POT1 Stimulates RecQ Helicases WRN and BLM to Unwind Telomeric DNA Substrates
2005; Elsevier BV; Volume: 280; Issue: 37 Linguagem: Inglês
10.1074/jbc.m505211200
ISSN1083-351X
AutoresPatricia L. Opresko, Penelope A. Mason, Elaine R. Podell, Ming Lei, Ian D. Hickson, Thomas R. Cech, Vilhelm A. Bohr,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoDefects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3 ′–5′-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3 ′ tail as it is exposed during unwinding. Defects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3 ′–5′-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3 ′ tail as it is exposed during unwinding. Deficiencies in human RecQ helicases, WRN, BLM, and RecQL4, are responsible for genomic instability disorders, namely Werner syndrome (WS) 4The abbreviations used are: WS, Werner syndrome; BS, Bloom syndrome; GST, glutathione S-transferase; ss, single strand; ds, double strand; TRF, telomere repeat binding factors; INV, invading strand; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; RPA, replication protein A; OB, oligonucleotide/oligosaccharide binding; Tel, telomere; nt, nucleotide; NE, nuclear extracts; ALT, alternative pathway for lengthening of telomeres.4The abbreviations used are: WS, Werner syndrome; BS, Bloom syndrome; GST, glutathione S-transferase; ss, single strand; ds, double strand; TRF, telomere repeat binding factors; INV, invading strand; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; RPA, replication protein A; OB, oligonucleotide/oligosaccharide binding; Tel, telomere; nt, nucleotide; NE, nuclear extracts; ALT, alternative pathway for lengthening of telomeres., Bloom syndrome (BS), and Rothmund-Thomson syndrome, respectively. WS patients display the early onset of many age-associated pathologies including graying and loss of hair, wrinkling of the skin, cataracts, type II diabetes, osteoporosis, cardiovascular disease, and a high frequency of sarcomas (1Martin G.M. Birth Defects Orig. Artic. Ser. 1978; 14: 5-39PubMed Google Scholar, 2Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (160) Google Scholar). BS patients exhibit severe growth retardation, immunodeficiency, sun-induced facial erythema, and a greatly increased predisposition to a wide range of cancers that develop early in life (3Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (578) Google Scholar). Cells derived from WS and BS patients display increased chromosomal rearrangements and deletions, defects in DNA replication and homologous recombination, and decreased replicative life spans (reviewed in Refs. 2Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (160) Google Scholar and 3Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (578) Google Scholar). Cellular phenotypes of WS and BS include features consistent with telomere dysfunction. Cells deficient in BLM helicase display increased telomere associations between homologous chromosomes (4Lillard-Wetherell K. Machwe A. Langland G.T. Combs K.A. Behbehani G.K. Schonberg S.A. German J. Turchi J.J. Orren D.K. Groden J. Hum. Mol. Genet. 2004; 13: 1919-1932Crossref PubMed Scopus (124) Google Scholar). Cells lacking WRN, or those that express a dominant-negative WRN mutant, exhibit increased telomere loss (5Bai Y. Murnane J.P. Hum. Genet. 2003; 113: 337-347Crossref PubMed Scopus (58) Google Scholar) and loss of telomeres from single sister chromatids (6Crabbe L. Verdun R.E. Haggblom C.I. Karlseder J. Science. 2004; 306: 1951-1953Crossref PubMed Scopus (492) Google Scholar). The forced expression of telomerase rescues both WS and BS primary fibroblasts from premature replicative senescence (7Wyllie F.S. Jones C.J. Skinner J.W. Haughton M.F. Wallis C. Wynford-Thomas D. Faragher R.G. Kipling D. Nat. Genet. 2000; 24: 16-17Crossref PubMed Scopus (286) Google Scholar, 8Davalos A.R. Campisi J. J. Cell Biol. 2003; 162: 1197-1209Crossref PubMed Scopus (88) Google Scholar). Furthermore, mice singly null for Wrn appear normal; however, the late generation mice null for both Wrn and Terc have shortened telomeres and develop pathologies resembling human WS (9Chang S. Multani A.S. Cabrera N.G. Naylor M.L. Laud P. Lombard D. Pathak S. Guarente L. DePinho R.A. Nat. Genet. 2004; 36: 877-882Crossref PubMed Scopus (381) Google Scholar). Mutations in Wrn and Blm in Terc null mice also enhance and accelerate the development of phenotypes associated with telomere dysfunction (10Du X. Shen J. Kugan N. Furth E.E. Lombard D.B. Cheung C. Pak S. Luo G. Pignolo R.J. DePinho R.A. Guarente L. Johnson F.B. Mol. Cell. Biol. 2004; 24: 8437-8446Crossref PubMed Scopus (186) Google Scholar). Collectively, these studies indicate that the RecQ helicases WRN and BLM are important for proper telomere maintenance and function. Telomeres are protein-DNA complexes that protect the ends of linear chromosomes, and consequences of telomere dysfunction include chromosome end fusions and genomic instability, apoptosis, or senescence (reviewed in Ref. 11Campisi J. Kim S. Lim C.S. Rubio M. Exp. Gerontol. 