The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m212798200
ISSN1083-351X
AutoresSudha Sharma, Joshua A. Sommers, Henry C. Driscoll, Laura A. Uzdilla, Teresa Wilson, Robert Brosh,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoExonuclease 1 (EXO-1), a member of the RAD2 family of nucleases, has recently been proposed to function in the genetic pathways of DNA recombination, repair, and replication which are important for genome integrity. Although the role of EXO-1 is not well understood, its 5′ to 3′-exonuclease and flap endonuclease activities may cleave intermediates that arise during DNA metabolism. In this study, we provide evidence that the Werner syndrome protein (WRN) physically interacts with human EXO-1 and dramatically stimulates both the exonucleolytic and endonucleolytic incision functions of EXO-1. The functional interaction between WRN and EXO-1 is mediated by a protein domain of WRN which interacts with flap endonuclease 1 (FEN-1). Thus, the genomic instability observed in WRN–/– cells may be at least partially attributed to the lack of interactions between the WRN protein and human nucleases including EXO-1. Exonuclease 1 (EXO-1), a member of the RAD2 family of nucleases, has recently been proposed to function in the genetic pathways of DNA recombination, repair, and replication which are important for genome integrity. Although the role of EXO-1 is not well understood, its 5′ to 3′-exonuclease and flap endonuclease activities may cleave intermediates that arise during DNA metabolism. In this study, we provide evidence that the Werner syndrome protein (WRN) physically interacts with human EXO-1 and dramatically stimulates both the exonucleolytic and endonucleolytic incision functions of EXO-1. The functional interaction between WRN and EXO-1 is mediated by a protein domain of WRN which interacts with flap endonuclease 1 (FEN-1). Thus, the genomic instability observed in WRN–/– cells may be at least partially attributed to the lack of interactions between the WRN protein and human nucleases including EXO-1. Werner syndrome (WS) 1The abbreviations used are: WS, Werner syndrome; dsDNA, double strand DNA; EXO-1, exonuclease 1; FEN-1, 5′-flap endonuclease/5′ to 3′-exonuclease; nt, nucleotide; PCNA, proliferating cell nuclear antigen; PVDF, polyvinylidene difluoride; RPA, replication protein A; ssDNA, single strand DNA. 1The abbreviations used are: WS, Werner syndrome; dsDNA, double strand DNA; EXO-1, exonuclease 1; FEN-1, 5′-flap endonuclease/5′ to 3′-exonuclease; nt, nucleotide; PCNA, proliferating cell nuclear antigen; PVDF, polyvinylidene difluoride; RPA, replication protein A; ssDNA, single strand DNA. is a hereditary premature aging disorder characterized by genome instability (1Martin G.M. Birth Defects Orig. Artic. Ser. 1978; 14: 5-39Google Scholar). WS cells display elevated chromosomal aberrations (2Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Google Scholar, 3Salk D. Bryant E. Hoehn H. Johnston P. Martin G.M. Adv. Exp. Med. Biol. 1985; 190: 305-311Google Scholar, 4Fukuchi K. Martin G.M. Monnat R.J.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Google Scholar), replication defects (3Salk D. Bryant E. Hoehn H. Johnston P. Martin G.M. Adv. Exp. Med. Biol. 1985; 190: 305-311Google Scholar, 5Martin G.M. Sprague C.A. Epstein C.J. Lab. Invest. 1970; 23: 86-92Google Scholar, 6Takeuchi F. Hanaoka F. Goto M. Akaoka I. Hori T. Yamada M. Miyamoto T. Hum. Genet. 1982; 60: 365-368Google Scholar, 7Hanaoka F. Yamada M. Takeuchi F. Goto M. Miyamoto T. Hori T. Adv. Exp. Med. Biol. 1985; 190: 439-457Google Scholar, 8Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell Res. 1992; 202: 267-273Google Scholar), abnormal recombination (9Cheng R.Z. Murano S. Kurz B. Shmookler R.R. Mutat. Res. 1990; 237: 259-269Google Scholar, 10Prince P.R. Emond M.J. Monnat R.J.J. Genes Dev. 2001; 15: 933-938Google Scholar), altered telomere dynamics (11Schulz V.P. Zakian V.A. Ogburn C.E. McKay J. Jarzebowicz A.A. Edland S.D. Martin G.M. Hum. Genet. 