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• W2078383934 abstract "Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using site-directed mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures. Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using site-directed mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures. Werner syndrome (WS) 2The abbreviations used are:WSWerner syndromeRQCRecQ conservedwHTHwinged helix-turn-helixHlchelicaseaaamino acidHRDChelicase RNaseD C-terminaldsDNAdouble strand DNAssDNAsingle strand DNAWRNWernerntnucleotideGSTglutathione S-transferaseEMSAelectrophoretic mobility shift assayBSAbovine serum albuminPBSphosphate-buffered salineHSQCheteronuclear single quantum coherenceDAPI4,6-diamidino-2-phenylindoleGFPgreen fluorescent protein is an autosomal recessive disorder characterized by premature aging after puberty with multiple symptoms, including graying and loss of hair, bilateral cataracts, atherosclerosis, diabetes mellitus, osteoporosis, hypogonadism, and cancer predisposition (1Goto M. Clin. Exp. Rheumatol. 2000; 18: 760-766PubMed Google Scholar). In addition, WS cells exhibit genomic instability (2Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (212) Google Scholar), replication defects (3Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell Res. 1992; 202: 267-273Crossref PubMed Scopus (187) Google Scholar), aberrant telomere maintenance (4Crabbe L. Verdun R.E. Haggblom C.I. Karlseder J. Science. 2004; 306: 1951-1953Crossref PubMed Scopus (498) Google Scholar), and a gene expression profile that resembles that of normal human aging (5Kyng K.J. May A. Kolvraa S. Bohr V.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12259-12264Crossref PubMed Scopus (133) Google Scholar). The WRN gene (also known as RECQ3) located at chromosome 8p11-12, encodes a nuclear ∼162-kDa protein (WRN), which exhibits a DNA-dependent ATPase, DNA helicase activity on double strand DNA (dsDNA) with 3′ → 5′-polarity (6Gray M.D. Shen J.C. Kamath-Loeb A.S. Blank A. Sopher B.L. Martin G.M. Oshima J. Loeb L.A. Nat. Genet. 1997; 17: 100-103Crossref PubMed Scopus (523) Google Scholar), and a functional N-terminal 3′ → 5′-exonuclease activity (7Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar). Werner syndrome RecQ conserved winged helix-turn-helix helicase amino acid helicase RNaseD C-terminal double strand DNA single strand DNA Werner nucleotide glutathione S-transferase electrophoretic mobility shift assay bovine serum albumin phosphate-buffered saline heteronuclear single quantum coherence 4,6-diamidino-2-phenylindole green fluorescent protein WRN belongs to a family of RecQ helicases that also includes RECQ1 (8Puranam K.L. Blackshear P.J. J. Biol. Chem. 1994; 269: 29838-29845Abstract Full Text PDF PubMed Google Scholar, 9Seki M. Miyazawa H. Tada S. Yanagisawa J. Yamaoka T. Hoshino S. Ozawa K. Eki T. Nogami M. Okumura K. Nucleic Acids Res. 1994; 22: 4566-4573Crossref PubMed Scopus (144) Google Scholar), RECQ2 (10Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1221) Google Scholar), RECQ4 (11Kitao S. Ohsugi I. Ichikawa K. Goto M. Furuichi Y. Shimamoto A. Genomics. 1998; 54: 443-452Crossref PubMed Scopus (238) Google Scholar), and RECQ5 (11Kitao S. Ohsugi I. Ichikawa K. Goto M. Furuichi Y. Shimamoto A. Genomics. 1998; 54: 443-452Crossref PubMed Scopus (238) Google Scholar). Functions of RecQ helicases based on cellular and biochemical evidence suggest their involvement in DNA replication, DNA repair, recombination, and telomere maintenance. Clinically, defects in certain RECQ genes have been linked to rare genetic disorders, including Bloom syndrome (RECQ2), Werner syndrome (RECQ3), and Rothmund-Thompson syndrome (RECQ4). One of the prominent characteristics common to all RecQ helicases is the sequence conservation of the seven helicase motifs centrally located in the respective proteins (12Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. Nucleic Acids Res. 1989; 17: 4713-4730Crossref PubMed Scopus (830) Google Scholar). In addition to the helicase domain, a majority of RecQ helicases shares another conserved sequence designated the RecQC-terminal (RQC) region (13Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (160) Google Scholar). The WRN RQC region is located between amino acid (aa) residues 872 and 1045 and is homologous to the part