The Werner Syndrome Protein Stimulates DNA Polymerase β Strand Displacement Synthesis via Its Helicase Activity
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m213103200
ISSN1083-351X
AutoresJeanine A. Harrigan, Patricia L. Opresko, Cayetano von Kobbe, Padmini S. Kedar, Rajendra Prasad, Samuel H. Wilson, Vilhelm A. Bohr,
Tópico(s)Plant Genetic and Mutation Studies
ResumoWerner syndrome is a hereditary premature aging disorder characterized by genomic instability. Genetic analysis and protein interaction studies indicate that the defective gene product (WRN) may play an important role in DNA replication, recombination, and repair. DNA polymerase β (pol β) is a central participant in both short and long-patch base excision repair (BER) pathways, which function to process most spontaneous, alkylated, and oxidative DNA damage. We report here a physical interaction between WRN and pol β, and using purified proteins reconstitute of a portion of the long-patch BER pathway to examine a potential role for WRN in this repair response. We demonstrate that WRN stimulates pol β strand displacement DNA synthesis and that this stimulation is dependent on the helicase activity of WRN. In addition, a truncated WRN protein, containing primarily the helicase domain, retains helicase activity and is sufficient to mediate the stimulation of pol β. The WRN helicase also unwinds a BER substrate, providing evidence that WRN plays a role in unwinding DNA repair intermediates. Based on these findings, we propose a novel mechanism by which WRN may mediate pol β-directed long-patch BER. Werner syndrome is a hereditary premature aging disorder characterized by genomic instability. Genetic analysis and protein interaction studies indicate that the defective gene product (WRN) may play an important role in DNA replication, recombination, and repair. DNA polymerase β (pol β) is a central participant in both short and long-patch base excision repair (BER) pathways, which function to process most spontaneous, alkylated, and oxidative DNA damage. We report here a physical interaction between WRN and pol β, and using purified proteins reconstitute of a portion of the long-patch BER pathway to examine a potential role for WRN in this repair response. We demonstrate that WRN stimulates pol β strand displacement DNA synthesis and that this stimulation is dependent on the helicase activity of WRN. In addition, a truncated WRN protein, containing primarily the helicase domain, retains helicase activity and is sufficient to mediate the stimulation of pol β. The WRN helicase also unwinds a BER substrate, providing evidence that WRN plays a role in unwinding DNA repair intermediates. Based on these findings, we propose a novel mechanism by which WRN may mediate pol β-directed long-patch BER. Werner syndrome (WS) 1The abbreviations used are: WS, Werner syndrome; BER, base excision repair; AP, apurinic/apyrimidinic; pol β, polymerase β; GST, glutathione S-transferase; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; DTT, dithiothreitol; BLM, Bloom syndrome protein; 4-NQO, 4-nitroquinoline 1-oxide; PCNA, proliferating cell nuclear antigen; UDG, uracil DNA glycosylase; aa, amino acids; APE1, AP endonuclease 1; dRP, 5′-deoxyribose phosphate. 1The abbreviations used are: WS, Werner syndrome; BER, base excision repair; AP, apurinic/apyrimidinic; pol β, polymerase β; GST, glutathione S-transferase; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; DTT, dithiothreitol; BLM, Bloom syndrome protein; 4-NQO, 4-nitroquinoline 1-oxide; PCNA, proliferating cell nuclear antigen; UDG, uracil DNA glycosylase; aa, amino acids; APE1, AP endonuclease 1; dRP, 5′-deoxyribose phosphate. is a rare, autosomal recessive premature aging disorder characterized by gray hair, wrinkled skin, cataracts, atherosclerosis, osteoporosis, diabetes mellitus (type 2), and an increased incidence of malignancies (1Martin G.M. Austad S.N. Johnson T.E. Nat. Genet. 