Biochemical Characterization of the DNA Substrate Specificity of Werner Syndrome Helicase
2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês
10.1074/jbc.m111446200
ISSN1083-351X
AutoresRobert Brosh, Juwaria Waheed, Joshua A. Sommers,
Tópico(s)Genomics and Chromatin Dynamics
ResumoWerner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, is a helicase/exonuclease that presumably functions in DNA metabolism. To understand the DNA structures WRN acts upon in vivo, we examined its substrate preferences for unwinding. WRN unwound a 3′-single-stranded (ss)DNA-tailed duplex substrate with streptavidin bound to the end of the 3′-ssDNA tail, suggesting that WRN does not require a free DNA end to unwind the duplex; however, WRN was completely blocked by streptavidin bound to the 3′-ssDNA tail 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the fork structure to initiate unwinding. WRN unwound two important intermediates of replication/repair, a 5′-ssDNA flap substrate and a synthetic replication fork. WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of the fork. For the 5′-flap structure, WRN specifically displaced the 5′-flap oligonucleotide, suggesting a role of WRN in Okazaki fragment processing. The ability of WRN to target DNA replication/repair intermediates may be relevant to its role in genome stability maintenance. Werner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, is a helicase/exonuclease that presumably functions in DNA metabolism. To understand the DNA structures WRN acts upon in vivo, we examined its substrate preferences for unwinding. WRN unwound a 3′-single-stranded (ss)DNA-tailed duplex substrate with streptavidin bound to the end of the 3′-ssDNA tail, suggesting that WRN does not require a free DNA end to unwind the duplex; however, WRN was completely blocked by streptavidin bound to the 3′-ssDNA tail 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the fork structure to initiate unwinding. WRN unwound two important intermediates of replication/repair, a 5′-ssDNA flap substrate and a synthetic replication fork. WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of the fork. For the 5′-flap structure, WRN specifically displaced the 5′-flap oligonucleotide, suggesting a role of WRN in Okazaki fragment processing. The ability of WRN to target DNA replication/repair intermediates may be relevant to its role in genome stability maintenance. Werner syndrome adenosine 5′-3-O-(thio)triphosphate double-stranded flap endonuclease 1 single-stranded Werner syndrome (WS)1 is an autosomal recessive disease that displays symptoms of premature aging after adolescence (1Martin G.M. Birth Defects Orig. Artic. Ser. 1978; 14: 5-39PubMed Google Scholar). WS is characterized by the early onset of age-related symptoms including gray hair, wrinkled skin, cataracts, atherosclerosis, diabetes, osteoporosis, and cancer. WS cells grown in culture display marked chromosomal instability characterized by elevated rates of chromosomal translocations and rearrangements (2Salk D., Au, K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (212) Google Scholar, 3Salk D. Bryant E. Hoehn H. Johnston P. Martin G.M. Adv. Exp. Med. 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Kato J.T. Gollahon K.A. Rabinovitch P.S. FASEB J. 2001; 15: 1224-1226Crossref PubMed Scopus (133) Google Scholar). The gene defective in WS, designated WRN, encodes a nuclear (17Matsumoto T. Shimamoto A. Goto M. Furuichi Y. Nat. Genet. 1997; 16: 335-336Crossref PubMed Scopus (161) Google Scholar) 1,432-amino acid protein with the seven conserved motifs found in the RecQ family of DNA helicases (18Yu 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 (1496) Google Scholar). WRN is a DNA-dependent ATPase and utilizes the energy from ATP hydrolysis to unwind double-stranded (ds)DNA (19Gray M.D. Shen J.C. Kamath-Loeb A.S. Blank A. Sopher B.L. Martin G.M. Oshima J. Loeb L.A. Nat. Genet. 