of the catalytic core of Escherichia coli RecQ for which the three-dimensional structure has revealed the presence of a winged helix-turn-helix (wHTH) motif (14Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (217) Google Scholar). Moreover, of the 17 WRN-interacting proteins, 14 that are functionally significant require aa 949-1092 or at least part of this region for the interaction of WRN with them (15Lee J.W. Harrigan J. Opresko P.L. Bohr V.A. Mech. Ageing Dev. 2005; 126: 79-86Crossref PubMed Scopus (82) Google Scholar). The wHTH motif in mammalian proteins has been demonstrated to be important for protein-DNA interactions as well as protein-protein interactions (16Mer G. Bochkarev A. Gupta R. Bochkareva E. Frappier L. Ingles C.J. Edwards A.M. Chazin W.J. Cell. 2000; 103: 449-456Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 17Fogh R.H. Ottleben G. Ruterjans H. Schnarr M. Boelens R. Kaptein R. EMBO J. 1994; 13: 3936-3944Crossref PubMed Scopus (86) Google Scholar, 18Clark K.L. Halay E.D. Lai E. Burley S.K. Nature. 1993; 364: 412-420Crossref PubMed Scopus (1098) Google Scholar, 19Gajiwala K.S. Chen H. Cornille F. Roques B.P. Reith W. Mach B. Burley S.K. Nature. 2000; 403: 916-921Crossref PubMed Scopus (274) Google Scholar, 20Littlefield O. Nelson H.C. Nat. Struct. Biol. 1999; 6: 464-470Crossref PubMed Scopus (140) Google Scholar). The wHTH motif mediates DNA-protein interaction through the “DNA recognition helix” of the helix core consisting of three α-helices. In general, the recognition helix interacts with the major groove of DNA as shown in the DNA-protein co-crystal structures of a variety of proteins involved in DNA metabolism (17Fogh R.H. Ottleben G. Ruterjans H. Schnarr M. Boelens R. Kaptein R. EMBO J. 1994; 13: 3936-3944Crossref PubMed Scopus (86) Google Scholar, 18Clark K.L. Halay E.D. Lai E. Burley S.K. Nature. 1993; 364: 412-420Crossref PubMed Scopus (1098) Google Scholar, 19Gajiwala K.S. Chen H. Cornille F. Roques B.P. Reith W. Mach B. Burley S.K. Nature. 2000; 403: 916-921Crossref PubMed Scopus (274) Google Scholar). Therefore, the presence of the wHTH motif in the catalytic core of E. coli RecQ and the possibility that the wHTH motif may also exist in the WRN RQC domain offer some explanation as to why the WRN RQC fragment (aa 949-1092) is the strongest DNA binding region followed by the helicase RNaseD C-terminal (HRDC) domain and the exonuclease domain (21von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The versatility of WRN to bind and act upon various DNA substrates in vitro such as dsDNA flanked by a 3′ ssDNA tail (6Gray M.D. Shen J.C. Kamath-Loeb A.S. Blank A. Sopher B.L. Martin G.M. Oshima J. Loeb L.A. Nat. Genet. 1997; 17: 100-103Crossref PubMed Scopus (523) Google Scholar, 22Suzuki N. Shimamoto A. Imamura O. Kuromitsu J. Kitao S. Goto M. Furuichi Y. Nucleic Acids Res. 1997; 25: 2973-2978Crossref PubMed Scopus (195) Google Scholar), synthetic replication fork (23Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), Holliday junction (21von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 24Constantinou A. Tarsounas M. Karow J.K. Brosh R.M. Bohr V.A. Hickson I.D. West S.C. EMBO Rep. 2000; 1: 80-84Crossref PubMed Scopus (337) Google Scholar), bubble (25Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (491) Google Scholar), triple helix (26Brosh Jr., R.M. Majumdar A. Desai S. Hickson I.D. Bohr V.A. Seidman M.M. J. Biol. Chem. 2001; 276: 3024-3030Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), G4 tetraplex (27Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), and telomeric D-loop (28Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kolvraa S. May A. Seidman M.M. Bohr V.A. Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) suggest that WRN may harbor a structure-specific DNA-binding motif, such as wHTH in the RQC domain, in addition to auxiliary DNA-interacting surfaces such as HRDC and exonuclease domains. Therefore, in order to understand the importance of the RQC domain in the DNA binding mechanism of WRN, site-directed mutagenesis was used to introduce missense mutations in the RQC domain of WRN to identify the critical aa mediating the WRN-DNA interaction. After identifying the critical aa that modulates the DNA binding activity of WRN, we demonstrated how this mutation affects the WRN helicase activity and the FEN-1 