Stimulation of Flap Endonuclease-1 by the Bloom's Syndrome Protein
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m309898200
ISSN1083-351X
AutoresSudha Sharma, Joshua A. Sommers, Leonard Wu, Vilhelm A. Bohr, Ian D. Hickson, Robert Brosh,
Tópico(s)Cancer-related Molecular Pathways
ResumoBloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5′-flap endonuclease/5′-3′ exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair. Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5′-flap endonuclease/5′-3′ exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair. Bloom's syndrome (BS) 1The abbreviations used are: BS, Bloom's syndrome; MBP, maltose-binding protein; BSA, bovine serum albumin; EtBr, ethidium bromide; ELISA, enzyme-linked immunosorbent assay; ss, single-stranded; nt, nucleotide.1The abbreviations used are: BS, Bloom's syndrome; MBP, maltose-binding protein; BSA, bovine serum albumin; EtBr, ethidium bromide; ELISA, enzyme-linked immunosorbent assay; ss, single-stranded; nt, nucleotide. is a rare genetic disorder characterized by growth deficiency, a sun-sensitive facial erythema, hypo- and hyperpigmented skin lesions, immunodeficiency, sub-fertility in females and infertility in males, susceptibility to diabetes, and a predisposition to the development of cancers of most types (1German J. Ellis N.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Molecular and Metabolic Basis of Inherited Disease. McGraw Hill, New York2000: 733-752Google Scholar). Cells from BS patients exhibit hyper-recombination (2Chaganti R.S. Schonberg S. German J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4508-4512Crossref PubMed Scopus (778) Google Scholar) and abnormalities in DNA replication that include an extended S phase and accumulation of an abnormal profile of replication intermediates compared with normal cells (3Gianneli F. Benson P.F. Pawsey S.A. Polani P.E. Nature. 1977; 265: 466-469Crossref PubMed Scopus (131) Google Scholar, 4Lonn U. Lonn S. Nylen U. Winblad G. German J. Cancer Res. 1990; 50: 3141-3145PubMed Google Scholar). BS cells exhibit chromosomal instability characterized by elevated rates of sister chromatid exchanges (SCEs), insertions, deletions, loss of heterozygosity, telomere associations, and quadriradials (for review, see Ref. 5Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). Despite the interest in the molecular pathology of BS, the fundamental defects underlying the genomic instability and cellular dysfunction characteristic of the disease are poorly understood.The gene mutated in BS, designated BLM, encodes a 1417 amino acid protein with a central region (residues 649–1000) containing seven conserved motifs characteristic of DNA helicases (6Ellis 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 (1204) Google Scholar). The region of BLM that contains these seven motifs exhibits ∼40% homology to other proteins in the RecQ family of DNA helicases. A 150 amino acid region of extended homology C-terminal to the helicase domain is found in most, but not all, RecQ helicases. Other members of the RecQ helicase family include Escherichia coli RecQ (7Nakayama K. Irino N. Nakayama H. Mol. Gen. Genet. 1985; 200: 266-271Crossref PubMed Scopus (123) Google Scholar), Saccharomyces cerevisiae Sgs1 (8Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (616) Google Scholar, 9Watt P.M. Louis E.J. Borts R.H. Hickson I.D. Cell. 1995; 81: 253-260Abstract Full Text PDF PubMed Scopus (376) Google Scholar), Schizosaccharomyces pombe Rqh1 (9Watt P.M. Louis E.J. Borts R.H. Hickson I.D. Cell. 1995; 81: 253-260Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 10Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (326) Google Scholar), and five human homologs (BLM, WRN, RecQ4, RecQ5, and RecQL) (5Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). Of the human RecQ homologs, three, including BLM, are associated with genetic disorders characterized by chromosomal instability. The WRN gene is mutated in Werner syndrome, a premature aging disorder with elevated cancer risk (11Yu 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: 258-262Crossref PubMed Scopus (1479) Google Scholar). Mutations in the RecQ4 gene have been found in persons with Rothmund-Thomson syndrome, another rare premature aging and cancer-predisposition disorder (12Kitao S. Shimamoto A. Goto M. Miller R.W. Smithson W.A. Lindor N.M. Furuichi Y. Nat. Genet. 