Biochemical and Kinetic Characterization of the DNA Helicase and Exonuclease Activities of Werner Syndrome Protein

The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration (1 mm) of Mg2+ and ATP with a Km of 140 μm for the ATP-Mg2+ complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg2+ concentration. Mn2+ and Ni2+ substituted for Mg2+ as a cofactor for WRN helicase, whereas Fe2+ or Cu2+ (10 μm) profoundly inhibited WRN unwinding in the presence of Mg2+.Zn2+ (100 μm) was preferred over Mg2+ as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn2+ strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn2+ binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1:1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding. The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration (1 mm) of Mg2+ and ATP with a Km of 140 μm for the ATP-Mg2+ complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg2+ concentration. Mn2+ and Ni2+ substituted for Mg2+ as a cofactor for WRN helicase, whereas Fe2+ or Cu2+ (10 μm) profoundly inhibited WRN unwinding in the presence of Mg2+.Zn2+ (100 μm) was preferred over Mg2+ as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn2+ strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn2+ binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1:1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding. Werner syndrome is a human autosomal recessive disorder that displays symptoms of premature aging and an increased incidence of cancer (1Martin G.M. Birth Defects Orig. Artic. Ser. 1978; 14: 5-39PubMed Google Scholar). Cellular phenotypes of Werner syndrome include genomic instability (2Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar, 3Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar, 4Salk D. Bryant E. Hoehn H. Johnston P. Martin G.M. Adv. Exp. Med. Biol. 1985; 190: 305-311Crossref PubMed Scopus (62) Google Scholar), aberrant recombination (5Cheng R.Z. Murano S. Kurz B. Shmookler R.R. Mutat. 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Miyamoto T. Hori T. Adv. Exp. Med. Biol. 1985; 190: 439-457Crossref PubMed Scopus (46) Google Scholar, 13Martin G.M. Sprague C.A. Epstein C.J. Lab. Investig. 1970; 23: 86-92PubMed Google Scholar, 14Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell Res. 1992; 202: 267-273Crossref PubMed Scopus (186) Google Scholar). The gene (WRN) defective in Werner syndrome encodes a protein that belongs to the RecQ family of DNA helicases (15Yu 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) that includes four other human helicases, including the genes defective in the chromosomal instability disorders Bloom syndrome (BLM) (16Ellis 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) and Rothmund-Thomson syndrome (RecQL4) (17Kitao 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). In addition to the conserved helicase motifs, WRN contains a region of similarity to the 3′ to 5′ exonuclease domain of Escherichia coli DNA polymerase I and RNase D (18Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (202) Google Scholar). In addition to its catalytic domains, WRN interacts with a number of proteins involved in DNA metabolism, suggesting important roles in cellular pathways of DNA replication, repair, and/or recombination (19Brosh Jr., R.M. Bohr V.A. Exp. Gerontol. 2002; 37: 491-506Crossref PubMed Scopus (68) Google Scholar, 20Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (159) Google Scholar).It is generally believed that RecQ helicases play an important role in the maintenance of genome stability (21Cobb J. Bjergbaek L. Gasser S. FEBS Lett. 2002; 529: 43-48Crossref PubMed Scopus (44) Google Scholar, 22Harrigan J.A. Bohr V.A. Biochimie (Paris). 