incision activity. Furthermore, we proved that the modulations of the WRN-coordinated functions were not a direct consequence of the altered three-dimensional structure of WRN via solution nuclear magnetic resonance. Mutagenesis of WRN—Site-directed mutagenesis was performed in order to change the wild-type aa residues, Lys-1008, Gln-1010, Lys-1016, and Arg-1020, to alanine in the region of WRN RQC using the QuikChange II site-directed mutagenesis kit (Stratagene). The sequences of primers used for site-directed mutagenesis are shown in TABLE ONE. PCR was performed using pGEX-KG-WRN-(949-1092) as a template, and PCR products were sequenced as described previously (21von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar).TABLE ONESequences of primers used for site-directed mutagenesisK1008A mutantForward5′-GT TTA TTT GGC ACT GGC GCG GAT CAA ACA GAG AG-3′Reverse5′-CA AAT AAA CCG TGA CCG CGC CTA GTT TGT CTC TC-3′Q1010A mutantForward5′-GC ACT GGC AAG GAT GCA ACA GAG AGT TGG TGG-3′Reverse5′-CG TGA CCG TTC CTA CGT TGT CTC TCA ACC ACC-3′K1016A mutantForward5′-GAG AGT TGG TGG GCG GCT TTT TCC CGT C-3′Reverse5′-CTC TCA ACC ACC CGC CGA AAA AGG GCA G-3′ Open table in a new tab Recombinant Proteins—All of E. coli WRN wild-type or mutant fragments were cloned into Gateway pDEST 15 (Invitrogen) according to the manufacturer's guidelines or pGEX-KG vectors, and the recombinant glutathione S-transferase (GST)-tagged proteins were purified as described previously (21von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). His-tagged recombinant flap endonuclease-1 (FEN-1) was prepared as described previously (29Sharma S. Sommers J.A. Wu L. Bohr V.A. Hickson I.D. Brosh Jr., R.M. J. Biol. Chem. 2004; 279: 9847-9856Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Protein concentrations were determined by using the following methods: Bradford assay (Bio-Rad), Silver Xpress staining (Invitrogen), Amido Black staining, and GelCode Blue staining (Pierce). Western blot analysis was also used to confirm the relative concentration of each protein using mouse anti-GST antibody and rabbit anti-mouse horseradish peroxidase-conjugated antibody. DNA Substrate Preparation—The fork substrates that have 19- and 34-bp duplex DNA with 3′ and 5′ single-stranded overhangs were prepared as described previously (23Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 30Opresko P.L. Laine J.P. Brosh Jr., R.M. Seidman M.M. Bohr V.A. J. Biol. Chem. 2001; 276: 44677-44687Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The bubble substrates containing a 12-nt bubble and the Holliday junction containing a 12-bp homologous core were prepared as described previously (25Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (491) Google Scholar). The 1-nt 5′ flap substrate was prepared using Tstem25 and FLAP26, and D-loop substrates were prepared as described previously (31Tom S. Henricksen L.A. Bambara R.A. J. Biol. Chem. 2000; 275: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 32van Brabant A.J. Ye T. Sanz M. German Spaceiiiqq J.L. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Crossref PubMed Scopus (200) Google Scholar, 33Brosh Jr., R.M. von Kobbe C. Sommers J.A. Karmakar P. Opresko P.L. Piotrowski J. Dianova I. Dianov G.L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar). Electrophoretic Mobility Shift Assay—EMSAs were performed as described previously (21von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) with modifications. 20-μl reactions contained 30 fmol of DNA substrates in a buffer containing 40 mm Tris (pH 7.0), 1 mm EDTA, 20 mm NaCl, 20 μg/ml bovine serum albumin (BSA), and 8% glycerol. As indicated in the figure legends, 5 ng of poly(dI-dC) was preincubated with WRN fragment on ice. The reaction mixtures were incubated on ice for 30 min on ice and electrophoresed in 5% native polyacrylamide gel at 4 °C. The electrophoresis was carried out in 1× TAE (40 mm Tris acetate, 1 mm EDTA (pH 8.0)) at constant voltage of 12.8 V/cm for 2 h, and the resulting band-shifts were visualized using PhosphorImager and ImageQuant software (Amersham Biosciences). Enzyme-linked Immunosorbent Assay—Purified recombinant FEN-1 protein was diluted to 1 ng/μl in carbonate buffer (0.016 m Na2CO3, 0.034 m NaHCO3 (pH 9.6)) and added to the appropriate wells of a 96-well microtiter plate (50 μl/well). The plate was