected LipofectAMINE (Life Technologies, Inc.), incubated 48 h. Transfected nontransfected used detect interaction exogenous endogenous washed once saline, lysed 0.5% Triton (50 mm Tris-HCl, pH 7.6, 150 NaCl, X-100, dithiothreitol) Complete protease inhibitor mixture (Roche Molecular Biochemicals), left standing 20 ice. lysates centrifuged, supernatants A-Sepharose CL-4B (Amersham Pharmacia Biotech) h 4 centrifuged. resultant 1.5 anti-FLAG M2 antibody (Sigma)-coated antiserum-coated immunoprecipitate protein-bound beads precipitated centrifugation, times lysis buffer, then suspended SDS-sample buffer. Samples fractionated 7% SDS-polyacrylamide gels. gels transferred polyvinylidene difluoride membranes (Millipore). detection proteins membrane immunoblotted (Sigma) anti-HA (MBL Laboratories) antibody, followed secondary horseradish peroxidase-conjugated anti-mouse IgG (Dako). Bands visualized ECL reagents Biotech). Interaction detected immunoblotting antiserum hWRN monoclonal (Santa Cruz Biotechnology) anti-rabbit England anti-goat MBP-mWHIP MBP BL21(DE3) purified amylose resin Biolabs). presence [35S]methionine TNT SP6-coupled reticulocyte lysate [35S]methionine-labeled bound binding (40 mmTris-HCl, glycerol, 0.1 EDTA, MgCl2, 2 twice devoid glycerol X-100 gel. gel fixed, soaked Amplify Biotech), dried, subjected autoradiography. Rabbit polyclonal antisera against generated immunizing rabbits coli. confirmed cross-react able both (data shown). poly-l-lysine coated 8-chamber culture slides lipofection. 24 after transfection rinsed PBS, fixed 4% paraformaldehyde PBS sucrose 10 min, permeabilized (20 HEPES, 7.4, 50 3 mmMgCl2, 300 sucrose) 5 min. After being each well overlaid blocking (0.1 citrate 6.0, skim milk, 0.05% NaN3) 37 °C, removed. (Eastman Kodak Co.) 1% bovine serum albumin 12 treated Texas red-conjugated goat (Vector Laboratories, Inc.) PBS. mounted Permafluor (Lipshaw-Immunon, Bio-Rad MRC-1024 confocal microscope. Yeast this study MR966 (MATa ura3–52 leu2–3, 112 trp-289 his1–7). yWHIP (YNL218w) deletion strain 966whip his1–7 whip::KanMX4) ywhip 966ws whip::KanMX4 sgs1Δ::AUR1) PCR-based replacement oligonucleotides designed delete whole coding sequence. pFA6a-KanMX4, kindly provided Dr. Philippsen, template generate intactkanMX4 flanked sequences toyWHIP. transform MR966, thesgs1 transformants selected YPAD 200 Geneticin. follows: oligo 1, 5′-CATCGGTTGCTTTCCTTGTGAAACTGTACGCGTTGTCTTAAACCGTACGCTGCAGGTCGAC-3′; 2, 5′-GAATTGTAGACGCGGCATTGAGAGAAGTATGGAAGACCCTTGACATCGATGAATTCGAGCTCG-3′. 966 sgs1Δ created transforming pNS1–27 digested KpnI andSacI (37Miyajima Transformants 0.5 Aureobasidin All checked PCR. measured micromanipulator Kennedy et al. (38Kennedy BK Gotta Sinclair DA. Mills McNabb D.S. Murthy Pak Laroche 381-391Abstract (325) Analysis Sensitivity—-Stationary phase diluted distilled water spotted 10-fold dilutions (105-102 cells) onto plates MMS, days, photographed. gain processes tried identify interact bait. designated Ubc9 Recently, established enzyme conjugating other (39Johnson E.S. Blobel 26799-26802Abstract (408) recently results showing covalently attached consists 660-amino polypeptides partial required loading PCNA elongating (40Tsurimoto Stillman 1990; 87: 1023-1027Crossref (199) A search data bases (Fig.1 A), (20–39 aa), eukaryotic WHIPs zinc finger motif (CX2CX11H3C), post-replicational RAD18 (Fig. B). Fig. andWRN mRNAs tissues. AlthoughWRN transcribed relatively tissue-specific manner,WHIP ubiquitously. confirm FLAG-tagged (FLAG-mWRN) (HA-mWHIP) immunoprecipitated antibody. immunoprecipitants Western blotting revealing co-precipitation (Fig.2 A). direct association pull-down assay translated address cell, anti-WHIP immunoprecipitation antisera. As C, co-immunoprecipitated WHIP. next determined where binds system. Deletion cells, β-galactosidase assayed. Positive constructs (1–271 aa) domain (78–219 but construct polypeptide corresponding (272–514 D). addition there four recQhomologues cells; RECQL1, BLM,RECQL4, RECQL5. mUbc9 mBLM mRECQL1 isozymes examined whether than WRN. Fig.3 did either isomer mBLM. context, interesting exogenously (Fig.3 Numerous maintenance focus-forming found recruiting RPA pre-replicative foci cell-free system, Xenopus leavis (41Yan C.Y. Kobayashi Newport 19: 375-378Crossref (129) reported plays role quite motifs similar those examine took advantage genetics, budding sole recQ asSGS1 (13Gangloff An original mutant allele oftop3 mutants. pleiotropic mother poor sporulation, reduction fidelity segregation during mitosis meiosis, mitotic 20Sinclair 22Yamagata 42Watt 81: 253-260Abstract (376) 43Gangloff de Massy Fabre 18: 1701-1711Crossref (107) hypersensitive instability vitroin interchanges chromosomes increased, abnormally sister chromatid exchanges present (44Chaganti Schonberg 1974; 71: 4508-4512Crossref (786) Because good model 4) 5) Scholar,37Miyajima no apparent MMS. whip/sgs1 Theywhip/sgs1 5). intensified 4). Although suggest normal conditions, functions pathway involving whereas damage-induced acts upstream Sgs1 issues must clarified future study. levels tissues necessarily correlated others (45Gangloff Soustelle 192-194Crossref (312) 46Onoda 2001; 264: 702-708Crossref (52) dual functions, is, suppress recombination conditions induce conditions. seems likely modulator when damaged. independently possessing Walker B motifs, substantially RuvB protein. encoded gene, MGS1(yWHIP), possess DNA-dependent ATPase single-stranded annealing (47Hishida Iwasaki Morishita Shinagawa press, 2001Google catalyze opposite reactions, unwinding double-stranded DNA, This fact may help explain alleviation sgs1disruptants gene.Figure 5Alleviation ofsgs1 yWHIPgene. Wild-type (A) wild-type, sgs1, ywhip/sgs1 (B) their sensitivity. Stationary 0.005% °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) conclusion, physical (Sgs1) indicate link might