2003; 85: 1185-1193Crossref PubMed Scopus (40) Google Scholar, 23Wu L. Hickson I.D. Mutat. Res. 2002; 509: 35-47Crossref PubMed Scopus (36) Google Scholar); however, the precise molecular and cellular functions of RecQ helicases are not well understood. Although the DNA substrate specificity of WRN helicase has been studied in some detail (24Brosh 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), the mechanism by which WRN catalyzes DNA unwinding is not known. WRN, like all other DNA helicases characterized to date, utilizes the energy from nucleotide hydrolysis to unwind double-stranded DNA (25Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 26Marians K.J. Struct. Fold. Des. 2000; 8: R227-R235Abstract Full Text Full Text PDF Scopus (34) Google Scholar, 27Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (459) Google Scholar, 28Singleton M.R. Wigley D.B. EMBO J. 2003; 22: 4579-4583Crossref PubMed Scopus (14) Google Scholar). Although the nucleotide preference of WRN helicase and exonuclease activities has been examined (29Kamath-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 (193) Google Scholar, 30Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (181) Google Scholar), little is known about the optimal solution conditions for WRN catalytic activities. Recent work from the Kowalczykowski laboratory (31Harmon F.G. Kowalczykowski S.C. J. Biol. Chem. 2001; 276: 232-243Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) demonstrated that E. coli RecQ helicase activity is sensitive to the ratio of magnesium ion to ATP concentration with an optimal ratio of 0.8 and a free magnesium ion concentration of 50 μm. In addition, E. coli RecQ helicase activity displayed a sigmoidal dependence on ATP concentration, suggesting multiple interacting ATP sites (31Harmon F.G. Kowalczykowski S.C. J. Biol. Chem. 2001; 276: 232-243Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, the assembly state of E. coli RecQ (32Xu H.Q. Deprez E. Zhang A.H. Tauc P. Ladjimi M.M. Brochon J.C. Auclair C. Xi X.G. J. Biol. Chem. 2003; 278: 34925-34933Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and other RecQ helicases (33Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 34Huang S. Beresten S. Li B. Oshima J. Ellis N.A. Campisi J. Nucleic Acids Res. 2000; 28: 2396-2405Crossref PubMed Scopus (130) Google Scholar, 35Janscak 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, 36Karow 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, 37Xue Y. Ratcliff G.C. Wang H. Davis-Searles P.R. Gray M.D. Erie D.A. Redinbo M.R. Biochemistry. 2002; 41: 2901-2912Crossref PubMed Scopus (58) Google Scholar) remains open to debate.Since little is known about the cofactor requirements for WRN helicase and exonuclease activities, we examined these parameters in this study. Evidence is presented that WRN helicase behaves similarly to E. coli RecQ with respect to optimal Mg2+:ATP ratio for DNA unwinding but displays distinct differences with respect to the effects of free Mg2+ ion and ATP concentrations on DNA unwinding activity. The ability of other divalent metals to substitute for magnesium in the WRN helicase reaction is metal-specific, and certain metal ions potently inhibited WRN helicase activity or stimulated WRN exonuclease activity. These results indicate that DNA metabolic processing by WRN helicase or exonuclease activities can be modulated by the availability of free metal ions.To better understand the WRN helicase mechanism, we utilized a fluorometric assay to monitor helicase-catalyzed unwinding of duplex DNA (38Bjornson K.P. Amaratunga M. Moore K.J. Lohman T.M. Biochemistry. 1994; 33: 14306-14316Crossref PubMed Scopus (94) Google Scholar). This assay uses the principle of fluorescence resonance energy transfer to observe the unwinding of duplex DNA in real time. A forked duplex DNA substrate, the preferred B-form DNA structure of WRN helicase (24Brosh 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) with fluorescein (F) 1The abbreviations used are: F, fluorescein; HF, hexachlorofluorescein; ssDNA, single-stranded DNA; nt, nucleotide(s). 