1999; 22: 82-84Crossref PubMed Scopus (566) Google Scholar). The linkage of mutations in human RecQ helicases with genomic instability, cancer, and premature aging suggests that this class of enzymes have important caretaker roles in specialized pathways of DNA metabolism (5Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar).The BLM protein is a DNA-dependent ATPase and unwinds B-form DNA in a 3′ to 5′ direction with respect to the strand that the helicase is presumed to translocate on (13Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). BLM is able to unwind an array of DNA structures including recombination intermediates (Holliday Junctions and D-loops) (14Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (418) Google Scholar, 15van Brabant A.J. Ye T. Sanz M. German III, J.L. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Crossref PubMed Scopus (196) Google Scholar) and alternate DNA structures (triplexes and tetraplexes) (16Brosh Jr., R.M. Majumdar A. Desai S. Hickson I.D. Bohr V.A. Seidman M.M. J. Biol. Chem. 2000; 276: 3024-3030Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 17Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Electron microscopic studies have provided evidence that BLM forms oligomeric ring-like structures in solution (18Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); however, the assembly state of BLM (and other RecQ helicases) that is important for DNA unwinding activity is open to debate, since recent evidence indicated that a catalytically active, truncated form of BLM was monomeric (19Janscak P. Garcia P.L. Hamburger F. Makuta Y. Shiraishi K. Imai Y. Ikeda H. Bickle T.A. J. Mol. Biol. 2003; 330: 29-42Crossref PubMed Scopus (116) Google Scholar). BLM has been shown to interact physically and/or functionally with a number of proteins (p53, Refs. 20Sengupta S. Linke S.P. Pedeux R. Yang Q. Farnsworth J. Garfield S.H. Valerie K. Shay J.W. Ellis N.A. Wasylyk B. Harris C.C. EMBO J. 2003; 22: 1210-1222Crossref PubMed Scopus (184) Google Scholar and 21Yang Q. Zhang R. Wang X.W. Spillare E.A. Linke S.P. Subramanian D. Griffith J.D. Li J.L. Hickson I.D. Shen J.C. Loeb L.A. Mazur S.J. Appella E. Brosh Jr., R.M. Karmakar P. Bohr V.A. Harris C.C. J. Biol. Chem. 2002; 277: 31980-31987Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar; WRN, Ref. 22von 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; MLH-1, Refs. 23Langland G. Kordich J. Creaney J. Goss K.H. Lillard-Wetherell K. Bebenek K. Kunkel T.A. Groden J. J. Biol. Chem. 2001; 276: 30031-30035Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar and 24Pedrazzi G. Perrera C. Blaser H. Kuster P. Marra G. Davies S.L. Ryu G.H. Freire R. Hickson I.D. Jiricny J. Stagljar I. Nucleic Acids Res. 2001; 29: 4378-4386Crossref PubMed Scopus (94) Google Scholar; RAD51, Ref. 25Wu L. Davies S.L. Levitt N.C. Hickson I.D. J. Biol. Chem. 2001; 276: 19375-19381Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar; RPA, Ref. 26Brosh 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 (254) Google Scholar; and TOPO3α, Refs. 27Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar, 28Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9639-9644Google Scholar, 29Wu L. Hickson I.D. Nucleic Acids Res. 2002; 30: 4823-4829Crossref PubMed Google Scholar) involved in various aspects of DNA metabolism (30Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (310) Google Scholar). In addition, BLM resides in complexes containing caretaker proteins that include BRCA1, ATM, MRE11-RAD50-NBS1, and Fanconi anemia (FA) proteins (31Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (95) Google Scholar, 32Meetei A.R. Sechi S. Wallisch M. Yang D. Young M.K. Joenje H. Hoatlin M.E. Wang W. Mol. Cell. Biol. 2003; 23: 3417-3426Crossref PubMed Scopus (291) Google Scholar). Based on the diverse set of DNA substrates upon which BLM can act and the multiple protein interactions involving BLM, it seems likely that BLM protein may serve multiple roles in DNA metabolism in vivo.Recently, we characterized interactions of WRN protein with two human members of the Rad2 nuclease family, FEN-1 (33Brosh Jr., R.M. Driscoll H.C. Dianov G.L. Sommers J.A. Biochemistry. 