1996; 13: 25-34Crossref PubMed Scopus (536) Google Scholar). The gene (WRN) defective in WS patients encodes a 1,432-amino acid protein that contains ATPase, 3′ → 5′ helicase, and 3′ → 5′ exonuclease activities (reviewed in Ref. 2Brosh Jr., R.M. Bohr V.A. Exp. Gerontol. 2002; 37: 491-506Crossref PubMed Scopus (68) Google Scholar). Cells from WS patients display an extended S phase (3Poot M. Gollahon K.A. Emond M.J. Silber J.R. Rabinovitch P.S. FASEB J. 2002; 16: 757-758Crossref PubMed Scopus (84) Google Scholar), undergo premature replicative senescence (4Martin G.M. Sprague C.A. Epstein C.J. Lab. Invest. 1970; 23: 86-92PubMed Google Scholar), and exhibit higher levels of DNA deletions, translocations, and chromosomal breaks (5Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar, 6Stefanini M. Scappaticci S. Lagomarsini P. Borroni G. Berardesca E. Nuzzo F. Mutation Res. 1989; 219: 179-185Crossref PubMed Scopus (42) Google Scholar). The increased genomic instability associated with WS cells suggests that WRN may function in DNA replication, recombination, and/or repair pathways. Sensitivity of WS cells to DNA-damaging agents provides additional evidence for a role of WRN in DNA repair. WS cells show marked hypersensitivity to 4-nitroquinoline 1-oxide (4-NQO), topoisomerase inhibitors, and DNA interstrand cross-linking agents (reviewed in Ref. 2Brosh Jr., R.M. Bohr V.A. Exp. Gerontol. 2002; 37: 491-506Crossref PubMed Scopus (68) Google Scholar). In addition, a mild sensitivity of WS cells to ionizing radiation (IR) has been demonstrated (7Yannone S.M. Roy S. Chan D.W. Murphy M.B. Huang S. Campisi J. Chen D.J. J. Biol. Chem. 2001; 276: 38242-38248Abstract Full Text Full Text PDF PubMed Google Scholar, 8Saintigny Y. Makienko K. Swanson C. Emond M.J. Monnat Jr., R.J. Mol. Cell. Biol. 2002; 22: 6971-6978Crossref PubMed Scopus (231) Google Scholar). In contrast, WS cells display no or little sensitivity to ultraviolet light, bleomycin, hydroxyurea, or alkylating agents (reviewed in Ref. 9Shen J. Loeb L.A. Mech. Ageing Dev. 2001; 122: 921-944Crossref PubMed Scopus (82) Google Scholar). However, a recent report shows that WRN–/– chicken cells are hypersensitive to the alkylating agent methyl methanesulfonate (MMS) (10Imamura O. Fujita K. Itoh C. Takeda S. Furuichi Y. Matsumoto T. Oncogene. 2002; 21: 954-963Crossref PubMed Scopus (86) Google Scholar). 4-NQO generates a wide spectrum of DNA adducts as well as oxidative damage (11Gebhart E. Bauer R. Schinzel M. Ruprecht K.W. Jonas J.B. Human Genet. 1988; 80: 135-139Crossref PubMed Scopus (138) Google Scholar, 12Ogburn C.E. Oshima J. Poot M. Chen R. Gollahon K.A. Rabinovitch P.S. Martin G.M. Human Genetics. 1997; 101: 121-125Crossref PubMed Scopus (162) Google Scholar), and IR produces a diverse range of DNA damage including strand breaks and oxidative base modifications (13Weinfeld M. Rasouli-Nia A. Chaudhry M.A. Britten R.A. Radiat. Res. 2001; 156: 584-589Crossref PubMed Google Scholar, 14Wallace S.S. Radiat. Res. 1998; 150: S60-S79Crossref PubMed Scopus (302) Google Scholar). Hypersensitivity of WS cells to these damaging agents suggests that WRN may participate in some aspect of oxidative DNA damage repair. Base excision repair (BER) is important for the repair of a wide variety of DNA damage including oxidized, alkylated, deaminated, and hydrolyzed bases (reviewed in Refs. 15Wilson D.M. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12754-12757Crossref PubMed Scopus (215) Google Scholar and 16Lindahl T. Mutat. Res. 2000; 462: 129-135Crossref PubMed Scopus (190) Google Scholar)). DNA glycosylases recognize damaged bases and cleave the glycosylic bond. The resulting apurinic/apyrimidinic (AP) site is then cleaved 5′ to the abasic site by an AP endonuclease (APE1), resulting in the formation of a 3′-hydroxyl and a 5′-deoxyribose phosphate (dRP). In mammalian cells, completion of BER occurs by short-patch (single nucleotide incorporation) or long-patch (replacement of 2–13 nucleotides) repair. In the short-patch pathway, DNA polymerase β (pol β) removes the dRP group and fills in the resulting gap, which is then sealed by a DNA