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• W2057808601 abstract "The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity. The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity. The transient formation of single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; HRDC, helicase and RNase D C-terminal; FRET, fluorescence resonance energy transfer; PAR, 4-(2-pyridylazo)resorcinol disodium salt; mantATP, 2′(3′)-O-(N-methlanthraniloyl)adenosine 5′-triphosphate; DTT, dithiothreitol; CD, circular dichroism. 1The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; HRDC, helicase and RNase D C-terminal; FRET, fluorescence resonance energy transfer; PAR, 4-(2-pyridylazo)resorcinol disodium salt; mantATP, 2′(3′)-O-(N-methlanthraniloyl)adenosine 5′-triphosphate; DTT, dithiothreitol; CD, circular dichroism. intermediate is essential to all aspects of DNA metabolism including DNA replication, recombination, and repair. The unwinding and separation of the individual strands of double-stranded DNA (dsDNA) is catalyzed by a class of specialized enzymes known as DNA helicases (1Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (665) Google Scholar, 2Soultanas P. Wigley D.B. Curr. Opin. Struct. Biol. 2000; 10: 124-128Crossref PubMed Scopus (98) Google Scholar). These enzymes function as molecular motors that use the energy released from the hydrolysis of ATP to unwind and translocate along DNA in a sequential fashion (3Singleton M.R. Wigley D.B. J. Bacteriol. 2002; 184: 1819-1826Crossref PubMed Scopus (170) Google Scholar, 4Singleton M.R. Wentzell L.M. Liu Y. West S.C. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13492-13497Crossref PubMed Scopus (169) Google Scholar, 5Betterton M.D. Jülicher F. Phys. Rev. Lett. 2003; 91: 258103Crossref PubMed Scopus (48) Google Scholar). These ubiquitous enzymes have been identified in all living organisms from virus to human. It appears that they evolved from a common ancestor (6Hall M.C. Matson S.W. Mol. Microbiol. 1999; 34: 867-877Crossref PubMed Scopus (268) Google Scholar). The RecQ helicase family is critical to the maintenance of genomic stability in prokaryotes and eukaryotes (7Chakraverty R.K. Hickson I.D. BioEssays. 1999; 21: 286-294Crossref PubMed Scopus (196) Google Scholar). Mutations of RecQ genes can lead to genomic instability and several human diseases including the Bloom and Werner syndromes (8Furuichi Y. Ann. N. Y. Acad. Sci. 2001; 928: 121-131Crossref PubMed Scopus (56) Google Scholar). Recently, it has been shown that the tumor suppressor BRCA1-associated protein, BACH1, which shares homologies with other members of the RecQ family, possesses ATPase and helicase activities (9Cantor S. Drapkin R. Zhang F. Lin Y. Han J. Pamidi S. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2357-2362Crossref PubMed Scopus (190) Google Scholar). The mutant BACH1 participates directly in breast and ovarian cancer development (9Cantor S. Drapkin R. Zhang F. Lin Y. Han J. Pamidi S. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2357-2362Crossref PubMed Scopus (190) Google Scholar). The RecQ helicase from Escherichia coli is the prototype helicase of this family (10Umezu K. Nakayama K. Nakayama H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5363-5367Crossref PubMed Scopus (226) Google Scholar) and has been shown to initiate homologous recombination as well as suppress illegitimate recombination (11Hanada K. Iwasaki M. Ihashi S. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5989-5994Crossref PubMed Scopus (33) Google Scholar, 12Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Crossref PubMed Scopus (234) Google Scholar). The RecQ helicase family members contain a helicase domain characterized by the presence of seven so-called “helicase” motifs necessary for using energy derived from ATP binding and hydrolysis to unwind DNA (13Bennett R.J. Keck J.L. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 79-97Crossref PubMed Scopus (85) Google Scholar, 14Caruthers J.M. McKay D.B. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (446) Google Scholar). Sequence analyses revealed that all of the RecQ helicases contain a C-terminal extension that can be further divided into two domains (Fig. 1A): the HRDC domain (helicase and RNase D C-terminal), which functions as an auxiliary DNA-binding domain (15Morozov V. Mushegian A.R. Koonin E.V. Bork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 16Liu Z. Macias M.J. Bottomley M.J. Stier G. Linge J.P. Nilges M. Bork P. Sattler M. Struct. Fold. Des. 1999; 7: 1557-1566Abstract Full Text Full Text PDF Scopus (118) Google Scholar), and the RecQ C-terminal domain that contains a conserved CXnCXnCDXC motif (in which X is any amino acid) among the RecQ family of helicases (Fig. 1A) of which the function is still not clear. Recently, the three-dimensional structure of a C-terminal truncated form of RecQ helicase has revealed that the enzyme folds into four subdomains, two of which combine to form the helicase region, whereas the others form zinc binding (Fig. 1B) and