2002; 41: 12204-12216Crossref PubMed Scopus (64) Google Scholar, 34Brosh 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), and EXO-1 (35Sharma S. Sommers J.A. Driscoll H.C. Uzdilla L. Wilson T.M. Brosh Jr., R.M. J. Biol. Chem. 2003; 278: 23487-23496Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Both of these enzymes are DNA structure-specific nucleases (36Lee B.I. Wilson D.M. J. Biol. Chem. 1999; 274: 37763-37769Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 37Lieber M.R. Bioessays. 1997; 19: 233-240Crossref PubMed Scopus (395) Google Scholar, 38Tran P.T. Erdeniz N. Dudley S. Liskay R.M. DNA Repair. 2002; 1: 895-912Crossref PubMed Scopus (98) Google Scholar) that have partially, but not completely, overlapping roles in DNA replication, recombination, and repair (39Qiu J. Qian Y. Chen V. Guan M.X. Shen B. J. Biol. Chem. 1999; 274: 17893-17900Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 40Sun X. Thrower D. Qiu J. Wu P. Zheng L. Zhou M. Bachant J. Wilson D.M. Shen B. DNA Repair (Amst.). 2003; 2: 925-940Crossref PubMed Scopus (22) Google Scholar, 41Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 42Tishkoff D.X. Amin N.S. Viars C.S. Arden K.C. Kolodner R.D. Cancer Res. 1998; 58: 5027-5031PubMed Google Scholar). WRN stimulates FEN-1 cleavage activity by a physical interaction with a C-terminal domain of the WRN protein that shares sequence homology with the BLM protein (34Brosh 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). The sequence homology between BLM and WRN in the region that mediates the physical and functional interaction between WRN and FEN-1/EXO-1 may point to a similar biochemical function of BLM to stimulate the Rad2 nuclease cleavage reaction. One hypothesis is that WRN or BLM may share responsibility of functioning with the Rad2 nucleases during replication to ensure efficient and accurate processing of specific DNA structures that arise during DNA synthesis. Alternatively, WRN and BLM may have separate roles in their interactions with cellular DNA nucleases, such as FEN-1 or EXO-1, that contribute to their unique forms of genomic instability as well as their distinctive cellular and clinical phenotypes. These two hypotheses are not mutually exclusive.FEN-1 haploinsufficiency in mice can lead to tumor progression (43Kucherlapati M. Yang K. Kuraguchi M. Zhao J. Lia M. Heyer J. Kane M.F. Fan K. Russell R. Brown A.M. Kneitz B. Edelmann W. Kolodner R.D. Lipkin M. Kucherlapati R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9924-9929Crossref PubMed Scopus (211) Google Scholar), suggesting that FEN-1, like BLM, is a tumor suppressor (44Henneke G. Friedrich-Heineken E. Hubscher U. Trends Biochem. Sci. 2003; 28: 384-390Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The cancer predisposition and replication defects of BS may be at least partially explained by the lack of a BLM-FEN-1 interaction, since FEN-1 has an essential role in DNA replication and repair. Evidence is presented in this study that BLM and FEN-1 are associated in a complex in human cells as demonstrated by their reciprocal co-immunoprecipitation. Purified recombinant BLM protein interacts physically with human FEN-1, and stimulates FEN-1 cleavage activities on 5′-flap and nicked duplex DNA substrates that are proposed intermediates in DNA replication and repair. The functional interaction is mediated by a C-terminal region of BLM that shares homology with the domain of WRN responsible for the physical/functional