ligase. If the dRP group is modified (most notably reduced) and refractory to removal by pol β dRP lyase activity, repair likely occurs through the long-patch pathway. In this alternative process, strand displacement and DNA synthesis by either pol β and/or pol δ/ϵ replaces several nucleotides. The resulting 5′-flap is excised by flap endonuclease (FEN-1), and the nick is sealed by a DNA ligase. Long-patch BER catalyzed by pol δ/ϵ also requires proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) to enhance polymerase activity. PCNA has also been shown to stimulate FEN-1 flap cleavage (17Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (660) Google Scholar). In addition, a number of accessory proteins have been reported to participate in and/or stimulate BER including poly(ADP-ribose) polymerase-1 (PARP-1) (reviewed in Ref. 18Dantzer F. Schreiber V. Niedergang C. Trucco C. Flatter E. De La R.G. Oliver J. Rolli V. Menissier-de Murcia J. de Murcia G. Biochimie (Paris). 1999; 81: 69-75Crossref PubMed Scopus (304) Google Scholar), p53 (19Zhou J. Ahn J. Wilson S.H. Prives C. EMBO J. 2001; 20: 914-923Crossref PubMed Scopus (277) Google Scholar), and replication protein A (RPA) (20DeMott M.S. Zigman S. Bambara R.A. J. Biol. Chem. 1998; 273: 27492-27498Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). WRN has been shown to interact physically and/or functionally with several proteins involved in long-patch BER including pol δ, PCNA, RPA, and FEN-1. WRN interacts physically with pol δ and recruits it to the nucleolus (21Szekely A.M. Chen Y.H. Zhang C. Oshima J. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11365-11370Crossref PubMed Scopus (102) Google Scholar). Furthermore, WRN has been shown to stimulate nucleotide incorporation by pol δ (22Kamath-Loeb A.S. Johansson E. Burgers P.M. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4603-4608Crossref PubMed Scopus (157) Google Scholar). A physical interaction between WRN and PCNA has been identified, however, a functional interaction has yet to be determined (23Lebel M. Spillare E.A. Harris C.C. Leder P. J. Biol. Chem. 1999; 274: 37795-37799Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). RPA stimulates the WRN helicase to unwind long duplex DNA substrates that WRN alone is not able to unwind (24Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 25Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar). WRN interacts physically with FEN-1 and stimulates its DNA flap cleavage activity (26Brosh 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, because these proteins participate in both replication and long-patch BER, an association with WRN does not reveal whether this interaction is important for one or both pathways. To distinguish a role for WRN in DNA repair, we investigated a potential interaction between WRN and pol β. We report here that WRN physically interacts with pol β. As protein-protein and protein-DNA interactions mediate the repair steps involved in BER, and pol β plays a central role in BER, we examined a potential functional role for this interaction by reconstituting a portion of the long-patch BER pathway. We find that pol β strand displacement synthesis is enhanced by WRN and this stimulation requires WRN helicase activity. Furthermore, a glutathione S-transferase (GST)-WRN fusion protein containing the helicase domain of WRN catalyzes unwinding and is sufficient to mediate the stimulation of pol β. We also demonstrate that WRN unwinds a BER intermediate suggesting a novel mechanism through which WRN may participate in pol β-mediated long-patch BER. [α-32P]ddATP, [α-32P]dCTP, ATP, and dNTPs were purchased from Amersham Biosciences. [γ-32P]ATP was purchased from PerkinElmer Life Sciences, polynucleotide kinase from New England BioLabs, and terminal deoxynucleotidyltransferase (TdT) from Promega. Purified recombinant proteins used in this study are presented in Fig. 1. AP endonuclease (APE1) (27Strauss P.R. Beard W.A. Patterson T.A. Wilson S.H. J. Biol. Chem. 1997; 272: 1302-1307Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), pol β (28Beard W.A. Wilson S.H. Methods Enzymol. 