1The abbreviations used are: F, fluorescein; HF, hexachlorofluorescein; ssDNA, single-stranded DNA; nt, nucleotide(s). covalently attached to the 3′ blunt end and hexachlorofluorescein (HF) attached to the 5′ blunt end of the duplex DNA molecule, was used for the kinetic analyses (Fig. 1). F is excited at a wavelength of 492 nm and emits a wavelength of 520 nm, which lies in the excitation spectrum of HF (Fig. 1A). Fluorescence resonance energy transfer occurs between F (donor) and HF (acceptor) when both are in close proximity during the duplex state of the substrate. However, upon helicase-catalyzed unwinding of the duplex and separation of the complementary strands, F and HF are no longer in close proximity, and the fluorescence emission from F excitation can be detected by a photosensor (Fig. 1B). This method of measuring helicase-catalyzed unwinding of duplex DNA using fluorescence stopped-flow instrumentation is valuable for kinetic analyses where data are collected continuously throughout the reaction in real time.We applied fluorescence resonance energy transfer technology to study the kinetics of WRN helicase activity under presteady state conditions in which the enzyme is saturated with an excess of DNA substrate (39Nanduri B. Byrd A.K. Eoff R.L. Tackett A.J. Raney K.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14722-14727Crossref PubMed Scopus (81) Google Scholar). The results from presteady state kinetic studies were used to determine the initial burst kinetics of DNA unwinding, enabling us to determine the rate of DNA unwinding by WRN under the assigned reaction conditions. Mechanistic information on the WRN helicase reaction was determined from these analyses and provides the first kinetic parameters of a human RecQ unwinding reaction based on real time measurements.MATERIALS AND METHODSProteins—Recombinant hexahistidine-tagged full-length WRN proteins (wild-type WRN or the exonuclease point mutant WRN-E84A) were overexpressed using a baculovirus/Sf9 insect system and purified as described previously (40Sharma S. Otterlei M. Sommers J.A. Driscoll H.C. Dianov G.L. Kao H.I. Bambara R.A. Brosh Jr., R.M. Mol. Biol. Cell. 2004; 15: 734-750Crossref PubMed Scopus (112) Google Scholar). A recombinant hexahistidine-tagged truncated form of WRN (amino-terminal 368 amino acids, designated N-WRN) was overexpressed using the baculovirus/Sf9 insect system and purified as described previously (41Brosh Jr., R.M. Karmakar P. Sommers J.A. Yang Q. Wang X.W. Spillare E.A. Harris C.C. Bohr V.A. J. Biol. Chem. 2001; 276: 35093-35102Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Purified recombinant UvrD and TraI helicases were kindly provided by Dr. Steve Matson (University of North Carolina at Chapel Hill). Purified recombinant BLM helicase (36Karow 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) was kindly provided by Dr. Ian Hickson (Cancer Research UK Laboratories). Recombinant RECQ1 helicase was overexpressed in insect cells using a baculovirus encoding recombinant human RECQ1 kindly provided by Dr. Alessandro Vindigni (International Centre for Genetic Engineering and Biotechnology) and purified as described elsewhere. 2Cui, S., Arosio, D., Doherty, K. M., Brosh, R. M., Jr., Falaschi, A., and Vindigni, A. (2004) Nucl. Acids Res., in press. DNA Substrates—PAGE-purified oligonucleotides (FLAP26 and TSTEM25 (24Brosh 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)) were purchased from Midland Certified Reagent Co. FLAP26 and TSTEM25 with F and HF, respectively, were obtained from Proligo. DNA substrates were prepared as described previously (24Brosh 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) with the exception that DNA substrates used for fluorometric assays were not 5′-32P end-labeled.Fluorometric Helicase Kinetic Assays—Fluorescence stopped-flow kinetic experiments were performed using Applied Photophysics SX.18MV stopped-flow reaction analyzer. The instrument was equipped with a 150-watt xenon lamp, and the monochromator was set to a slit width of 1 mm. Fluorescein was excited at a wavelength of 492 nm, and the fluorescence emission was monitored at wavelengths greater than 520 nm with the 51300 cut-on filter from Oriel Corp. Experiments were carried out in the two-syringe mode where WRN and ATP were preincubated at 37 °C in one syringe for 1 min, while the DNA substrate was preincubated at 37 °C in the second syringe. Each syringe contained 30 mm HEPES (pH 7.4), 5% glycerol, 40 mm KCl, the indicated concentration of MgCl2, and 100 ng/μl bovine serum albumin. Concentrations of WRN, ATP, and DNA fork substrate in the syringe were double that of the indicated final concentration in the reaction. Equal volumes (60 μl) of