interaction with FEN-1/EXO-1. The physical and functional interaction of human RecQ helicases, including BLM and WRN, with human Rad2 nucleases is likely to be important for the roles of these proteins in the maintenance of genome stability.MATERIALS AND METHODSProteins—Hexahistidine-tagged recombinant human BLM protein was overexpressed in S. cerevisiae and purified as described elsewhere (18Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). MBP fusion peptides of BLM were expressed in BL21 (DE3) cells and purified as described previously (28Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9639-9644Google Scholar). Recombinant histidine-tagged WRN protein was overexpressed using a baculovirus/Sf9 insect system and purified as described elsewhere (34Brosh 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).FEN-1, encoded by a plasmid kindly provided by Dr. M. Lieber (University of Southern California), was overexpressed in E. coli by isopropyl-1-thio-β-d-galactopyranoside (0.5 mm) induction of mid-log phase cells for 4 h at 37 °C. The bacterial cell pellet (10 g) containing overexpressed FEN-1 protein was resuspended in 50 ml of FEN-1 Purification Buffer (25 mm HEPES-KOH, pH 7.9, 100 mm KCl, 10% glycerol) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 2 μg/ml chymostatin, 2 μg/ml pepstatin A, 2 μg/ml aprotinin, 2 μg/ml leupeptin). Lysozyme was added to a concentration of 100 μg/ml, and the lysate was incubated on ice for 20 min. The suspension was then sonicated briefly, quickly frozen on dry ice, and allowed to thaw on ice, before clarification by centrifugation at 40,000 × g for 15 min at 4 °C. Imidazole (5 mm final concentration) was added to the supernatant, and the material was loaded onto a 5 ml HisTrap column (Amersham Biosciences) using an AKTA FPLC system (Amersham Biosciences). The column was washed successively with FEN-1 Purification Buffer containing 40 and 100 mm imidazole, respectively. FEN-1 was eluted with FEN-1 Purification Buffer containing 250 mm imidazole. Fractions collected were analyzed by SDS-polyacrylamide gel electrophoresis, and those fractions containing FEN-1 were pooled and dialyzed against 25 mm HEPES-KOH, pH 7.9, 100 mm KCl, 10% glycerol, and 1 mm dithiothreitol. Aliquots of recombinant FEN-1 were frozen in liquid nitrogen and stored at –80 °C. The purified FEN-1 recombinant protein was judged to be 98% pure from analysis on Coomassie-stained SDS-polyacrylamide gels.BLM-FEN-1 Co-immunoprecipitation Experiments—HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. GM08505, an SV40-transformed fibroblast cell line from a Bloom's syndrome patient obtained from Coriell Institute for Medical Research, was grown in MEM-Eagle supplemented with 15% fetal bovine serum at 37 °C in 5% CO2. Nuclear extracts were prepared from exponentially growing HeLa and GM08505 cells as described previously (45Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9142) Google Scholar). For co-immunoprecipitation experiments, nuclear extract (1 mg of protein) was incubated with either rabbit polyclonal anti-FEN-1 antibody (1: 400, Ref. 34Brosh 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) or anti-BLM antibodies IHIC33 (1:50) raised in rabbits against a chimeric protein consisting of MBP fused to residues 1–449 of BLM (IHIC33, as described in Ref. 28Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9639-9644Google Scholar) in buffer D (50 mm HEPES, pH 7.5, 100 mm KCl, 10% glycerol) for 4 h at 4 °C. The mixture was subsequently tumbled with 20 μl of protein G-agarose (Roche Applied Science) at 4 °C overnight. The beads were then washed three times with buffer D supplemented with 0.1% Tween 20. Proteins were eluted by boiling in SDS sample buffer, and half of the eluate was resolved on 10% polyacrylamide Tris-glycine SDS gels, and transferred to polyvinylidene difluoride membranes (Amersham Biosciences). The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20, and probed for BLM and FEN-1 using IHIC33 (1:500) and anti-FEN-1 (1:5000) rabbit