1996; 97: 750-754Google Scholar), and hypersensitivity to DNA-damaging agents (12Ogburn C.E. Oshima J. Poot M. Chen R. Hunt K.E. Gollahon K.A. Rabinovitch P.S. Martin G.M. Hum. Genet. 1997; 101: 121-125Google Scholar, 13Lebel M. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13097-13102Google Scholar, 14Poot M. Gollahon K.A. Rabinovitch P.S. Hum. Genet. 1999; 104: 10-14Google Scholar, 15Pichierri P. Franchitto A. Mosesso P. Palitti F. Mutat. Res. 2000; 456: 45-57Google Scholar, 16Poot M. Yom J.S. Whang S.H. Kato J.T. Gollahon K.A. Rabinovitch P.S. FASEB J. 2001; 15: 1224-1226Google Scholar). The gene defective in WS, designated WRN (17Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Google Scholar), encodes a protein with DNA helicase (18Gray 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: 100-103Google Scholar, 19Suzuki N. Shimamoto A. Imamura O. Kuromitsu J. Kitao S. Goto M. Furuichi Y. Nucleic Acids Res. 1997; 25: 2973-2978Google Scholar) and exonuclease (20Shen J.C. Gray M.D. Oshima J. Kamath-Loeb A.S. Fry M. Loeb L.A. J. Biol. Chem. 1998; 273: 34139-34144Google Scholar, 21Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Google Scholar, 22Kamath-Loeb A.S. Shen J.C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Google Scholar) activities which presumably functions in DNA metabolism to preserve genome integrity. To understand the basis of WS, a number of groups have investigated WRN protein interactions (for review, see Ref. 23Brosh Jr., R.M. Bohr V.A. Exp. Gerontol. 2002; 37: 491-506Google Scholar). The collective work indicates that WRN interacts functionally with proteins implicated in replication and DNA repair including replication protein A (RPA), p53, Ku, and polymerase δ. These studies have enabled researchers to speculate about pathways of DNA metabolism in which WRN might participate; however, the precise functions of the WRN gene product in vivo are not well understood. Recently we reported a novel interaction of WRN protein with the human 5′-flap endonuclease/5′ to 3′-exonuclease (FEN-1) (24Brosh 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: 5791-5801Google Scholar), a DNA structure-specific nuclease implicated in DNA replication, recombination, and repair (25Lieber M.R. Bioessays. 1997; 19: 233-240Google Scholar). WRN protein stimulates FEN-1 cleavage activity by a physical interaction with a COOH-terminal domain of the WRN protein (24Brosh 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: 5791-5801Google Scholar). Another member of the RAD2 family to which FEN-1 belongs is human exonuclease 1 (EXO-1) (26Wilson D.M. Carney J.P. Coleman M.A. Adamson A.W. Christensen M. Lamerdin J.E. Nucleic Acids Res. 1998; 26: 3762-3768Google Scholar). Like FEN-1, EXO-1 is a structure-specific endonuclease as well as an exonuclease (27Lee B.I. Wilson D.M. J. Biol. Chem. 1999; 274: 37763-37769Google Scholar, 28Tran P.T. Erdeniz N. Dudley S. Liskay R.M. DNA Repair. 2002; 1: 895-912Google Scholar). EXO-1 has been implicated in a number of DNA metabolic pathways including mismatch correction, mitotic and meiotic recombination, and double strand break repair (29Fiorentini P. Huang K.N. Tishkoff D.X. Kolodner R.D. Symington L.S. Mol. Cell. Biol. 1997; 17: 2764-2773Google Scholar, 30Qiu J. Qian Y. Chen V. Guan M.X. Shen B. J. Biol. Chem. 1999; 274: 17893-17900Google Scholar, 31Tsubouchi H. Ogawa H. Mol. Biol. Cell. 2000; 11: 2221-2233Google Scholar, 32Moreau S. Morgan E.A. Symington L.S. Genetics. 2001; 159: 1423-1433Google Scholar, 33Schmutte C. Sadoff M.M. Shim K.S. Acharya S. Fishel R. J. Biol. Chem. 2001; 276: 33011-33018Google Scholar, 34Lewis L.K. Karthikeyan G. Westmoreland J.W. Resnick M.A. Genetics. 2002; 160: 49-62Google Scholar). A role for EXO-1 in Okazaki fragment processing during DNA replication has been suggested based on its structure-specific endonuclease and RNase H activities that are similar to those of FEN-1 (30Qiu J. Qian Y. Chen V. Guan M.X. Shen B. J. Biol. Chem. 1999; 274: 17893-17900Google Scholar). Functional overlap between EXO-1 and FEN-1 (Rad27 in Saccharomyces cerevisiae) has been proposed from observations in yeast that exoI: rad27 double mutants are inviable (35Tishkoff D.X. Boerger A.L. Bertrand P. Filosi N. Gaida G.M. Kane M.F. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7487-7492Google Scholar) and that overexpression of yeast EXO-1 or human EXO-1 complements cellular phenotypes of rad27 mutants (30Qiu J. Qian Y. Chen V. Guan M.X. Shen B. J. Biol. Chem. 1999; 274: 17893-17900Google Scholar, 35Tishkoff D.X. Boerger A.L. Bertrand P. Filosi N. Gaida G.M. Kane M.F. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7487-7492Google Scholar, 36Tishkoff D.X. Amin N.S. Viars C.S. Arden K.C. Kolodner R.D. Cancer Res. 1998; 58: 5027-5031Google Scholar). The evident sequence homology and similar biochemical activities of EXO-1 and FEN-1, as well as the potential functional redundancy of the two nucleases, suggested to us that WRN might also interact functionally with EXO-1. We have found this to be the case. Evidence is presented that WRN interacts physically with human EXO-1 and stimulates the endonucleolytic and exonucleolytic cleavage activities of EXO-1. The functional interaction is independent of WRN catalytic function and mediated by a COOH-terminal region of WRN which also interacts with FEN-1. Thus, WRN modulates the cleavage activities of both human EXO-1 and FEN-1 by a direct protein interaction, suggesting that either structure-specific nuclease may act together with WRN during DNA replication, recombination, or repair. The physical and functional interaction of WRN with these human nucleases is likely to be important for the cellular role of WRN in the maintenance of genome integrity. Proteins—Hexahistidine-tagged recombinant human EXO-1 protein was overexpressed using a baculovirus/insect system and purified as described elsewhere. 2L. A. Uzdilla, J. P. Carney, P. Hungspreugs, D. M. Wilson, and T. M. Wilson, manuscript in preparation. The purified EXO-1 protein was judged to be >97% pure from analysis on Coomassie-stained SDS-polyacrylamide gels (see Fig. 2A). Baculovirus constructs for full-length recombinant hexahistidine-tagged WRN proteins (wild-type, WRN-K577M, WRN-E84A) or a truncated version of WRN containing only the amino-terminal 368 amino acids of the protein (designated N-WRN1–368) were kindly provided by Drs. M. Gray (University of Washington, Seattle) and J. Campisi (Lawrence Berkeley National Laboratory, Berkeley, CA). Amplified WRN-encoding baculovirus was used to infect Sf9 cells for overexpression of WRN protein as described elsewhere (37Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Google Scholar). A recombinant hexahistidine-tagged carboxyl-terminal fragment of WRN (residues 940–1432, designated C-WRN940–1432) was overexpressed in Escherichia coli and purified as described previously (38Cooper M.P. Machwe A. Orren D.K. Brosh Jr., R.M. Ramsden D. Bohr V.A. Genes Dev. 2000; 14: 907-912Google Scholar). Recombinant human FEN-1 was purified as described previously (24Brosh 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: 5791-5801Google Scholar). Human PCNA and RPA were graciously provided by Dr. M. Kenny (Albert Einstein College of Medicine, Bronx, NY). WRN-EXO-1 Coimmunoprecipitation Experiments—HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. Nuclear extract was prepared as described previously (39Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar). For coimmunoprecipitation, HeLa nuclear extract (1.5 mg of protein) was incubated with goat anti-WRN polyclonal antibody (1:40; Santa Cruz Biotechnology) in buffer D (50 mm HEPES pH 7.5, 100 mm KCl, 10% glycerol) for 4 h at 4 °C and tumbled with 20 μl of protein G-agarose (Roche Applied Science) at 4 °C overnight. Beads were washed three times with buffer D supplemented with 0.1% Tween 20. Proteins were eluted by boiling treatment in SDS sample buffer, resolved on 8% polyacrylamide Tris-glycine SDS gels, and transferred to PVDF membranes (Amersham Biosciences). The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20 and probed using rabbit polyclonal antibody against a recombinant human EXO-1 nuclease domain fragment characterized previously (27Lee B.I. Wilson D.M. J. Biol. Chem. 