2001; 36: 1619-1637Crossref PubMed Scopus (306) Google Scholar). Human telomeres consist of 5–15 kb of TTAGGG tandem repeats and terminate in a 3′ single strand (ss) G-rich tail that serves as the substrate for telomerase-mediated elongation in an open telomere state. In one proposed capped or closed state, this tail loops back and invades the telomeric duplex tract, forming an intra-telomeric D-loop and a large lasso-like t-loop structure (12Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. De Lange T. Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar). This t-loop is stabilized by a protein complex that includes telomere repeat binding factors (TRF) 1 and 2, which bind duplex (TTAGGG)n DNA and regulate telomere length and access of the 3′ tail to telomerase and/or nucleases (13Smogorzewska A. van Steensel B. Bianchi A. Oelmann S. Schaefer M.R. Schnapp G. De Lange T. Mol. Cell. Biol. 2000; 20: 1659-1668Crossref PubMed Scopus (625) Google Scholar, 14van Steensel B. De Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1051) Google Scholar). Human POT1 (protection of telomeres-1) protein (15Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (801) Google Scholar) is also an important regulator of telomere length (16Armbruster B.N. Linardic C.M. Veldman T. Bansal N.P. Downie D.L. Counter C.M. Mol. Cell. Biol. 2004; 24: 3552-3561Crossref PubMed Scopus (65) Google Scholar, 17Colgin L.M. Baran K. Baumann P. Cech T.R. Reddel R.R. Curr. Biol. 2003; 13: 942-946Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 18Loayza D. De Lange T. Nature. 2003; 423: 1013-1018Crossref PubMed Scopus (538) Google Scholar) and binds specifically to telomeric ssDNA (19Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar, 20Loayza D. Parsons H. Donigian J. Hoke K. De Lange T. J. Biol. Chem. 2004; 279: 13241-13248Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). POT1 associates with TRF1 and TRF2 through the interaction with other telomeric proteins including TPP1 (formerly termed PIP1, PTOP, or TINT1) and TIN2 (reviewed in Refs. 21De Lange T. Nat. Rev. Mol. Cell Biol. 2004; 5: 323-329Crossref PubMed Scopus (337) Google Scholar and 22Colgin L. Reddel R. Curr. Biol. 2004; 14: R901-R902Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Collectively, these proteins cooperate with telomeric DNA structure in proper telomere maintenance and capping. The precise roles of RecQ helicases in telomere maintenance are unclear; however, studies suggest they likely function in recombination and/or replication at telomeric ends. In budding and fission yeast, RecQ helicases function in an alternative pathway for lengthening of telomeres (ALT) that occurs via recombination in telomerase-negative cells (23Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Crossref PubMed Scopus (222) Google Scholar, 24Mandell J.G. Goodrich K.J. Bahler J. Cech T.R. J. Biol. Chem. 2004; 280: 5249-5257Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 25Mandell J.G. Bahler J. Volpe T.A. Martienssen R.A. Cech T.R. Genome Biol. 2005; 6: R1-R15Crossref PubMed Google Scholar). WRN and BLM have been found to associate with telomeres in human ALT cell lines (26Opresko 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 (269) Google Scholar), and WRN was found at telomeres in S-phase human primary fibroblasts (6Crabbe L. Verdun R.E. Haggblom C.I. Karlseder J. Science. 2004; 306: 1951-1953Crossref PubMed Scopus (492) Google Scholar). WRN and BLM are 3′–5′ DNA helicases that are capable of dissociating telomeric D-loop structures (4Lillard-Wetherell K. Machwe A. Langland G.T. Combs K.A. Behbehani G.K. Schonberg S.A. German J. Turchi J.J. Orren D.K. Groden J. Hum. Mol. Genet. 2004; 13: 1919-1932Crossref PubMed Scopus (124) Google Scholar, 26Opresko 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 (269) Google Scholar). The WRN protein also contains a 3′–5′-exonuclease that cooperates with the helicase activity to release the invading tail of a telomeric D-loop (26Opresko 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 (269) Google Scholar). In addition, these helicases unwind G-quadruplex DNA structures that form readily in telomeric sequences (27Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (486) Google Scholar). Therefore, the RecQ helicases are thought to participate in telomere maintenance by resolving complex structures at telomeric ends to facilitate DNA replication or recombination pathways. The WRN and BLM helicases are poorly processive and likely function with co-factor proteins in a complex at the telomeres. TRF2 protein physically binds to WRN and BLM and stimulates these helicases to unwind relatively short duplex substrates that are telomeric or nontelomeric (4Lillard-Wetherell K. Machwe A. Langland G.T. Combs K.A. Behbehani G.K. Schonberg S.A. German J. Turchi J.J. Orren D.K. Groden J. Hum. Mol. Genet. 