polyclonal antibodies, respectively, followed by detection with a goat-anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology). BLM and FEN-1 on immunoblots were detected using ECL Plus (Amersham Biosciences).MBP-BLM-Amylose Pull-down Experiments—Maltose-binding protein (MBP) fusion peptides were expressed in BL21(DE3) cells (New England Biolabs) transformed with the pMAL-C2 expression plasmids containing various portions of the BLM cDNA (MBP-BLM-(1–447), MBP-BLM-(966–1417)) (28Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9639-9644Google Scholar). BL21(DE3) cells transformed with plasmids encoding MBP or MBP-HuR (kind gift from Dr. M. Gorospe, NIA, NIH) were used for control experiments. Overnight transformed bacterial cultures were used to inoculate 50 ml of LB supplemented with 2% glucose and 100 μg/ml ampicillin at a 1:100 dilution, and the cultures were grown at 37 °C to an OD600 of ∼0.5. Isopropyl-1-thio-β-d-galactopyranoside was then added to a final concentration of 0.4 mm, and the cultures were allowed to grow for 3 h before being chilled on ice for 30 min. After centrifugation at 10,000 rpm in a Beckman JA10 rotor, the cell pellet was resupended in 1 ml of Lysis buffer (20 mm Tris-HCl, pH 7.4, 300 mm NaCl, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm mercaptoethanol, 10% glycerol) supplemented with complete protease inhibitor mixture (Roche Applied Science) at the manufacturer's recommended concentration. Cells were lysed by sonication and the lysate was clarified by centrifugation at 42,000 rpm in a Beckman 70 TI rotor for 30 min at 4 °C. For the pull-down assay, 200 μl of the clarified lysate was incubated for 2 h at 4 °C with 50 μl of amylose resin (New England Biolabs) that had been pre-washed twice with Lysis buffer. MBP fusion protein bound resin was washed three times with Lysis buffer and the washed beads were incubated for 2 h at 4 °C with 50 μl of binding buffer (50 mm Tris, pH 8.0, 10% glycerol, 100 mm NaCl, 0.01% Nonidet P-40, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 5 μg/ml leupeptin) containing 100 ng of purified recombinant FEN-1. Resin was washed three times with binding buffer supplemented with 0.1% Tween 20, and proteins were eluted by boiling with SDS Sample buffer and resolved on 8–16% gradient polyacrylamide Tris-glycine SDS gels. After transferring the proteins to a polyvinylidene difluoride membrane, the membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20, and probed with either anti-FEN-1 or anti-BLM antibodies as described above.ELISA for Detection of BLM-FEN-1 Protein-Protein Interaction— Purified recombinant BLM and WRN proteins were diluted to a concentration of 1 ng/μl in carbonate buffer (0.016 m Na2CO3, 0.034 m NaHCO3, pH 9.6), and were added to appropriate wells of a 96-well microtiter plate (50 μl/well), which was incubated at 4 °C. Bovine serum albumin (BSA) was used in the coating step for control reactions. The samples were aspirated, and the wells were blocked for 2 h at 30 °C with Blocking buffer (phosphate-buffered saline, 0.5% Tween 20 and 3% BSA). The procedure was repeated. FEN-1 was diluted to 1.0 ng/μl in Blocking buffer, and was added to the appropriate wells of the ELISA plate (50 μl/well), which was incubated for 1 h at 30 °C. For ethidium bromide (EtBr) treatment, 50 μg/ml EtBr was included in the incubation with FEN-1 during the binding step in the corresponding wells. The samples were aspirated, and the wells were washed five times before addition of anti-FEN-1 antibody diluted 1:10,000 in Blocking buffer and incubated at 30 °C for 1 h. Following three washings, horse-radish peroxidase-conjugated anti-rabbit secondary antibody (1:10,000) was added to the wells, and the samples were incubated for 30 min at 30 °C. After washing five times, any WRN or BLM bound to the FEN-1 was detected using OPD substrate (Sigma). The reaction was terminated after 3 min with 3 n H2SO4, and absorbance readings were taken at 490 nm.ELISA Data Analysis—The fraction of the immobilized BLM or WRN bound to the microtiter well that was specifically bound by FEN-1 protein was determined from the ELISA. A Hill plot was used to analyze the data as described previously (26Brosh 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 (254) Google Scholar).Oligonucleotide Substrates—PAGE-purified oligonucleotides (Midland Certified Reagent Co.) used for preparation of DNA substrates were as described previously (33Brosh Jr., R.M. Driscoll H.C. Dianov G.L. Sommers J.A. Biochemistry. 