1997; 17: 100-103Crossref PubMed Scopus (523) Google Scholar, 20Suzuki N. Shimamoto A. Imamura O. Kuromitsu J. Kitao S. Goto M. Furuichi Y. Nucleic Acids Res. 1997; 25: 2973-2978Crossref PubMed Scopus (195) Google Scholar). Using a conventional helicase directionality substrate with 2 radiolabeled oligonucleotides annealed to opposite ends of a long ssDNA molecule, it was determined that WRN helicase unwinds dsDNA with a 3′ → 5′-polarity with respect to the single strand that it is inferred to bind (21Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar). WRN is also a 3′ → 5′-exonuclease (21Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar, 22Huang S., Li, B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar, 23Kamath-Loeb A.S. Shen J.C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), consistent with the presence of three conserved exonuclease motifs homologous to the exonuclease domain of Escherichia coli DNA polymerase I and RNase D in the protein sequence (24Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (204) Google Scholar). The catalytic activities of WRN suggest that the protein plays an important role in a pathway of genome stability, but precisely what pathway of DNA metabolism is defective in WS is not well understood. The involvement of three human RecQ family members (WRN, BLM, and RECQL4) in inherited disorders (WS, Bloom syndrome, and Rothmund-Thomson syndrome, respectively) characterized by genomic instability, premature aging, and cancer suggests that the helicase function is likely to be important in the molecular pathology of disease (for review, see Ref. 25Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Crossref PubMed Scopus (190) Google Scholar). A reasonable hypothesis is that specific DNA substrates may be acted upon by the helicase during replication, repair, or recombination to confer genomic stability. A role of WRN in DNA replication has been suggested by its recovery from cells in a replication complex (26Lebel M. Spillare E.A. Harris C.C. Leder P. J. Biol. Chem. 1999; 274: 37795-37799Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) as well as its interactions with other proteins of the replication machinery which include replication protein A (21Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar, 27Brosh R.M., Jr. 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 (257) Google Scholar), flap endonuclease 1 (FEN-1) (28Brosh R.M., Jr. 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), polymerase δ (29Kamath-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, 30Szekely 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), topoisomerase I (26Lebel M. Spillare E.A. Harris C.C. Leder P. J. Biol. Chem. 1999; 274: 37795-37799Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), and proliferating cell nuclear antigen (26Lebel M. Spillare E.A. Harris C.C. Leder P. J. Biol. Chem. 1999; 274: 37795-37799Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Among the replication defects observed in cells from WS patients, the delayed S phase progression (8Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell Res. 1992; 202: 267-273Crossref PubMed Scopus (187) Google Scholar) suggests that WRN may play a direct role in the metabolism of certain structures that potentially interfere with the progression of the replication fork. Sequence-specific DNA structures such as triplexes and tetraplexes potentially block DNA synthesis. WRN is able to resolve these structures (31Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 32Brosh R.M., Jr. Majumdar A. Desai S. Hickson I.D. Bohr V.A. Seidman M.M. J. Biol. Chem. 2001; 276: 3024-3030Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33Mohaghegh P. Karow J.K. Brosh R.M., Jr. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (491) Google Scholar), and most recently it was shown that WRN can alleviate the pausing of yeast polymerase δ at sites of tetraplexes by eliminating the secondary structure (34Kamath-Loeb A.S. Loeb L.A. Johansson E. Burgers P.M.J. Fry M. J. Biol. Chem. 2001; 276: 16439-16446Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). WRN also actively unwinds the four-stranded X structure (33Mohaghegh P. Karow J.K. Brosh R.M., Jr. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (491) Google Scholar), a model for the Holliday junction recombination intermediate. Recent models propose that Holliday junctions can arise at sites of stalled DNA replication (35McGlynn P. Lloyd R.G. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8235-8240Crossref PubMed Scopus (141) Google Scholar, 36McGlynn P. Lloyd R.