sample from both syringes were mixed to initiate the reaction, which took place at 37 °C. One thousand data points were collected from monitoring 20 μl of each kinetic time course reaction. For converting the output data from volts to percent unwinding, a time course with the same set up was performed except with only the fluorescein oligonucleotide instead of the fluorescent forked duplex substrate. Data were then normalized by defining the voltage obtained with the fluorescein oligonucleotide as 100% unwinding.Radiometric Helicase Assays—Helicase assay reactions (20 μl) contained 30 mm HEPES (pH 7.4), 5% glycerol, 40 mm KCl, 100 ng/μl bovine serum albumin, 0.8 nm DNA fork substrate, and the indicated concentrations of MgCl2 and/or specified metal, ATP, and WRN. For time course assays imitating the fluorometric assay, WRN and ATP were preincubated in a tube together, while DNA was preincubated separately at 37 °C for 1 min. Reactions were initiated by mixing the contents of both tubes together, and 20-μl samples were quenched at the indicated times. For metal inhibition or substitution assays, the metal and WRN were preincubated on ice for 3 min before the initiation of a 15-min reaction at 37 °C by adding DNA fork substrate. Reactions were quenched with 20 μl of stop buffer (35 mm EDTA, 0.6% SDS, 25% glycerol, 0.04% bromphenol blue, 0.04% xylene cyanol) containing a 10-fold excess of unlabeled oligonucleotide of the same sequence as the labeled strand of the DNA fork substrate. 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 ImageQuant software (Amersham Biosciences). The percent helicase substrate unwound was calculated by the formula: percent unwinding = 100 × (P/(S + P)) where P is the product and S is the residual substrate. The values of P and S have been corrected after subtracting background values in controls having no enzyme and heat-denatured substrate, respectively. Helicase data represent the mean of at least three independent experiments with mean ± S.D. shown by error bars.Radiometric Exonuclease Assays—Exonuclease assay reactions (20 μl) contained the same components as the radiometric helicase assays except that Zn2+ was substituted for Mg2+ where indicated. The metal was preincubated with WRN for 3 min on ice before initiating the 15-min reaction at 37 °C with radiolabeled fork substrate. Each reaction was quenched with 10 μl of stop buffer (80% formamide, 0.5× Tris borate-EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue) and heated at 95 °C for 10 min. Products were resolved on denaturing 14% polyacrylamide gels and visualized by a PhosphorImager.RESULTSTo better understand the catalytic functions of the Werner syndrome protein and how they may be important in DNA metabolism, we investigated the cofactor requirements for WRN helicase and exonuclease activities. In addition, we examined the kinetics of WRN-catalyzed DNA unwinding to better understand its helicase mechanism.Fluorescence Resonance Energy Transfer for Measuring WRN Helicase Unwinding of Duplex DNA—To begin to understand the mechanism of duplex DNA unwinding catalyzed by the WRN helicase, we performed kinetic analyses of the unwinding reaction using a fluorometric helicase assay and a more traditional gel-based assay that uses a radiolabeled DNA substrate. The advantage of the fluorometric assay is that helicase activity can be measured in real time, enabling one to determine kinetic parameters that rely on fast kinetic analysis in the millisecond scale. The conventional gel-based assay enabled us to conveniently determine optimal divalent cation and ATP concentrations for WRN helicase activity using smaller amounts of enzyme and to substantiate our helicase data from fluorescence assays to verify that we were, in fact, measuring DNA unwinding. Combining these approaches enabled us to investigate the kinetics of WRN helicase activity under optimized reaction conditions.The DNA substrate that we elected to use for these studies was based on our previous work (24Brosh 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) that defined the minimal length requirements for the 5′ and 3′ single-stranded DNA (ssDNA) tails of forked