1995; 262: 98-107Crossref PubMed Scopus (159) Google Scholar), uracil DNA glycosylase (UDG) (29Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar), wild-type WRN (30Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar), the WRN helicase mutant K577M (K-WRN) (30Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar), and FEN-1 (26Brosh 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) were purified as described previously. Recombinant GST-WRN fusion proteins were overexpressed in Escherichia coli and purified using glutathione beads (Amersham Biosciences) as described previously (26Brosh 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). Bands migrating below GST-WRN500–946 (∼75 kDa) were determined to be degradation products by Western blot analysis using anti-GST antibodies. Telomerase-immortalized wild-type (WT, hTERT-GM01604), and Werner syndrome (WS, hTERT-AG03141) cell lines (a gift from Jerry Shay) were cultured in 150-mm dishes until nearly confluent. Cells were incubated in a lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 25 mm NaF, 0.1 mm sodium orthovanadate, 0.2% Triton X-100, 0.3% Nonidet P-40) containing protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 5 μg/ml leupeptin) on ice for 30 min and then centrifuged at 14,000 rpm for 30 min at 4 °C. The protein concentration was determined using the Bio-Rad protein assay, with bovine serum albumin (BSA) as a standard. For co-immunoprecipitation, an equal amount (1 mg of protein) of cell lysate was mixed with 0.7 μg of affinity-purified anti-pol β polyclonal antibody, anti-WRN polyclonal antibody (SC-5629, Santa Cruz Biotechnology), or rabbit nonimmune IgG, respectively. The mixture was incubated with rotation for 4 h at 4 °C. The protein-antibody immunocomplexes were absorbed onto protein A-Sepharose/protein G-agarose beads. The proteins were eluted in sodium dodecyl sulfate (SDS) sample buffer, separated by 4–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and immunoblotted using an anti-WRN monoclonal antibody (1:200 dilution, BD Transduction Laboratories). The same blot was stripped by incubating with buffer containing 62.5 mm Tris-HCl, pH 6.8, 100 mm β-mercaptoethanol, and 1% SDS for 30 min at 50 °C, followed by Western analysis using anti-pol β monoclonal antibody, 18 S (31Srivastava D.K. Evans R.K. Kumar A. Beard W.A. Wilson S.H. Biochemistry. 1996; 35: 3728-3734Crossref PubMed Scopus (17) Google Scholar). HeLa nuclear extracts (NE) pull-down assays with GST and a GST-C-terminal WRN fragment (GST-WRN949–1432) with HeLa NE were performed essentially as described elsewhere (26Brosh 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, 32von 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 (110) Google Scholar). Briefly, GST or the GST-WRN fragment was bound to glutathione beads and then incubated with HeLa NE (∼1 mg). After washing, proteins were eluted in sample buffer by boiling and electrophoresed on 10% polyacrylamide SDS gels and transferred to polyvinylidene difluoride membranes. GST-C-terminal WRN fragment and bound proteins were detected by Amido Black staining of the membrane. pol β bound to GST-WRN949–1432 was detected by Western blot with mouse monoclonal anti-pol β antibodies (Trevigen, 1:5000). GST or GST-WRN fragments were prebound to glutathione beads and then incubated with purified pol β (5 μg) for 1.5 h at 4 °C. Following several washes with phosphate-buffered saline (1×) and 0.1% Tween 20, bound proteins were resuspended in sample buffer and analyzed by Western blot as described above. Co-immunoprecipitation experiments were performed essentially as described previously (33Kedar P.S. Kim S.J. Robertson A. Hou E. Prasad R. Horton J.K. Wilson S.H. J. Biol. Chem. 2002; 277: 31115-31123Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Briefly, purified WRN (1.5 μm) and pol β (1.5 μm) proteins were incubated in a binding buffer (25 mm Tris-HCl, pH 8.0, 10% glycerol, 100 mm