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8227-8234Crossref PubMed Scopus (159) Google Scholar), and WRN may play a role in the subsequent processing of this structure during replication restart, perhaps in a recombinational repair pathway. To understand better the substrate specificity of WRN helicase, we have examined the ability of WRN to unwind a panel of defined duplex DNA substrates using an in vitro strand displacement assay. Helicase activity assays were performed to identify the DNA duplex substrates most efficiently unwound by WRN. This system enabled us to assay carefully WRN interactions with the substrate in a functional context and to determine which features of the DNA substrate are important for WRN to initiate the unwinding reaction efficiently. It was reported previously that oligonucleotide duplex substrates with both 3′- and 5′-(ss)DNA tails are unwound preferentially compared with similar substrates with only 3′-tails (32Brosh R.M., Jr. Majumdar A. Desai S. Hickson I.D. Bohr V.A. Seidman M.M. J. Biol. Chem. 2001; 276: 3024-3030Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33Mohaghegh P. Karow J.K. Brosh R.M., Jr. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (491) Google Scholar). One aspect of this study entailed defining the minimum length requirements for the 5′- and 3′-ssDNA tails of forked duplex substrates required by WRN for efficient unwinding. In addition, we have used helicase substrates with specifically positioned steric blocks to probe the preference of WRN for forked duplex structures. The replication defects and hypersensitivity to certain DNA-damaging agents of WS cells suggest a direct role of the protein in the processes of replication and/or repair. One important DNA structural intermediate in both of these processes is the 5′-ssDNA flap substrate. Flap structures may arise during Okazaki fragment processing in replication or strand displacement during DNA repair pathways such as base excision repair. Recently, we demonstrated that WRN interacts with human FEN-1 (28Brosh R.M., Jr. 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), a DNA repair/replication enzyme that processes 5′-ssDNA flap structures. In the current study, we have shown that WRN efficiently unwinds the 5′-flap DNA substrate. By its action at flap structures, WRN may facilitate strand displacement by an advancing DNA polymerase and processing of unannealed 5′-ssDNA flaps. The catalytic unwinding activity of WRN may be important at specific structures of the replication fork. Although the synthetic replication fork lacks the ssDNA loading region of a typical helicase substrate, WRN effectively unwound the structure in a specific manner by directing its helicase activity in the direction of the fork, leaving the two duplex arms intact. This characteristic feature of DNA unwinding by WRN might be important at the site of a stalled replication fork or in some other aspect of DNA metabolism at the site of new DNA synthesis. The preference of WRN to unwind dsDNA substrates with junctions suggests that WRN may act upon these structures during the processes of DNA replication, recombination, or repair. The cellular defects and genomic instability of WS may arise from persistent DNA structures that fail to be acted upon by WRN helicase. Recombinant WRN protein used in this study was purified as previously described (28Brosh R.M., Jr. 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, 37Orren D.K. Brosh R.M., Jr. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar). PAGE-purified oligonucleotides, obtained from Midland Certified Reagent Company, are listed in Table I. DNA duplex substrates were prepared as described previously (38Henricksen L.A. Tom S. Liu Y. Bambara R.A. J. Biol. Chem. 2000; 275: 16420-16427Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and are shown in Tables II andIII.Table IOligonucleotide sequences for DNA substrate (5′ to 3′) Open table in a new tab Table IIDNA substrates (part 1) used in this work Open table in a new tab Table IIIDNA substrates (part 2) used in this work Open table in a new tab Helicase assay reaction mixtures (20 μl) contained 30 mm Hepes pH 7.6, 5% glycerol, 40 mm KCl, 0.1 mg/ml bovine serum albumin, 8 