1999; 274: 37763-37769Google Scholar) (1:5,000; courtesy of Dr. D. M. Wilson III (NIA, NIH)) followed by goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) or probed for WRN using mouse anti-WRN monoclonal antibody (1:250; BD Pharmingen) followed by goat-anti-mouse IgG-horseradish peroxidase (Vector Laboratories). EXO-1 or WRN on immunoblot was detected using ECL Plus (Amersham Biosciences). Coimmunoprecipitation of purified WRN and EXO-1 was performed in the presence of binding buffer (50 mm Tris, pH 8.0, 10% glycerol, 100 mm NaCl, 0.01% Nonidet P-40) containing protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 5 μg/ml leupeptin). In a 100-μl reaction volume, 500 ng of purified WRN and 500 ng of EXO-1 were incubated with goat anti-WRN polyclonal antibody (1:40) in binding buffer for 4 h at 4 °C. The protein complex was adsorbed on to protein G-agarose beads by incubating the mixture overnight at 4 °C with gentle rotation. The beads were washed three times with binding buffer, eluted by boiling with SDS sample buffer, and resolved on 8–16% gradient SDS-polyacrylamide Tris-glycine gels. After transferring the proteins to PVDF membranes, the membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20 and probed with either anti-EXO-1 or anti-WRN antibodies as described above. Far Western Blotting—Far Western blotting was conducted as described previously (40Brosh 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: 23500-23508Google Scholar). Briefly, 0.2–1.0 μg of each protein was electrophoresed on 8–16% SDS-polyacrylamide gels, transferred to PVDF membranes, and processed as described previously (40Brosh 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: 23500-23508Google Scholar) with the exception that specified blocked membranes were incubated in the presence of 0.5 μg/ml EXO-1. Membranes were washed, and conventional Western analysis was then performed to detect the presence of EXO-1 using rabbit anti-EXO-1 polyclonal antibody (described above) (1:5,000). Goat anti-rabbit IgG-horseradish peroxidase conjugate was used as secondary antibody at a 1:10,000 dilution and detected using ECL Plus. Oligonucleotide Substrates—PAGE-purified oligonucleotides (Midland Certified Reagent Co.) (Table I) were used for preparation of substrates. Substrates were prepared as described previously (41Tom S. Henricksen L.A. Bambara R.A. J. Biol. Chem. 2000; 275: 10498-10505Google Scholar). Briefly, 10 pmol of the appropriate flap oligonucleotide was 5′-radiolabeled with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs) using the manufacturer's protocol. Unincorporated nucleotide was removed using a Sephadex G-25 spin column (Amersham Biosciences). The radiolabeled oligonucleotide was annealed to 25 pmol of the appropriate template oligonucleotide by heating at 95 °C, then cooling down from 70 to 24 °C. If necessary, 50 pmol of an upstream oligonucleotide was then annealed to the duplex substrate by heating at 37 °C for 1 h and slowly cooling to 24 °C. Two nicked duplex substrates were prepared: 1) nicked duplex A using FLAP00A, TSTEM, and U25; and 2) nicked duplex B using FLAP00B, TSTEM2, and U21. The monodeoxynucleotide (nt) flap was prepared with FLAP01, TSTEM, and U25. For the monoribodeoxynucleotide flap, FLAP01RNA, TSTEM, and U25 were used. The 10-nt flap was created with FLAP10, TSTEM2, and U21. The blunt ended duplex substrate was made using TSTEM-COMP and TSTEM.Table IOligonucleotide sequences for DNA substrates (5′ to 3′)TemplateLengthSequencentTSTEM44GCACTGGCCGTCGTTTTACGGTCGTGACTGGGAAAACCCTGGCGTSTEM242CCAGTGAATTCGAGCTCGGTACCCGCTAGCGGGGATCCTCTAFlapsFLAP00A19GTAAAACGACGGCCAGTGCFLAP00B21TACCGAGCTCGAATTCACTGGFLAP0120AGTAAAACGACGGCCAGTGCFLAP01RNA20AGTAAAACGACGGCCAGTGCFLAP1031ATTGGTTATTTACCGAGCTCGAATTCACTGGPrimersTSTEMCOMP44CGCCAGGGTTTTCCCAGTCACGACCGTAAAACGACGGCCAGTGCU2121TAGAGGATCCCCGCTAGCGGGU2424CGCCAGGGTTTTCCCAGTCACGACU2525CGCCAGGGTTTTCCCAGTCACGACC Open table in a new tab For the nicked duplex substrate with a 3′-end label on the upstream primer, 10 pmol of U24 was annealed to 25 pmol of TSTEM and end labeled with [α-32P]dCTP and Klenow fragment (New England Biolabs) at 25 °C for 20 min followed by an additional incubation for 20 min at 25 °C with 50 μm unlabeled dCTP. Unincorporated nucleotide was removed by passing over two Sephadex G-25 columns. Klenow was then heat inactivated at 95 °C followed by slow cooling to permit the oligonucleotides to reanneal. 