NaCl, and 0.01% Nonidet P-40) containing protease inhibitors: 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 5 μg/ml leupeptin. Mixtures (50 μl) of pol β and WRN were incubated with either an anti-pol β polyclonal antibody (affinity-purified IgG) (34Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), an anti-WRN polyclonal antibody (sc-5629, Santa Cruz Biotechnology), or preimmune IgG by rotation for 4 h at 4 °C. Protein/antibody complexes were then absorbed onto protein A-Sepharose/protein G-agarose and incubated overnight at 4 °C with rotation. The beads were washed four times with binding buffer containing protease inhibitors. Western blot analysis was performed as described above. The detection of protein interactions was performed by ELISA essentially as described previously (35Brosh 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 (255) Google Scholar). Wells were coated with pol β (75 ng) or BSA (negative control) for 16 h at 4 °C. Wells were washed and incubated with blocking buffer (phosphate-buffered saline, 2% BSA, 0.1% Tween 20) for 1 h at 37 °C. Binding of WRN (75 ng) or BSA was performed for 2 h at 37 °C. Following washing, primary antibody (anti-rabbit WRN, Novus, 1:1000) was added and incubated for 1 h at 37 °C. Wells were washed and secondary antibody (anti-rabbit IgG-horseradish peroxidase, Vector, 1:10,000) was added and incubated for 1 h at 37 °C. Bound WRN was detected using o-phenylenediamine dihydrochloride (OPD) as a substrate. The reaction was terminated by the addition of 3 m H2SO4. Absorbance was read at 490 nm. For the ethidium bromide controls, ethidium bromide was added to the binding step at a concentration of 10 μg/ml. Forked Substrate—Helicase activity was detected by the displacement of a 32P-labeled 5′ oligonucleotide (5′-(T)15GAGTGTGGTGTACATGCACTAC-3′) from its partial duplex with the complementary unlabeled oligonucleotide (5′-GTAGTGCATGTACACCACACTC(T)15-3′). Reactions were performed as described previously (36Opresko 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). Briefly, 5′ end-labeled DNA substrate (0.5 nm) was incubated in helicase reaction buffer (40 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 5 mm dithiothreitol (DTT), 2 mm ATP, and 0.1 mg/ml BSA) in a final volume of 20 μl. Reactions were initiated by the addition of WRN, incubated at 37 °C for 15 min, and terminated by the addition of 3× stop dye to a final concentration of 1× along with a 10× molar excess of unlabeled competitor oligonucleotide. Products were run on a 12% native polyacrylamide gel, visualized using a PhosphorImager, and quantitated using Image-Quant software. Uracil-containing Substrate—The 34-mer uracil-containing DNA (37Prasad R. Lavrik O.I. Kim S.J. Kedar P. Yang X.P. Vande Berg B.J. Wilson S.H. J. Biol. Chem. 2001; 276: 32411-32414Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) was 3′ end-labeled with terminal deoxynucleotidyl transferase (TdT) and [α-32P]ddATP. Prior to the helicase reaction, the substrate (20 nm) was pretreated with 2 nm UDG in 50 mm HEPES, pH 7.5, 20 mm KCl, and 2 mm DTT for 20 min at 37 °C. Helicase reactions (10 μl) contained 5 nm pretreated DNA substrate in helicase reaction buffer, with WRN, pol β, FEN-1, and dNTPs as indicated. Reactions were initiated by adding APE1 and incubating for 25 min at 37 °C. Reactions were terminated and analyzed as described above. Exonuclease activity was detected by the degradation of a 32P-labeled 5′ oligonucleotide (oligo 5), which was annealed to an unlabeled partial duplex complementary oligonucleotide (oligo 6) as described (36Opresko 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). Reactions were performed as described previously (36Opresko 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). Briefly, 5′ end-labeled DNA substrate (0.5 nm) was incubated in a standard reaction buffer containing 40 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 5 mm DTT, 2 mm ATP, and 0.1 mg/ml BSA in a final volume of 10 μl. Reactions were initiated by the addition of