winged-helix motifs (17Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (212) Google Scholar). The zinc atom is bound by four conserved cysteine residues located at a platform composed of α-helices17 and 18. The cysteine residue Cys380 (labeled as C1) is located at the beginning of the α-helix 17. Cys400 (labeled as C3) and Cys403 (labeled as C4) are at the beginning and the middle of the α-helix 18, respectively, whereas Cys397 (labeled as C2) is located in the loop linking the two helices (Fig. 1B). In addition, the zinc finger motif may be further stabilized by three hydrogen bonds formed among the conserved residues of phenylalanine (Phe374), arginine (Arg381), and asparagine (Asp401) (Fig. 1B). Previous studies have established that the zinc finger domains and other metal-binding protein domains are involved in diverse functions including protein-DNA interactions, protein folding, and protein-protein interactions (18Berg J.M. Shi Y. Science. 1996; 271: 1081-1085Crossref PubMed Scopus (1646) Google Scholar). To elucidate the roles played by the zinc atom and the zinc finger motif in RecQ helicase, mutant RecQ molecules were engineered by site-directed mutagenesis within the zinc finger motif. Biochemical characterizations of these mutants showed that the zinc binding motif is essential to efficient DNA binding and stabilization of the three-dimensional structure of RecQ molecules. [α-32P]ATP was obtained from Amersham Biosciences. 4-(2-Pyridylazo)resorcinol disodium salt (PAR), EDTA, 2-mercaptoethanol, ATP, and α-chymotrypsin were obtained from Sigma. Chelex® 100 resin was purchased from Bio-Rad. The N-methylanthraniloyl derivatives of adenine nucleotides were synthesized according to Hiratsuka (19Hiratsuka T. Biochim. Biophys. Acta. 1982; 719: 509-517Crossref PubMed Scopus (51) Google Scholar) and purified on DEAE-cellulose using a gradient of triethylammonium bicarbonate. Expression of Wild-type Enzyme and Construction of the Zinc Finger Mutant of RecQ Helicase—The E. coli RecQ helicase containing a N-terminal His tag was expressed in E. coli and purified by nickelchelating and anion-exchange chromatography as described previously (20Xu 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). Mutations were made by a two-step polymerase chain reaction method (21Iwaasa M. Umeda S. Ohsato T. Takamatsu C. Fukuoh A. Iwasaki H. Shinagawa H. Hamasaki N. Kang D. J. Neurochem. 2002; 82: 30-37Crossref PubMed Scopus (6) Google Scholar). The outside primers were QFN (5′-GGAATTCCATATGGTGAATGTGGCGCAGGCGGAAGTGTTG-3′) and QRX (5′-CCGCTCGAGCTACTCTTCGTCATCGCCATCAACATG-3′). The primers used to introduce mutations are QMF1 (5′-CAGGAGCCGAACGGGAACAACGATATCTGC-3′) and QMF2 (5′-GCAGATATCGTTGTTCCCGTTCGGCTCCTG-3′). The mutations resulting in coding changes are underlined. The mutant polymerase chain reaction products were digested with NdeI and XhoI, and the 1.85-kb fragment was subcloned into pET-15b. Following the mutagenesis, the entire gene was sequenced to ensure that no additional mutations were created. The primers used to construct other mutants that could not be purified to homogeneity are not shown. Quantification of Zinc Ion Bound to E. coli RecQ Helicase—The zinc content of the wild-type and the zinc finger mutant of RecQ helicase was measured by the PAR assay as described by Hunt et al. (22Hunt J.B. Neece S.H. Ginsburg A. Anal. Biochem. 1985; 146: 150-157Crossref PubMed Scopus (209) Google Scholar). PAR has a low absorbance at 500 nm in the absence of zinc ion. However, in the presence of zinc ion, the absorbance at 500 nm increases dramatically due to the formation of the PAR2·Zn2+ complex. To more precisely quantify the zinc content of both wild-type and mutant helicases, all of the buffers were treated with Chelex 100 resin. The enzymes were dialyzed against the EDTA-free Chelex-treated buffer passed over a 10-cm column of Chelex-100 and reconcentrated. To facilitate zinc release, the enzymes (1 nmol in a volume of 20 μl) were first denatured with Chelex-treated 7 m guanidine HCl and then transferred to a 1-ml cuvette and the volume was adjusted to 0.9 ml with buffer A (20 mm Tris-HCl at pH 8.0, 150 mm NaCl). PAR was added into the cuvette for a final concentration of 100 μm. The absorbance at 500 nm was measured. The quantity of zinc ion was determined from a standard curve of ZnCl2 samples in a range of concentrations using the sample preparation procedure as described above with the RecQ helicase omitted. The zinc ion concentration was also determined using the absorbance coefficient for the (PAR)2·Zn2+ complex (ϵ500nm = 6.6 × 104m–1 cm–1). The zinc-demetalated RecQ helicase was obtained by dialysis of purified RecQ helicase against buffer B (10% glycerol, 300 mm NaCl, 20 mm Tris-HCl at pH 8.0, 10 mm EDTA, 1 mm DTT) overnight at 4 °C (17Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (212) Google Scholar). Fluorescence Measurements—Fluorescence spectra were determined using Fluoro Max-2 spectrofluorimeter (Jobin Yvon, Spex Instruments S.A., Inc.) as described by Levin et