50 pmol of FLAP00 was then added and annealed by heating at 37 °C for 1 h followed by slow cooling. EXO-1 Incision Assay—20-μl reactions contained 0.5 nm DNA substrate (unless otherwise noted) and the indicated concentrations of WRN and/or EXO-1 in 30 mm HEPES pH 7.6, 5% glycerol, 40 mm KCl, 0.1 mg/ml bovine serum albumin, and 8 mm MgCl2. WRN was mixed with the substrate on ice prior to the addition of EXO-1. Reactions were incubated at 37 °C for 15 min (unless indicated otherwise), terminated with the addition of 10 μl of formamide dye (80% formamide (v/v), 0.1% bromphenol blue, and 0.1% xylene cyanol), and heated to 95 °C for 5 min. Products were resolved on 20% polyacrylamide, 7 m urea denaturing gels. A PhosphorImager was used for detection, and the ImageQuant software (Molecular Dynamics) was used for quantitation of the reaction products. The percent incision was calculated as described previously (24Brosh 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: 5791-5801Google Scholar). WRN Helicase Assay—20-μl reactions contained 0.5 nm 10-nt 5′-flap DNA substrate (except where indicated) and the indicated concentrations of WRN and/or EXO-1 in the same reaction buffer as used for EXO-1 incision assays except for the additional presence of 2 mm ATP. Reactions were incubated at 37 °C for 15 min, terminated with the addition of 20 μl of helicase stop solution (50 mm EDTA, 40% glycerol, 0.1% bromphenol blue, 0.1% xylene cyanol) containing a 10-fold excess of unlabeled oligonucleotide with the same sequence as the labeled strand. The products of the helicase reactions were resolved on nondenaturing 12% polyacrylamide gels. Radiolabeled DNA species in polyacrylamide gels were visualized using a PhosphorImager and quantitated using the ImageQuant software. The percent helicase substrate unwound was calculated as described previously (42Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Google Scholar). Physical Interaction between WRN and EXO-1—The previously reported physical interaction between WRN and FEN-1 (24Brosh 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: 5791-5801Google Scholar) suggested to us that EXO-1, a RAD2 family member and structure-specific nuclease like FEN-1 (27Lee B.I. Wilson D.M. J. Biol. Chem. 1999; 274: 37763-37769Google Scholar, 28Tran P.T. Erdeniz N. Dudley S. Liskay R.M. DNA Repair. 2002; 1: 895-912Google Scholar), might interact directly with WRN. To explore this possibility, we tested for the coimmunoprecipitation of WRN and EXO-1 from human nuclear extracts (Fig. 1). Using 200 μg of protein from HeLa nuclear extract, we detected EXO-1 (Fig. 1, lane 1) that comigrated with the purified recombinant human EXO-1 used in this study (Fig. 1, lane 4) by Western blot analysis. An antibody directed against WRN protein reproducibly immunoprecipitated EXO-1 from 1.5 mg of HeLa nuclear extract as detected by Western blot with an anti-EXO-1 antibody (Fig. 1, lane 2). The immunoprecipitation of WRN from HeLa nuclear extract with anti-WRN antibody was verified by Western blot analysis using anti-WRN antibody (data not shown). Preimmune IgG failed to precipitate either EXO-1 (lane 3) or WRN (data not shown) in control experiments. These results provide evidence that WRN and EXO-1 are associated with each other in human nuclei, suggesting that the two proteins may physically interact with each other. To explore the specificity and nature of the suggested WRN-EXO-1 interaction, coimmunoprecipitation experiments were performed with the purified recombinant proteins. Using an antibody directed against WRN, both WRN and EXO-1 were immunoprecipitated (Fig. 1, B and C, lane 5). WRN and EXO-1 were also coimmunoprecipitated in the presence of ethidium bromide confirming that the