incubated at 4 °C overnight. The wells were washed with 1× phosphate-buffered saline (PBS). For blocking, 200 μl of blocking buffer (1× PBS, 0.5% Tween 20, and 3% BSA) was added to each well and incubated for 1 h at 37 °C. After washing twice with 300 μl of 1 × PBS, 50 μl of either purified recombinant GST RQC or K1016A mutant fragments was applied to each well for binding to FEN-1 at various concentrations, and samples were incubated at 37 °C for 1 h. Purified recombinant GST protein was used as a control for RQC or K1016A mutant. BSA was also used as a control with RQC, K1016A mutant, or BSA alone to measure nonspecific binding. For ethidium bromide (EtBr) treatment, 50 μg/ml EtBr was included in the incubation with FEN-1 and RQC or K1016A mutant during the incubation for binding. After the incubation, the wells were washed five times with 1× PBS. Anti-GST mouse antibody (Santa Cruz Biotechnology) was added at 1:500 in blocking buffer, and the samples were incubated at 37 °C for 1 h. The wells were washed five times and incubated with anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000) in blocking buffer for 1 h at 37°C. After washing five times, 50 μl of developing buffer (0.05 m phosphate/citrate buffer (pH 5.0), 0.06% H2O2) containing o-phenylenediamine dihydrochloride (1 tablet/10 ml of developing buffer; Sigma) was added. The reaction was terminated after 5 min by adding 25 μl of 3 m H2SO4, and the absorbance was measured at 490 nm. The fraction of RQC or K1016A mutant specifically bound to FEN-1 protein was determined from the enzyme-linked immunosorbent assay, and the Hill plot was used for the calculation of Kd values as described previously (34Brosh Jr., R.M. Li J.L. Kenny M.K. Karow J.K. Cooper M.P. Kureekattil R.P. Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Flap Endonuclease-1 Incision Assay—FEN-1 incision assay was performed as described previously (29Sharma S. Sommers J.A. Wu L. Bohr V.A. Hickson I.D. Brosh Jr., R.M. J. Biol. Chem. 2004; 279: 9847-9856Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) with modifications. 20-μl reactions contained 10 fmol of DNA substrate, 8 fmol of FEN-1, and the indicated amounts of RQC or the Lys-1016 mutant in a buffer containing 150 mm HEPES (pH 7.5), 200 mm KCl, 40 mm MgCl2, 500 μg/ml BSA, and 25% glycerol. The RQC or the Lys-1016 mutant was mixed with the substrate and buffer on ice prior to the addition of FEN-1. Reactions were incubated at 37 °C for 15 min and terminated with 10 μl of stop solution (80% formamide (v/v), 0.1% bromphenol blue, and 0.1% xylene cyanol). After heating reaction mixtures to 95 °C for 5 min, they were electrophoresed in 20% polyacrylamide, 7 m urea denaturing gels. Helicase Assay—The reaction conditions for helicase assays and time course experiments were performed as described previously (35Choudhary S. Sommers J.A. Brosh Jr., R.M. J. Biol. Chem. 2004; 279: 34603-34613Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 20-μl reactions contained 30 mm HEPES (pH 7.4), 5% glycerol, 40 mm KCl, 100 ng/μl BSA, 0.8 nm DNA substrate, 2 mm MgCl2, and 2 mm ATP. Reactions were incubated for 15 min (unless otherwise indicated) at 37 °C and terminated using 20 μl of stop buffer (35 mm EDTA, 0.6% SDS, 25% glycerol, 0.04% bromphenol blue, 0.04% xylene cyanol) containing a 10-fold excess of unlabeled oligonucleotide of the same sequence as the labeled strand of the DNA fork substrate. Reaction mixtures were electrophoresed in nondenaturing 12% polyacrylamide gels, and the results were analyzed using PhosphorImager and ImageQuant software (Amersham Biosciences). NMR Sample Preparation—The cloning and expression of RQC motif of the WRN protein for NMR studies have been described previously (36Sun J.Z. Feng H.Q. Lin G.X. Zeng W. Hu J.S. J. Biomol. NMR. 2005; 32 (in press)Google Scholar). Briefly, the plasmids encoding the wild-type and the K1016A mutant WRN protein were used as templates to subclone the KpnI and XhoI fragment containing the 144-residue RQC domain of WRN into pET32d, the mutant pET32a (Novagen) in which the GGTATG sequence right after the thrombin cleavage site was mutated to introduce a KpnI site. These plasmids, pET32d-RQC, were transformed into the E. coli strain BL21 (Novagen), grown in M9 minimal medium containing either 15NH4Cl and unlabeled d-glucose or 15NH4Cl and d-[13C6]glucose. The