WRN and incubated for 15 min at 37 °C. Reactions were terminated by the addition of an equal volume of formamide stop dye (80% formamide, 0.5× Tris borate/EDTA buffer, 0.1% bromphenol blue, and 0.1% xylene cyanol). Products were heat-denatured at 90 °C for 5 min and run on 14% denaturing polyacrylamide gels. Radioactive products were visualized using a PhosphorImager. Reconstitution of the repair reaction was performed essentially as described previously (37Prasad R. Lavrik O.I. Kim S.J. Kedar P. Yang X.P. Vande Berg B.J. Wilson S.H. J. Biol. Chem. 2001; 276: 32411-32414Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Prior to assembly of the repair reaction, uracil-containing DNA substrate (400 nm) was pretreated with 40 nm UDG in 50 mm HEPES, pH 7.5, 20 mm KCl, and 2 mm DTT for 20 min at 37 °C (Fig. 5A). Repair reactions (10 μl) contained 100 nm pretreated DNA substrate in 50 mm HEPES, pH 7.5, 20 mm KCl, 2 mm DTT, 10 mm MgCl2, 4 mm ATP (except where noted), 20 μm each of dATP, dGTP, dTTP, 2 μm dCTP, 2.3 μm [α-32P]dCTP, and UDG (10 nm). The presence of WRN, K-WRN, GST-WRN500–946, or FEN-1 is indicated in the figure legends. Reactions were initiated by the addition of 10 nm APE1 and 5 nm pol β. The reaction mixture was incubated for 25 min at 37 °C and terminated by the addition of an equal volume of stop dye (95% formamide, 20 mm EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol). Following incubation at 95 °C for 5 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel and visualized using a PhosphorImager. Under these reaction conditions, the incised abasic site produced by the sequential actions of UDG and APE1 was determined to be ∼90% (data not shown). Percent of total nucleotides incorporated was calculated as: % of total = (amount of radioactivity associated with each dNMP addition/total radioactivity) × 100. Reactions (10 μl) contained 50 mm HEPES, pH 7.5, 20 mm KCl, 2 mm DTT, 10 mm MgCl2, 1 mm each dATP, dGTP, dTTP, dCTP, and pol β and WRN as indicated in the figure legend. The reaction was initiated by the addition of 5 nm 5′ end-labeled 15-mer (5′-CTGCAGCTGATGCGC-3′) annealed to a 34-mer unlabeled complementary oligonucleotide (5′-GTACCCGGGGATCCGTACGGCGCATCAGCTGCAG-3′) and incubated at 37 °C for 15 or 30 min. The reaction mixture was terminated by the addition of an equal volume of stop dye (95% formamide, 20 mm EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol). Reaction products were incubated at 90 °C for 5 min, run on 20% denaturing polyacrylamide gels, and visualized using a PhosphorImager. Physical Interaction and Complex Formation between WRN and pol β —To investigate a potential role for WRN in the repair of oxidative DNA damage, we first performed co-immunoprecipitation assays to identify an interaction between WRN and pol β. Using antibodies against pol β, we co-immunoprecipitated WRN from normal (Fig. 2A, lane 1) but not WS cells (Fig. 2A, lane 2). We also co-precipitated pol β from wild-type cells using antibodies against WRN (Fig. 2B, lane 1) but not from WS cells (Fig. 2B, lane 2). As the C terminus of WRN is important for mediating interactions with many of its protein partners, including pol δ (21Szekely A.M. Chen Y.H. Zhang C. Oshima J. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11365-11370Crossref PubMed Scopus (102) Google Scholar), we used a pull-down assay with a C-terminal WRN fragment (GST-WRN949–1432) to investigate a possible association between WRN and pol β in HeLa nuclear extracts. pol β co-precipitated with GST-WRN949–1432 (Fig. 2C, right panel, lane 4). This association with GST-WRN949–1432 was specific as GST alone did not precipitate pol β (Fig. 2C, right panel, lane 3). These results suggest that WRN and pol β are in the same complex in vivo. To test whether WRN and pol β physically interact, we performed co-immunoprecipitation experiments with purified, recombinant proteins. As shown in Fig. 3A, WRN and K-WRN (a WRN ATPase/helicase mutant, Fig. 1A) were precipitated by pol β antibodies (Fig. 3A, lanes 1 and 2), but