protein interaction is not mediated through DNA (Fig. 1, B and C, lane 6). Approximately 20% of the input EXO-1 protein was precipitated by the WRN antibody. The purified proteins failed to be immunoprecipitated by goat preimmune serum (Fig. 1, B and C, lane 4) or in the absence of antibody (Fig. 1, B and C, lane 2). In control experiments, EXO-1 failed to be immunoprecipitated by anti-WRN antibody when WRN protein was omitted from the binding mixture (Fig. 1, B and C, lane 3), attesting to the specificity of the WRN antibody used for the coimmunoprecipitation studies. These results indicate a direct physical interaction between WRN and EXO-1 and support the evidence that WRN and EXO-1 interact with each other in vivo. We next sought to determine whether the COOH-terminal region of WRN responsible for the physical and functional interaction with FEN-1 also interacted physically with EXO-1. To test for this interaction, we performed far Western blotting experiments. For these studies, we tested the ability of full-length WRN protein, a COOH-terminal fragment of WRN (C-WRN940–1432), and an NH2-terminal fragment of WRN (N-WRN1–368) to bind EXO-1. These proteins are shown in a Coomassie-stained SDS-polyacrylamide gel in Fig. 2A. The proteins were resolved on SDS-polyacrylamide gels and transferred to PVDF membranes that were subsequently blocked, incubated in buffer alone or buffer containing EXO-1, and washed extensively. The blots were then probed with an antibody against EXO-1. As shown in Fig. 2B, EXO-1 bound to full-length WRN and the COOH-terminal fragment of WRN (lanes 1 and 2) but did not bind to the NH2-terminal fragment of WRN (lane 3). In the control experiment, when a similar membrane was incubated with buffer lacking EXO-1 and probed with anti-EXO-1 antibody, no bands corresponding to the position of full-length WRN, COOH-terminal or NH2-terminal WRN were detected (Fig. 2C, lanes 1, 2, and 3). In control experiments, two faint bands (fast and slow migrating) were detected in lane 4 containing bovine serum albumin, but this signal was caused by nonspecific reactivity of the EXO-1 antibody, as these bands were also detected on membranes that were incubated in the presence or absence of EXO-1 protein. These results together with the immunoprecipitation experiments demonstrate that WRN and EXO-1 interact physically and that the COOH-terminal region of the WRN protein alone is capable of binding to EXO-1. WRN Stimulates EXO-1 Cleavage of a 1-Nucleotide 5′-Flap Substrate—The physical interaction between WRN and EXO-1 and the ability of WRN to stimulate FEN-1 cleavage by a protein interaction (24Brosh 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: 5791-5801Google Scholar) suggested that WRN might stimulate the incision activities of human EXO-1. Under the same reaction conditions, a 1-nt 5′-flap substrate was susceptible to EXO-1 cleavage that generated the 1-nt product (Fig. 3A, lane 2), whereas FEN-1 cleavage of the same substrate resulted primarily in the 2 nt product, and to a lesser extent the 1-nt product (Fig. 3A, lane 6), consistent with previous observations (24Brosh 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: 5791-5801Google Scholar). In the presence of WRN (4 nm), both EXO-1 and FEN-1 cleavage reactions were stimulated to yield the same respective cleavage products (Fig. 3A, lanes 3 and 7). Using a limiting concentration of EXO-1 (0.125 nm), the cleavage reaction was stimulated 8-fold by 8 nm WRN (Fig. 3B, lanes 5 and 6, and 3C). Using a 2-fold higher concentration of EXO-1 (0.25 nm), 10% of the DNA substrate was cleaved (Fig. 3B, lane 8, and 3C). At this EXO-1 level, WRN stimulated EXO-1 cleavage to 43% of the substrate incised (Fig. 3B, lane 9, and 3C). In comparison, 0.25 nm EXO-1 did not appreciably incise the 5′-32P end-labeled blunt duplex 44-bp substrate, and the presence of 8 nm WRN resulted in only a small stimulation of incision activity (∼1.5%) using 0.25 nm EXO-1; however, at a higher concentration of EXO-1 (1 nm), the blunt duplex DNA substrate was degraded from its 5′-end, and the EXO-1 reaction