duplex substrates required by WRN for efficient unwinding. Based on these results, we examined WRN helicase activity on an optimal forked DNA substrate with a 19-bp duplex flanked by tail lengths of 25 nucleotides (3′) and 26 nucleotides (5′). As shown in Fig. 1, F was positioned on the 3′ blunt end, whereas HF was positioned on the 5′ blunt end. Since the 3′ to 5′ exonuclease activity of WRN can attack the 3′ blunt end of a forked duplex DNA molecule (42Opresko 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 (85) Google Scholar), it was a concern to optimize WRN helicase activity while minimizing or obstructing its exonuclease activity. Under conditions of DNA unwinding (presence of ATP), the DNA oligonucleotides of the helicase substrate lacking the F and HF moieties were not appreciably degraded by WRN exonuclease activity as judged by quantitation of DNA products resolved on denaturing gels (24Brosh 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). However, it was possible that a minimal amount of fluorescent signal may have been attributed to a small population of DNA helicase substrates whose fluorescein molecules had been removed by WRN exonuclease activity. Denaturing gel analysis of WRN reaction products from a radiolabeled helicase substrate possessing the F and HF moieties demonstrated that WRN exonuclease activity was completely blocked from attacking the DNA substrate even in the absence of ATP (data not shown). From these results, we were able to conclude that the F/HF conjugates on the blunt end of the helicase substrate effectively blocked WRN exonuclease activity. Hence we did not have to be concerned about the interference of any fluorescent signal attributed to the incision of the F label on the 3′ blunt end by WRN exonuclease activity.To substantiate the fluorescence-based helicase assay, we collected and compared data from fluorometric and radiometric assays. This was accomplished by performing WRN helicase reactions under identical conditions using either assay and then comparing the results from fluorescence measurements with those from native gel analysis of radiolabeled products obtained by chemical quench at specific time points throughout the 200-s time course. For the radiometric assay, the fluorescent DNA substrate (with covalently attached F and HF moieties) was 5′-32P end-labeled. Throughout the fluorescence-based time course, we observed the output voltage signal increase with time that was presumably due to the increase in the number of excited F molecules that were no longer in close proximity to HF upon unwinding of the duplex substrate by WRN. The fluorescence data were normalized by defining the voltage obtained with only the F-labeled single-stranded oligonucleotide as 100% unwinding of the substrate (43Xu H.Q. Zhang A.H. Auclair C. Xi X.G. Nucleic Acids Res. 2003; 31: 1-7PubMed Google Scholar). This enabled us to convert the voltage output data to percent unwinding. The results from these parallel kinetic experiments using 0.8 nm DNA substrate and 12 nm WRN are shown in Fig. 2A. Helicase data points from the radiometric assay (large filled circles) were overlaid on the normalized fluorometric helicase data and found to agree well. This comparison substantiated the fluorometric assay for measuring WRN helicase activity.Fig. 2Kinetic analyses of DNA unwinding by WRN helicase using a fluorometric stopped-flow assay. Helicase reaction mixtures contained 0.8 nm forked DNA substrate, 2 mm ATP, 8 mm MgCl2, and the indicated concentrations of WRN helicase. A, correlation of kinetic data between fluorometric stopped-flow analyses and radiometric quenched-flow analyses of WRN helicase reactions containing 12 nm WRN. The data from the radiometric assay (large filled circles) were overlaid with the data from the fluorometric assay. The plot shows percent unwinding against time (s). A representative gel of the radiometric time course assay is shown: lane 1, no enzyme (NE) control; lanes 2–14, reaction time points quenched at 5, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, and 180 s, respectively; lane 15, heat-denatured substrate control. B, fluorometric unwinding data as a