mmMgCl2, 2 mm ATP, 10 fmol of DNA duplex substrate (0.5 nm DNA substrate concentration), and the indicated amounts of WRN. For helicase reactions containing streptavidin, 15 nm streptavidin was preincubated with the DNA substrate with all reaction components except WRN for 10 min at 37 °C. Helicase reactions were initiated by the addition of WRN and then incubated at 37 °C for 15 min unless otherwise indicated. WRN concentrations used were 0.19, 0.38, 1.9, and 3.8 nmmonomer. Reactions were quenched with 10 μl of loading buffer (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% (19:1 acrylamide:bisacrylamide) polyacrylamide gels except where indicated in the figure legends. Radiolabeled DNA species in polyacrylamide gels were visualized using a PhosphorImager and quantitated using the ImageQuant software (Molecular Dynamics). The percent helicase substrate unwound was calculated by the 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. Helicase data represent the mean of at least three independent experiments with standard deviations shown by error bars. To determine the DNA substrate requirements for efficient unwinding of B form dsDNA by WRN helicase, we have tested a series of related DNA substrates (0.5 nm final DNA substrate concentration) incubated with various concentrations of WRN protein using a strand displacement assay. The products of the helicase reaction were analyzed by native PAGE to determine which substrates were most efficiently unwound. Under the reaction conditions used for these studies, the DNA oligonucleotides, both those unwound and those remaining in duplex state, were not appreciably degraded by nuclease activity of WRN as judged by quantitation of DNA products resolved on denaturing gels (data not shown). Thus, the percent of DNA substrate unwound (% displacement) is determined by the amount of intact oligonucleotide released divided by the total amount of DNA substrate in the reaction. Our initial studies were focused on determining the importance of the length of the noncomplementary 5′-ssDNA tail of a forked dsDNA substrate for the efficiency of unwinding by WRN (Table II, substrates 1–5). Because WRN has been reported to unwind dsDNA with a 3′-ssDNA overhang, the length of the 5′-ssDNA tail may also be an important factor in the WRN helicase reaction. WRN-catalyzed unwinding of a 19-bp DNA duplex with a 25-nucleotide 3′-overhang (substrate 1) is relatively inefficient, with appreciable strand displacement (20–25%) during the 15-min time course only detected at the highest WRN protein concentrations tested (1.9, 3.8 nm WRN) (Fig.1). Thus a 3.8-fold greater amount of WRN monomer (1.9 nm) compared with DNA substrate (0.5 nm) is required for detectable unwinding, suggesting that unwinding is stoichiometric. Using a DNA substrate with the same duplex region but flanked by a single noncomplementary nucleotide on the terminus of the 5′-tail (substrate 2), WRN unwound a significantly greater amount of substrate, 40 and 58% displacement at WRN concentrations of 1.9 and 3.8 nm, respectively. Using a DNA duplex substrate with a 5-nucleotide noncomplementary 5′-ssDNA tail (substrate 3), the unwinding reaction was even more significantly improved. In this case, lower concentrations of WRN (0.19 and 0.38 nm) were sufficient to unwind 10 and 28% of the dsDNA substrate, respectively. At these protein concentrations, WRN unwound ∼5.5-fold more of substrate 3 compared with substrates 2 and 1, indicating that the enzyme is more efficient at unwinding dsDNA substrates with a 5-nucleotide 5′-ssDNA flanking region compared with substrates with only a 1-nucleotide 5′-noncomplementary tail or no tail at all. At the two highest concentrations of WRN (1.9 and 3.8 nm), substrate 3 bearing the 5-nucleotide 5′-ssDNA tail was efficiently unwound by WRN, achieving 70–80% displacement. Slightly higher levels of unwinding were attained at the two highest WRN concentrations with substrate 4, which has the 10-nucleotide 5′-tail. A DNA duplex with a long 5′-ssDNA tail of 26 nucleotides (substrate 5) was unwound significantly better (∼12-fold at a WRN concentration of 0.18 nm) than the DNA substrate with only the 3′-ssDNA tail (substrate 1), clearly indicating the preference of WRN for the forked