was stimulated by WRN in a dose-dependent manner (data not shown). These results indicate that EXO-1 incision at the internally positioned 1-nt flap was primarily responsible for the removal of the unpaired nucleotide rather than 5′ to 3′-exonuclease digestion from the end of the blunt duplex substrate. WRN stimulation of EXO-1 cleavage of the 1-nt flap substrate was also observed in reactions containing 0.5 nm EXO-1 (Fig. 3B, lanes 10 and 11), although the level of stimulation was not as great because a plateau of incision activity (55%) was approached in the reactions containing EXO-1 and WRN (Fig. 3C). 8 nm WRN alone did not catalyze significant cleavage of the 1-nt flap DNA substrate (Fig. 3B, lane 12), consistent with previous observations (24Brosh 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: 5791-5801Google Scholar). We also observed a significant stimulation of EXO-1 cleavage of a single ribonucleotide 5′-flap substrate in the presence of WRN using a limiting amount of EXO-1 (0.125 nm). 3S. Sharma, J. A. Sommers, H. C. Driscoll, L. Uzdilla, T. M. Wilson, and R. M. Brosh, Jr., unpublished data. We subsequently studied EXO-1 cleavage as a function of WRN concentration. A limiting amount of EXO-1 (0.125 nm) was used such that cleavage of the 1-nt flap substrate was very low (∼3%) (Fig. 3D). A 3-fold stimulation of EXO-1 cleavage was detected at a WRN concentration of 2 nm (Fig. 3D). In the presence of 4 nm WRN, EXO-1 cleavage increased to 22% incision (Fig. 3D), a 7-fold stimulation of EXO-1 cleavage in the 15-min incubation. At 8 nm WRN, product formation began to plateau at ∼30% (Fig. 3D). Kinetic analysis of the EXO-1 cleavage reaction on the 1-nt 5′-flap DNA substrate demonstrated that the presence of WRN profoundly affected the rate of EXO-1 incision (Fig. 4). In these studies, we used a concentration of WRN (8 nm) which was previously determined to achieve maximal stimulation of EXO-1 cleavage (Fig. 3D). The concentration of EXO-1 (0.125 nm) used resulted in a low, but reproducibly detectable incision of ∼2% of the 0.5 nm DNA substrate in a 15-min reaction in the absence of WRN (Fig. 3D). Stimulation of EXO-1 incision by WRN was detected at time points as short as 1–2 min (Fig. 4). Up to 9 min, EXO-1 cleavage in the absence of WRN was ∼1%; however, in the presence of WRN, EXO-1 incised 19% of the DNA substrate at the 9 min time point. Regression analysis of the linear regions of the slopes yielded reaction rates of 25.9 and 0.7 pmol of product/min for the WRN + EXO-1 and EXO-1 reactions, respectively. This difference represented a 37-fold increase in the rate of EXO-1 cleavage when WRN is present. At 15 min, the EXO-1 cleavage reaction conducted in the presence of WRN achieved a plateau of ∼28% substrate incised. In contrast, EXO-1 alone cleaved only 1.5% of the substrate by the end of 15 min. These results demonstrate conclusively that WRN stimulates the rate of EXO-1 cleavage. Catalytic Activities of WRN Are Not Required for Stimulation of EXO-1 Incision—Our previous work demonstrated that a protein domain of WRN, devoid of catalytic activities and which interacts physically with FEN-1, mediated the functional interaction between WRN and FEN-1 (24Brosh 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: 5791-5801Google Scholar). However, it was conceivable that WRN might affect the cleavage activity of EXO-1 by a mechanism distinct from that of the WRN-FEN-1 interaction. The three catalytic activities of WRN (ATPase, helicase, exonuclease) might influence the functional interaction with EXO-1. To address this possibility, we tested the effects of full-length recombinant WRN proteins with site-directed mutations in the active sites of its catalytic domains on EXO-1 cleavage. WRN-K577M mutant protein, devoid of ATPase or helicase activity (18Gray 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: 100-103Google Scholar, 43Brosh 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: 18341-18350Google Scholar), was capable of stimulating EXO-1 cleavage of the 1-nt flap DNA substrate similarly to
Referência(s)