function of WRN protein concentration.View Large Image Figure ViewerDownload (PPT)Next we examined WRN helicase activity using the fluorometric assay at a fixed concentration of forked duplex DNA substrate (0.8 nm) and a range of WRN protein concentrations. At each WRN concentration, fluorescence data were obtained for a time course of 200 s. For each WRN protein concentration, the fluorescent signal (voltage) increased with respect to time. At lower WRN concentrations (0.6 and 3 nm), output voltage continued to increase throughout the time course, whereas a signal plateau was approached by 100 s in reactions containing higher WRN concentrations (16 or 32 nm). The fluorescent traces from reactions containing 16 and 32 nm WRN were superimposable, suggesting that the DNA substrate was saturated with WRN enzyme at these concentrations. The data were normalized and converted to percent unwinding with respect to time as shown in Fig. 2B. Initial rates of unwinding were determined for each time course and are listed in Table I. As WRN protein concentration increased, initial rates of unwinding also increased. However, this increase in initial rates was seen only up to the saturation point (i.e. 16 nm WRN).Table IKinetics of WRN-catalyzed DNA unwinding as a function of WRN protein concentration[WRN]Initial rate of unwindingnmnm bp/s0.60.0197630.167260.2128120.3648160.5624320.5624 Open table in a new tab Optimal ATP-Mg2+Concentration for WRN Helicase Activity—Before initiating a kinetic analysis using the fluorometric assay, we were interested in optimizing the reaction conditions for WRN helicase activity, which had not been done previously. Since the ATP-Mg2+ complex is the source of chemical energy for helicase-catalyzed unwinding and therefore a critical component of the helicase reaction, we focused our efforts to determine optimal concentrations of Mg2+ and ATP for WRN helicase activity. Initially we sought to determine an estimated ratio between ATP and Mg2+ where the greatest initial rate of WRN unwinding would be detected and then to determine the optimal ATP-Mg2+ concentration. For these purposes, we used the radiometric assay because significantly smaller reaction volumes can be used compared with the fluorometric assay. The optimal Mg2+:ATP ratio was determined by maintaining ATP at a fixed concentration of 2 mm and titrating in various concentrations of MgCl2. A 20-s time course was performed at each MgCl2 concentration, and the initial rates of unwinding were plotted as a function of the [Mg2+]: [ATP] ratio (Fig. 3). WRN helicase activity increased fairly linearly with Mg2+:ATP ratios up to 1; greater Mg2+:ATP ratios of up to 4 did not significantly further increase or decrease WRN unwinding. A Mg2+:ATP ratio of 1 was determined to be optimal for WRN helicase activity. This was confirmed by carrying out the same set of experiments using 5 mm ATP, which gave a profile similar to that obtained with 2 mm ATP (Fig. 3). Each set of data was best fit to a sigmoidal curve, which displayed the optimum at a Mg2+:ATP ratio of 1.Fig. 3Dependence of WRN helicase activity on Mg2+ ion concentration. Helicase reaction mixtures contained 0.8 nm forked DNA substrate, 5 nm WRN, and 2 mm ATP with increasing concentrations of MgCl2 from 0 to 8 mm (closed circles) or 5 mm ATP with increasing concentrations of MgCl2 from 0 to 20 mm (open circles). The radiometric assay was used to measure initial rates of WRN helicase activity with quench points at 6, 10, 15, and 20 s. Initial rates of unwinding (mean value of three independent experiments with S.D. indicated by error bars) are plotted as a function of [Mg2+]:[ATP] (mm:mm) ratio. Each data set was best fit to a sigmoidal curve as shown with a solid line for 2 mm ATP and a dashed line for 5 mm ATP.View Large Image Figure ViewerDownload (PPT)We next performed experiments to determine the optimal concentration of ATP-Mg2+ for WRN helicase activity. Twenty-second time courses were performed for a series of WRN helicase reactions in which the ATP-Mg2+ complex, maintained at a 1:1 ratio, was increased. As shown in Fig. 4, initial rates of WRN helicase activity increased with an initial hyperbol