2004; 13: 1919-1932Crossref PubMed Scopus (124) Google Scholar, 28Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). However, these helicases require replication protein A (RPA) to completely unwind longer duplex substrates in vitro (29Brosh 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, 30Brosh 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, 31Opresko 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). RPA interacts physically with WRN and BLM (29Brosh 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, 32Shen J.C. Lao Y. Kamath-Loeb A. Wold M.S. Loeb L.A. Mech. Ageing Dev. 2003; 124: 921-930Crossref PubMed Scopus (50) Google Scholar) and binds to the partially unwound single strands to prevent their reannealing upon helicase dissociation. POT1 resembles RPA in that it also interacts with ssDNA via oligonucleotide/oligosaccharide binding (OB) folds, but it differs in having high specificity for telomeric DNA sequences (15Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (801) Google Scholar, 19Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar, 20Loayza D. Parsons H. Donigian J. Hoke K. De Lange T. J. Biol. Chem. 2004; 279: 13241-13248Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 33Lei M. Podell E.R. Baumann P. Cech T.R. Nature. 2003; 426: 198-203Crossref PubMed Scopus (161) Google Scholar). Therefore, we asked whether POT1 could facilitate WRN and BLM unwinding of longer telomeric substrates. We tested two POT1 splice variants: the full-length version (v1) and a less abundant C-terminal truncated version (v2) (19Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar). Both POT1 variants stimulated the WRN and BLM helicases to unwind telomeric forked duplex and D-loop structures that were poor substrates for these helicases alone. In contrast to RPA, optimal stimulation was dependent on the presence of telomeric sequence in the duplex regions of the substrates. Furthermore, we observed that WRN and BLM physically interact with POT1. Proteins—Recombinant histidine-tagged wild type WRN protein and the exonuclease-dead WRN mutant (X-WRN, E84A) were purified using a baculovirus/insect cell expression system as described previously (26Opresko 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 (269) Google Scholar, 34Orren 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). Recombinant histidine-tagged BLM was overexpressed in Saccharomyces cerevisiae and purified as described previously (35Karow 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). Recombinant human POT1 variant 1 and variant 2 proteins were purified using a baculovirus/insect cell expression system as described previously (19Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar). Splice variant 1 is the full-length protein, and variant 2 comprises the N-terminal half, including the two OB folds responsible for DNA binding (19Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar, 36Baumann P. Podell E. Cech T.R. Mol. Cell. Biol. 2002; 22: 8079-8087Crossref PubMed Scopus (140) Google Scholar). The purity of these protein preparations was analyzed by SDS-PAGE and Coomassie staining as shown in Fig. 1A. Human RPA was kindly provided by Dr. Mark Kenny (Albert Einstein Cancer Center, New York). Bacterial UvrD helicase was a generous gift from Dr. Steve Mattson (University of North Carolina, Chapel Hill). Human recombinant APE1-purified protein was provided by Dr. David Wilson (National Institute on Aging, Baltimore) (37Erzberger J.P. Barsky D. Scharer O.D. Colvin M.E. Wilson III, D.M. Nucleic Acids Res. 1998; 26: 2771-2778Crossref PubMed Scopus (89) Google Scholar). DNA Substrates—The forked duplex substrates consisted of 15-mer ssDNA tails followed by 34 bp of duplex DNA that contained either the (TTAGGG)4 sequence or nontelomeric sequence and were constructed as described previously (28Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 31Opresko 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). Substrates were radiolabeled at the 5′-end of the forked side. The D-loop Tel DS substrate containing the (TTAGGG)4 sequence in the 33-bp duplex portion of the invading strand was constructed by annealing the BT, BB, and invading strand (INV) oligonucleotides as described previously (26Opresko 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 (269) Google Scholar). Variant D-loops were similarly constructed, and all D-loops contained a 5′-end radiolabeled INV strand. The D-loop Tel SS contained the (TTAGGG)4 sequence in the displaced single strand region of the D-loop instead and was