2002; 41: 12204-12216Crossref PubMed Scopus (64) Google Scholar). Briefly, the appropriate oligonucleotide (10 pmol) was 5′-radiolabeled with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs) using the manufacturer's protocol. Unincorporated nucleotides were removed using a G25 Sephadex spin column (Amersham Biosciences). The radiolabeled oligonucleotide was annealed to the appropriate template oligonucleotide (25 pmol) by heating at 95 °C, and then cooling from 70 °C to 24 °C. If necessary, an upstream oligonucleotide (50 pmol) was then annealed to the duplex substrate by heating at 37 °C for 1 h and slowly cooling to 24 °C. Radiolabeled three-stranded DNA substrate molecules were verified for the presence of all three oligonucleotides by comparing the mobility of the labeled DNA substrate with the corresponding labeled control duplex and single-stranded oligonucleotides on native polyacrylamide gels. Three-stranded DNA substrate preparations were determined to be >98% homogeneous. The nicked duplex substrate was prepared using FLAP00, TSTEM25, and U25. The 1-nt flap substrate was prepared using FLAP01, TSTEM25, and U25. The 26-nt 5′-flap substrate was prepared using FLAP26, TSTEM25, and U25 (33Brosh Jr., R.M. Driscoll H.C. Dianov G.L. Sommers J.A. Biochemistry. 2002; 41: 12204-12216Crossref PubMed Scopus (64) Google Scholar). Annealed oligonucleotide substrates were used directly for the in vitro biochemical reactions.FEN-1 Incision Assays—Reactions (20 μl) contained 10 fmol of DNA substrate (except where indicated), and the specified amounts of BLM and/or FEN-1 in 30 mm HEPES (pH 7.6), 5% glycerol, 40 mm KCl, 0.1 mg/ml BSA, and 8 mm MgCl2. BLM 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 (unless indicated otherwise), and were terminated by the addition of 10 μl of formamide stop solution (80% formamide (v/v), 0.1% bromphenol blue, and 0.1% xylene cyanol), and then 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 ImageQuant software (Molecular Dynamics) was used for quantification of the level of reaction products. Percent incision was calculated as described previously (34Brosh 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) from the equation: % incision = [P/(S + P)] × 100, where P is the sum of the intensity of the bands representing incision products, and S the intensity of the band representing the intact oligonucleotide. Background values from the no enzyme controls were subtracted out. Cleavage data represent the mean of at least three independent experiments with standard deviations shown by error bars.BLM Helicase Assays—Reactions (20 μl) contained 10 fmol of 26-nt 5′-flap DNA substrate, 2 mm ATP, and the indicated amounts of BLM in the same reaction buffer used for FEN-1 incision assays. Reactions were incubated at 37 °C for 15 min, and were terminated by the addition of 20 μl of Helicase Stop Solution (50 mm EDTA, 40% glycerol, 0.1% bromphenol blue, 0.1% xylene cyanol) containing 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 quantified using ImageQuant software (Molecular Dynamics). The percent helicase substrate unwound was calculated as described previously (46Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) using the following formula: % unwinding = 100 × (P/(S + P)], where P is the product and S is the substrate. The values of P and S have been corrected after subtracting background values in the no enzyme and heat-denatured substrate controls, respectively.RESULTSPhysical Int
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