substrate. Substrate 5 was unwound better than the fork duplex with the 5-nucleotide (substrate 3) or 10-nucleotide (substrate 4) 5′-ssDNA tails at 0.19 and 0.38 nm WRN protein concentrations. At higher WRN concentrations (1.9, 3.8 nm), substrates 3, 4, and 5 were unwound nearly equally well, suggesting that a forked duplex with 5–10 nucleotides of 5′-ssDNA tail is optimally unwound by WRN. Importantly, the helicase data on this family of substrates clearly show that a forked duplex is unwound substantially better than a 3′-tailed duplex. Because the forked duplex is the preferred substrate for WRN helicase, we next addressed the importance of the length of the noncomplementary 3′-ssDNA tail of a DNA duplex substrate that is flanked by a long 5′-ssDNA tail of 26 nucleotides (Table II, substrates 5–8). A DNA substrate possessing only a single noncomplementary 3′-nucleotide (substrate 6) was essentially not acted upon by the WRN helicase (Fig. 2), similar to a previously published report (21Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar) and our own data that WRN does not unwind a duplex DNA substrate flanked by only a 5′-ssDNA tail. However, if the DNA substrate possessed a 5-nucleotide 3′-ssDNA tail (substrate 7), significantly greater amounts of DNA unwinding were detected at WRN concentrations of 0.19–3.8 nm. At the highest WRN protein concentrations tested, 1.9 and 3.8 nm, substrate 7 was unwound 44 and 54%, respectively, a 20-fold increase compared with substrate 6. Using a 10-nucleotide 3′-tailed substrate (substrate 8), a significantly greater amount of DNA substrate was unwound compared with substrate 7 at WRN protein concentrations of 0.19–3.8 nm. The maximal difference between substrates 7 and 8 was ∼2-fold at a WRN concentration of 0.38 nm. The 25-nucleotide 3′-ssDNA tailed substrate (substrate 5) was unwound very similarly compared with substrate 8 at all WRN levels. Altogether the helicase data indicate that a 3′-ssDNA tail of 5 nucleotides is the minimal length for efficient unwinding of a forked substrate by WRN and that a 10-nucleotide 3′-ssDNA tail can be judged to be optimal. The results from WRN helicase assays using forked duplex substrates with a long 25-nucleotide 3′-ssDNA tail and increasing lengths of 5′-ssDNA tail indicated that 5–10 nucleotides is the minimal length of 5′-ssDNA tail necessary for optimal unwinding (Fig. 1). Likewise, the results from WRN helicase assays using forked duplex substrates with a long 26-nucleotide 5′-ssDNA tail and increasing lengths of 3′-ssDNA tail indicated that a forked duplex with a 5-nucleotide 3′-ssDNA tail is unwound by WRN, but a 10-nucleotide 3′-ssDNA tail is necessary for optimal unwinding (Fig. 2). Taken together, these results would suggest that WRN might efficiently unwind a forked duplex substrate with 3′- and 5′-ssDNA tail lengths of 10 nucleotides. We tested WRN on such a forked duplex (substrate 9) and found this to be the case (Fig. 2). Approximately 43% of the substrate was unwound at a WRN concentration of 0.38 nm (Fig. 2). At WRN concentrations of 1.9 and 3.8 nm, 68 and 85% of the substrate was unwound, respectively. WRN-catalyzed DNA unwinding was dependent on ATP hydrolysis because the poorly hydrolyzable analog ATPγS failed to support the unwinding reaction (data not shown). These data indicate that WRN can efficiently unwind a forked duplex with 3′- and 5′-ssDNA tails of 10 nucleotides. Comparable levels of unwinding were detected for substrates 4, 5, 8, and 9, suggesting that a forked duplex with 3′- and 5′-ssDNA tails of 10 nucleotides is unwound as efficiently as the duplex with tails of 25 nucleotides (3′) and 26 nucleotides (5′). The reported 3′ → 5′ directionality of WRN helicase (21Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar) may be a consequence of unidirectional translocation on ssDNA in a 3′ → 5′ direction; alternatively, the polarity of DNA unwinding may be determined by the binding specificity of WRN protein for a ssDNA/dsDNA junction characterized by a 3′-ssDNA overhang. The latter explanation was recently proposed to be responsible for the 3′ → 5′ directionality of a recombinant Sgs1 protein fragment (39Bennett R.J. Keck J.L. Wang J.C. J. Mol. Biol. 1999; 289: 235-248Crossref PubMed Scopus (114) Google Scholar). To address this issue for WRN, we examined DNA binding by WRN to a number of DNA substrates used in this study, including the 3′-tailed duplex (substrate 1) and the forked duplex (substrate 5). Gel shift analysis of protein-DNA mixtures that had been incubated in the presence of ATPγS or in the absence of nucleotide did not demonstrate a major level of stable binding to any of the duplex DNA substrates (data not shown). We were able to detect reproducibly 1–2% of the forked duplex substrate 5 molecules cross-linked to 3.8 nmWRN when the samples were treated with 0.25% glutaraldehyde after the binding incubation (data not shown). Under these conditions, WRN consistently failed to be cross-linked to substrates 1 or 2, suggesting that WRN may bind slightly preferentially to forked structures compared with the 3′-tailed substrate lacking a flanking 5′-tail (substrate 1) or bearing a single nucleotide 5′-tail (substrate 2), as detected by this assay. To probe the importance of DNA structural elements for the WRN unwinding function, we tested WRN helicase activity on dsDNA substrates with specifically positioned biotin-streptavidin complexes (Table III, substrates 10–12 and 19). The streptavidin-biotin complex is an extremely strong interaction (Kd ∼ 10−15m) (40Green N.M. Adv. Protein Chem. 1975; 29: 85-133Crossref PubMed Scopus (1612) Google Scholar), and the diameter of a streptavidin tetramer, ∼45 Å (41Hendrickson W.A. Pahler A. Smith J.L. Satow Y. Merritt E.A. Phizackerley R.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2190-2194Crossref PubMed Scopus (571) Google Scholar), has previously been shown to block dsDNA unwinding by T7 Gene 4 and DnaB helicases when it is positioned on the single strand on which the enzyme translocates (42Hacker K.J. Johnson K.A. Biochemistry. 1997; 36: 14080-14087Crossref PubMed Scopus (107) Google Scholar, 43Kaplan D.L. J. Mol. Biol. 2000; 301: 285-299Crossref PubMed Scopus (108) Google Scholar). Initially, we tested a substrate similar to the DNA duplex with a 25-nucleotide 3′-ssDNA overhang (substrate 1) which also has a biotin moiety conjugated to the terminal 3′-nucleotides of the ssDNA overhang (substrate 10). Streptavidin was effectively bound to the DNA substrate throughout the time course of the experiment as detected by a gel-shifted species when streptavidin was present (data not shown). WRN unwound substrate 10 nearly equally well in the presence or absence of streptavidin, as demonstrated by only a small reduction in the level of unwinding when streptavidin was present (Fig.3). These results suggest that WRN is not required to load onto the end of the protruding 3′-ssDNA of the DNA substrate to unwind the duplex. We next tested the same 3′-tailed substrate, only the biotin was positioned 6 nucleotides upstream of the ssDNA/dsDNA junction (substrate 11). In this case, WRN was blocked from unwinding the substrate in the presence of streptavidin (Fig.4). In contrast to these results, WRN unwound up to 40% of the DNA substrate in the absence of streptavidin. The fact that streptavidin only had a minor effect on WRN helicase activity on substrate 10 under the identical reaction conditions (Fig.3) indicated that the strong inhibitory effect of streptavidin on the WRN helicase reaction is specific for substrate 11 and caused by the position of the bound biotin. We also tested WRN in a streptavidin displacement assay under reaction conditions identical to those of the helicase assay and found that WRN completely failed to displace streptavidin bound to the same biotin-conjugated oligonucleotide (TSTEM25BINT) used to construct substrate 11 (data not shown). These results suggest that streptavidin bound to the biotin-conjugate positioned 6 nucleotides upstream of the ssDNA/dsDNA junction interferes with the progression of WRN helicase into the duplex region. Because WRN was unable to displace streptavidin from ssDNA, the helicase inhibition by the internally positioned streptavidin complex suggests that streptavidin may have posed a block to translocation of WRN along the 3′-ssDNA tail to the ssDNA/dsDNA junction on substrate 11. Alternatively, the streptavidin moiety may have deterr
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