thioredoxin-histidine-tagged RQC fusion protein was purified by HiTrap-Sepharose chelating columns (Amersham Biosciences). After the fusion protein was cleaved with thrombin (Sigma), the RQC protein was separated from the thioredoxin-histidine tag, uncleaved fusion protein, and other proteins using a second chelating column. The purification procedure yielded 2-6 mg of labeled protein from 1 liter of culture with a purity of about 95%. The NMR samples used were 0.45 mm15N- and 13C/15N-labeled protein dissolved in 25 mm potassium phosphate (pH 7.4), 25 mm NaCl, 4 mmd10-dithiothreitol, 1 mmd16-EDTA, 92.5% H2O, 7.5% D2O. NMR Spectroscopy—NMR experiments were recorded at 22 °C on a Bruker DRX-600 spectrometer equipped with z-shielded gradient triple-resonance cryoprobe. All data were processed with the NMRPipe package (37Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) and analyzed using the PIPP, CAPP, and STAPP programs (38Garret D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Res. 1991; 95: 214-220Google Scholar). The sequential backbone assignments of the RQC domain were made using triple-resonance methodology (39Bax A.G.S. Acc. Chem. Res. 1993; 26: 31-138Crossref Scopus (796) Google Scholar). The two-dimensional spin-echo difference experiments, HNCG_arom and HN(CO)CG_arom, were used to identify 1HN-15N resonances of aromatic residues and the following residues (40Hu J.S. Bax A. J. Biomol. NMR. 1997; 9: 323-328Crossref PubMed Scopus (62) Google Scholar), which were used as starting points for automatic sequential backbone resonance (1HN, 15N, 13Cα, and 13Cβ) assignments when combined with three-dimensional HNCACB, CBCA- (CO)NH, and HNCA experiments. Three-dimensional HNCO, HNHA, and HNCOCO (41Hu J.S. Bax A. J. Am. Chem. Soc. 1996; 118: 8170-8171Crossref Scopus (71) Google Scholar) experiments were performed to obtain 1Hα and carbonyl (13C′) resonance assignments and to simultaneously obtain ϕ restraints. 1Hα and 1Hβ resonances for the majority of residues were obtained by using the three-dimensional HBHA(CO)NH experiment. The two-dimensional HNCG_arom and HN(CO)CG_arom were used to determine intra-residue 3JNCγ and 3JC′ Cγ coupling constants (40Hu J.S. Bax A. J. Biomol. NMR. 1997; 9: 323-328Crossref PubMed Scopus (62) Google Scholar). Transfection and Immunofluorescence Assays—SV40-transformed human kidney epithelial (293T) cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) and 10% fetal bovine serum. For expression as GFP fusion protein in mammalian cells, cells growing on coverslips were transfected with wild-type pEGFPC3-WRN (aa 1-1432) or pEGFPC3-WRN-K1016A mutant (aa 1-1432) containing a single missense mutation at Lys-1016 using the PolyFect transfection reagent (Qiagen). After 40 h, the cells were fixed and permeabilized with 3.5% paraformaldehyde (Sigma), 0.2% Triton X-100 (Sigma) in 1× PBS for 15 min each at room temperature. Then the coverslips were washed three times with PBS followed by incubation with blocking buffer (0.1% Tween 20, 2% BSA in 1× PBS) for 1 h at room temperature. Finally, coverslips were mounted on Vectashield containing DAPI (Vector Laboratories) and viewed under a AxioVision 3.1 microscope (Zeiss) with deconvolution (42von Kobbe C. Karmakar P. Dawut L. Opresko P. Zeng X. Brosh Jr., R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 22035-22044Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A Putative wHTH Motif in the RQC Domain of WRN—The three-dimensional crystal structure of E. coli RecQ helicase has revealed the presence of a wHTH motif (14Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (217) Google Scholar). Thus, we hypothesized that WRN may also contain a wHTH motif responsible for mediating the major DNA binding activity of WRN. In support of a similar DNA-binding motif in WRN RQC, secondary structure predictions revealed a helix motif in the WRN RQC that directly matched the putative DNA recognition helix of E. coli RecQ (supplement 1). Sequence homologies between the putative DNA recognition helix in WRN and the other RecQ helicases were examined using multiple-sequence alignment software, ClustalW, and four amino acids were selected for site-directed mutagenesis (Fig. 1, arrows). Conserved as a positively charged amino acid in 5 of 7 RecQ helicases, the WRN Lys-1008 located outside the putative DNA recognition helix was