constructed by annealing the BT, BBmx, and INVtel oligonucleotides. The D-loop Non Tel contained only nontelomeric sequence and was constructed by annealing the BT, BBmx, and INVmx strands. The following oligonucleotides were used: INVtel, 5′-CGTGACCAGGACGTGAGTCTGGAGTGCAGAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGACAATCATCCTGACTGCAGACCGAGCTTGA; INVmx, 5′-CACCATCCAGTTCTCTTTTGAGAACTGGATGGTGTATCACATTGCGTTGATGGGACCGTTAACGCTC; and BBmx, 5′-TCAAGCTCGGTCTGCAGTCAGGATGATTGTGAGCGTTAACGGTCCCATCAACGCAATGTGATATCTGCACTCGAGACTCACGTCCTGGTCACG. Helicase and Exonuclease Reactions—Reactions were performed in standard reaction buffer (31Opresko 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), unless otherwise indicated. DNA substrate and protein concentrations were as indicated in the figure legends. The reactions were initiated by the addition of WRN or BLM protein and were incubated at 37 °C for 15 min. For the reactions containing WRN (total volume = 30 μl), a 10-μl aliquot was mixed with formamide stop dye and run on a 14% denaturing polyacrylamide gel. The remainder of the reactions containing WRN (20 μl) or the reactions containing BLM helicase (20 μl) were added to 10 μl of 3× native stop dye supplemented with 75 μg/ml proteinase K and a 10× molar excess of unlabeled competitor oligonucleotide (28Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). The products were deproteinized for 30 min at 37 °C and were then run on 8 or 12% native polyacrylamide gels as indicated in the figure legends. Products were visualized using a PhosphorImager, and quantitation was performed using ImageQuant software (Amersham Biosciences). For the reactions with WRN on the forked telomeric substrate, the percent of unreacted substrate and the percent of each major product band in the native gels (as defined by displaced oligonucleotide size, Fig. 2, A and E) were calculated as a fraction of the total radioactivity in the reaction lane. For the reactions with WRN and the various D-loop substrates, the percent of displaced near full-length products (longest) was calculated as a function of total displaced products in the lane (sum of the longest product band and the collective shorter product bands) in the native gels (Fig. 3A). For the reactions with BLM helicase or X-WRN, the percent of total product displacement was quantitated as a fraction of total radioactivity in the reaction lane as described previously (28Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Values were corrected for background in the no enzyme control and heat-denatured substrate lanes.FIGURE 3POT1 variants stimulate WRN to release an intact telomeric tail from a D-loop structure. A shows a schematic of the D-loop substrates with (TTAGGG)4 sequence (thick line) in either the duplex portion or in the single strand region or a D-loop with nontelomeric sequence. D-loop Tel ds, lanes 1–6 (B) and lanes 1–5 (C); D-loop Tel ss, lanes 7–12 (B) and 6–10 (C); D-loop Non-tel, lanes 13–18 (B) and lanes 11–15 (C). The substrate (0.5 nm) was incubated with WRN protein (5 nm) as indicated for 15 min at 37 °C. Increasing concentrations of POT1v1 were added at 5, 15, or 40 nm to the reactions in lanes 3–5 and 9–11, and 15–17 (B), respectively, and lanes 3–5 and 8–10, and 13–15, respectively (C). Numbers indicate the molar ratio of POT1 variant to WRN. Reaction products were separated on an 8% native gel (B) or a 14% denaturing gel (C). ▴, heat-denatured substrate. D, the percent of displaced near full-length products (longest, indicated by the arrow) was calculated as a function of total displaced products in B (see "Experimental Procedures") and plotted against the molar ratio of POT1v1 to WRN proteins; Tel ds, ▪ and dashed line; Tel ss, • and dotted line; Non-tel, ▴ and solid line. Values represent the mean, and error bars represent the S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ELISA Detection of Protein Interactions—ELISA was conducted as described previously (30Brosh 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), with some modification. The blocking and binding steps were performed in phosphate-buffered saline containing 3% BSA and 0.1% Tween 20. Wells were coated with 75 ng of purified POT1v1 or POT1v2 diluted in carbonate buffer (50 μl) or with BSA as a background control by incubation for 2 h at 37°C. Independent wells were also coated with WRN, BLM, or APE1 as positive controls. After blocking, various concentrations of WRN, BLM, or APE1 (negative control) protein were added (50 μl) to the wells coated with POT1v1 or POT1v2 (see Fig. 5 legend) and incubated for 2 h at 37°C. Following washes, primary antibody (1:1,500, anti-rabbit IgG against WRN (Novus); 1:1000, anti-rabbit IgG against BLM (Abcam); or 1:100 anti-rabbit IgG against APE1 (Trevigen)) was added and incubated for 1 h at 