not when pol β was omitted (Fig. 3A, lane 3) or when the immunoprecipitation was performed with preimmune IgG (Fig. 3A, lane 4). When WRN antibodies were used for the co-immunoprecipitation experiments, pol β was effectively precipitated (Fig. 3B, lanes 1 and 2). However, when WRN was omitted (Fig. 3B, lane 3) or preimmune IgG was used (Fig. 3B, lane 4) pol β was not detected. To further confirm a direct association between WRN and pol β, we performed ELISA with purified proteins. pol β specifically bound to WRN and not to the BSA control (Fig. 3C). This interaction was not mediated by DNA since the binding was not affected by the presence of ethidium bromide (Fig. 3C, bar 5). These results demonstrate that WRN and pol β interact directly. To map the regions of WRN to which pol β binds, we incubated a series of GST-WRN fragments with purified pol β (Fig. 3D). Consistent with the pull-down experiments presented in Fig. 2C, purified pol β bound to GST-WRN949–1432 (Fig. 3D, lane 4). pol β also bound to GST-WRN500–946, the helicase domain (Fig. 3D, lane 3). However, pol β did not bind to GST-WRN239–499 (Fig. 3D, lane 2) or GST alone (Fig. 3D, lane 1). These results suggest that pol β preferentially interacts with the C terminus of WRN (aa 949–1432) but also binds to the helicase domain (aa 500–946). The Effect of pol β on WRN Activities—Based on the physical interaction between WRN and pol β, we next tested whether pol β functionally altered WRN helicase or exonuclease activities. On a forked 22-bp duplex DNA substrate, increasing concentrations of pol β did not influence WRN helicase activity (Fig. 4A). Similarly, WRN exonuclease activity on a forked oligonucleotide containing a 34-bp duplex was not affected by pol β (Fig. 4B). These results suggest that under the reaction conditions employed, pol β does not modulate WRN helicase or exonuclease activities. WRN Stimulates pol β DNA Strand Displacement Synthesis—To determine whether WRN influences pol β-mediated long-patch BER, we reconstituted a portion of the long-patch BER reaction using a 34-bp oligonucleotide substrate containing a uracil lesion at position 16 and the following purified human enzymes: UDG, APE1, pol β, and FEN-1 (Fig. 5A). Without the addition of DNA ligase, 16-mer oligonucleotide products represent incorporation of a single [α-32P]dCMP nucleotide and short-patch BER. Longer products represent strand displacement DNA synthesis and long-patch BER intermediates. pol β synthesizes primarily the 16-nucleotide product (Fig. 5B, lane 1), which represents one-nucleotide gap-filling. The addition of FEN-1 produced a modest stimulation in strand displacement synthesis (Fig. 5B, lane 2) as has been published previously (37Prasad R. Lavrik O.I. Kim S.J. Kedar P. Yang X.P. Vande Berg B.J. Wilson S.H. J. Biol. Chem. 2001; 276: 32411-32414Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). When WRN was added, a comparatively greater stimulation of pol β strand displacement synthesis was seen (Fig. 5B, lane 3). When FEN-1 and WRN were added together, the greatest stimulation resulted (Fig. 5B, lane 4). These results suggest that WRN, particularly when present with FEN-1 in this in vitro system, produces a strong stimulation of strand displacement synthesis by pol β. Consistent with the above results, quantitation of the number of nucleotides incorporated by pol β alone indicated primarily short-patch BER intermediates (90% of the synthesis, Fig. 5C). The presence of either FEN-1 or WRN alone decreased the number of short-patch intermediates to ∼70%, while increasing the long-patch intermediates to ∼30% (Fig. 5C). The addition of both FEN-1 and WRN resulted in ∼55% of long-patch intermediates (Fig. 5C). Furthermore, FEN-1 stimulated predominately long-patch intermediates at the 2 nucleotide position (∼17%) while addition of WRN produced mostly 4 nucleotide products (∼13%) (Fig. 5D). WRN also increased pol β-mediated strand displacement DNA products to 5 and 6 nucleotides in l
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