chosen to serve as a control to missense mutations made inside the putative DNA recognition helix. Likewise, the polar Gln-1010 located outside the putative DNA recognition helix was chosen as a second control. Inside the putative DNA recognition helix of WRN RQC, we found only two positively charged aa residues, Lys-1016 and Arg-1020, which are likely to be involved in protein-DNA interactions. Significantly, these residues are conserved as a positive charged aa in 6 of 7 and 4 of 7 RecQ helicases, respectively. After the confirmation of a single missense mutation at each four aa residues, the wild-type RQC and RQC mutant proteins were prepared. The R1020A mutant could not be purified to homogeneity as other WRN mutant fragments and was excluded from our studies (data not shown). Lys-1016 of WRN Plays a Significant Role in Binding Fork and Bubble DNA Substrates—To compare relative DNA binding affinities of these mutant RQC proteins, it was critical to use precisely the same amount of each protein. Therefore, each protein preparation and the relative amount of each protein fragment were analyzed carefully, and the quantitative results were in close agreement using several different methods: Bradford assay, Amido Black staining, GelCode Blue staining (Fig. 2A), and silver staining (Fig. 2C). In addition, Western blot analysis was performed to verify the relative concentrations of wild-type RQC, K1008A, Q1010A, and K1016A (Fig. 2B) and Hlc, HlcRQC, and Hlc-K1016A proteins (Fig. 2D). Because the RQC domain was demonstrated to be the strongest DNA binding region of WRN (21von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), we examined which of the three amino acids, Lys-1008, Gln-1010, or Lys-1016, was important for the WRN-DNA interaction. The wild-type RQC and mutants K1008A, Q1010A, and K1016A were used to perform EMSAs with various DNA substrates, including the fork, bubble, D-loop, or the Holliday junction substrates. These substrates were selected to reflect the types of DNA structures that WRN might be expected to encounter in vivo (21von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). When radiolabeled fork substrate was used, both the wild-type RQC and Q1010A mutant showed nearly identical concentration-dependent DNA mobility shifts (Fig. 3A). The EMSA results using the K1008A mutant generated band-shift patterns similar to that of the wild-type RQC (data not shown). However, when the K1016A mutant was used with the same fork DNA substrate, minimal band-shifts were observed, suggesting that Lys-1016 plays a significant role in the WRN-DNA interaction (Fig. 3B). By using a bubble DNA substrate, the K1016A mutant displayed significantly reduced DNA binding, whereas the K1008A mutant or the Q1010A mutant generated band-shifts similar to the wild-type RQC (Fig. 3, C and D; K1008A mutant data not shown). Each of these experiments was performed multiple times, and band-shifts obtained from these independent experiments were quantified by using ImageQuant and plotted with standard deviations as shown in supplemental 2A (fork substrate) and 2B (bubble substrate). A Single Missense Mutation at Lys-1016 of WRN Results in Decreased Stimulation of FEN-1 Incision Activity—We showed previously that WRN RQC (aa 949-1092) strongly stimulates the incision activity of FEN-1 in vitro (33Brosh Jr., R.M. von Kobbe C. Sommers J.A. Karmakar P. Opresko P.L. Piotrowski J. Dianova I. Dianov G.L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar). To investigate how the WRN-DNA interaction might affect the functional stimulation of FEN-1 cleavage, incision assays were performed by using 1-nt flap substrates in the presence of wild-type RQC or the K1016A mutant (Fig. 4A). Wild-type RQC stimulated FEN-1 incision activity (Fig. 4A, lanes 5-7) similar to previous results (33Brosh Jr., R.M. von Kobbe C. Sommers J.A. Karmakar P. Opresko P.L. Piotrowski J. Dianova I. Dianov G.L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar). However, there was little or no stimulation of FEN-1 incision activities using the K1016A mutant (Fig. 4A, lanes 8-10). The inc" @default.
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• W2078383934 title "Modulation of Werner Syndrome Protein Function by a Single Mutation in the Conserved RecQ Domain" @default.
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