37 °C. Wells were washed, and secondary antibody (1:10,000, anti-rabbit IgG-horseradish peroxidase; Vector Laboratories) was added and incubated for 1 h at 37°C. After washes, bound WRN, BLM, or APE1 was detected with o-phenylenediamine dihydrochloride followed by termination with 3 m H2SO4. Absorbance was read at 490 nm. Values were normalized to the positive control (signal from wells coated with WRN, BLM, or APE1). To ensure that the interaction was not mediated by DNA, control reactions included either 5 μg/ml DNase I (Calbiochem) or 10 μg/ml ethidium bromide during the binding step. To determine the dissociation constant (Kd) for the WRN-POT1v2 and BLM-POT1v2 complexes, the fraction of immobilized POT1v2 bound by WRN or BLM was calculated, and the data were analyzed by Hill plot and Scatchard binding theory, as described previously (30Brosh 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). For this analysis and the plots in Fig. 5, D and E, the relative absorbance values were corrected for background in the BSA alone control. GST-POT1-Sepharose Pull-down Assay—GST-tagged POT1v1 and POT1v2 were expressed in baculovirus/insect cell cultures as described previously (19Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar). 500-ml cultures were harvested and resuspended in 8 ml of 25 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, including protease inhibitors. Samples were lysed by sonication and incubated with 20 μg/ml DNase I (Calbiochem) for 30 min on ice. Samples were then cleared by centrifugation. GST alone or GST-POT1v1 or -POT1v2 from 1 ml of lysates was bound to glutathione beads and then incubated with HeLa nuclear extracts (400 ul) prepared as described previously (28Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 38von 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). After washing, total protein was eluted in sample buffer by boiling and was analyzed by SDS-PAGE and Western blot analysis. Loading of the GST-tagged POT1 variants were determined by Amido Black staining. Membranes were probed with a mouse monoclonal anti-WRN antibody (1:500, BD Transduction Laboratories) or a rabbit polyclonal anti-BLM antibody (1:1,000, Abcam). POT1 Stimulation of the WRN Helicase Activity—To determine whether POT1 could promote WRN helicase unwinding, we first tested full-length POT1 (splice variant 1). Coomassie-stained gels of the purified proteins used for this study, and the subsequent experiments described in this paper, are shown in Fig. 1A as described previously (19Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar, 28Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). For the helicase substrate we used a 34-bp forked duplex structure that contained 15-mer ssDNA tails at one end (Fig. 1B). Both the WRN helicase and exonuclease are active on this substrate (31Opresko 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). Therefore, we first tested an exonuclease-dead WRN mutant (X-WRN) to measure directly the effects on the WRN unwinding activity. X-WRN contains a point mutation (E84A) that inactivates the exonuclease activity (39Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (377) Google Scholar). The duplex region of the telomeric fork consists of the (TTAGGG)4 sequence followed by 10 bp of unique sequence, whereas the telomeric sequence is scrambled in the mixed fork (Fig. 1B). Minimal displacement of either the telomeric forked duplex (Fig. 1B, lane 2) or the mixed forked duplex (lane 9) was achieved by X-WRN alone. However, the incubation of X-WRN with increasing concentrations of full-length POT1v1 resulted in up to a 10-fold increase in displacement of the telomeric fork, from 3 to 35% (Fig. 1B, lanes 2–5, and C). However, POT1v1 did not simulate X-WRN to unwind the mixed forked duplex (Fig. 1B, lanes 10–12), in contrast to RPA (lane 21). Time course analyses indicated that an 8-fold molar excess of POT1v1 increased the estimated rate of X-WRN strand displacement dramatically by 10-fold, from 0.01 bp/min/X-WRN (monomer) to 0.1 bp/min/X-WRN (monomer), of the telomeric fork (Fig. 1D). These data indicate that POT1v1 directly stimulates the WRN helicase to unwind the 34-bp telomeric substrate. Next we examined the activity of wild type WRN in the presence of full-length POT1v1. On the 34-bp forked duplex, the WRN helicase initiates unwinding at the forked end, whereas the 3′–5′-exonuclease initiates digestion at the blunt end (31Opresko 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) (Fig. 7C). Therefore, the reactions were run both on a native gel to visualize the unwound products (Fig. 2A) and on a denaturing gel (Fig. 2B) to better visualize the exonuclease products. As reported earlier (31Opresko P.L. Laine J.P. Brosh Jr., R.M. Seidman M.M. Bo
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