al. (23Levin M.K. Gurjar M.M. Patel S.S. J. Biol. Chem. 2003; 278: 23311-23316Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Some results were further confirmed by using PiStar-180 spectrometer (Applied Photophysics). In a 10 × 10 × 40-mm3 quartz cuvette, 0.5 μm RecQ protein in 1 ml of reaction buffer was excited at 280 nm and the fluorescence emission was monitored at 350 nm. mantATP binding to protein was measured by exciting the RecQ protein at 280 nm and measuring the fluorescence of mantATP at 440 nm because of FRET. The observed fluorescence intensity, Fobs, was corrected for inner filter and sample dilution effect according to Equation 1, F=Fobs×10[(AEX+AEM)/2]×V0+ViV0 (Eq. 1) where F is the corrected fluorescence intensity, V0 is the initial sample volume, Vi is the total volume of titrant added, and AEX and AEM are the absorbance values of the solution at excitation and emission wavelengths, respectively. The statistical thermodynamic model used to describe the ATP-RecQ complex formation is based on the observation that RecQ helicase has one ATP-binding site (17Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (212) Google Scholar). Therefore, the one-to-one binding model was used to establish the mantATP binding to RecQ. We have chosen the macroscopic interaction constant defined in Equation 2, Kd=[Ef][Df]/[C] (Eq. 2) where Ef represents the concentration of free enzyme, Df represents the concentration of free mantATP, Kd is the equilibrium interaction constant, and C is the concentration of the ATP-RecQ complex formed. The concentrations of total enzyme, ET, and total mantATP, DT, can be written in terms of Ef and Df as shown in Equations 3 and 4. ET=Ef+C (Eq. 3) DT=Df+C (Eq. 4) From Equations (Eq. 2), (Eq. 3), (Eq. 4), the expression of C is obtained as shown in Equation 5. C=(DT+ET+Kd)−(DT+ET+Kd)2−4ETDT2 (Eq. 5) The value of Kd can be obtained by fitting the fluorescence intensity values to Equation 6, F=FS+fDDT+fCC (Eq. 6) where FS is the starting fluorescence of the reaction mixture, fD is the fluorescence coefficient of free mantATP, and fC is the fluorescence coefficient of complex formed. Limited Proteolytic Digestion—The wild-type, mutant, and Zn2+-extracted RecQ helicases at a concentration of 15 μm and in a total volume of 30 μl were digested with α-chymotrypsin (Sigma) for 2 and 10 min at room temperature. The ratio between the protease and RecQ was 1:100 for each helicase. Aliquots (15 μl corresponding to 1 μg of protein) from the reaction were quenched with 15 μl of gel-loading buffer (250 mm Tris-HCl at pH 6.8, 3.4% SDS, 1.1 m 2-mercaptoethanol, 20% glycerol, and 0.01% bromphenol blue). The samples were boiled for 2 min and then analyzed by SDS-PAGE gel. Agarose Gel Mobility Shift Assay—Gel mobility shift assays were performed as described previously (24Dou S.X. Wang P.Y. Xu H.Q. Xi X.G. J. Biol. Chem. 2004; 279: 6354-6363Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The reaction mixture (25 μl) contained 20 mm Tris-HCl at pH 7.6, 1 mm DTT, 0.1 mg/ml bovine serum albumin, 2 mm MgCl2, and 1 pmol (molecules) of the linearized DNA substrate (3 kb). The reaction was allowed to proceed for 10 min on ice and was terminated by the addition of 10 μl of loading buffer (80% glycerol, 0.1% bromphenol blue). The complexes were separated by electrophoresis through 0.8% agarose gel in TAE (Tris-acetate-EDTA) buffer at 100 V for 1.5 h and were visualized by ethidium bromide staining. DNA Binding Assay under Equilibrium Condition—The binding of RecQ helicase to DNA was analyzed by fluorescence anisotropy using a Beacon 2000 fluorescence polarization spectrophotometer (PanVera) as described previously (24Dou S.X. Wang P.Y. Xu H.Q. Xi X.G. J. Biol. Chem. 2004; 279: 6354-6363Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). An appropriate quantity of fluorescein-labeled ssDNA or dsDNA was added to a standard titration buffer (150 μl of total volume) in a temperature-controlled cuvette at 25 °C. The anisotropy of the fluorescein-labeled DNA was measured successively until it stabilized. An appropriate quantity of RecQ helicase then was added. The anisotropy then was measured continuously until it reached a stable plateau. To determine the concentration of the helicase-DNA complex, fluorescence signals observed in these RecQ helicase titrations were subtracted by those observed in the absence of enzyme. The increase in sample volume during the titration was taken into account in the analysis of the data. The reported values represent the averages of two to three measurements. The ssDNA used in this assay is a 36-mer 5′-fluorescein-labeled synthetic oligonucleotide (5′-F-AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-3′). The dsDNA was generated with its complementary sequence (5′-ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTCT-3′). Circular Dichroism Analysis—All of the spectra were collected in buffer C (20 mm Tris-HCl, 25 mm NaCl, 0.1 mm EDTA) in a 0.2-mm cuvette at 20 °C on a Jobin-Yvon V dichrograph spectrophotometer. The protein samples were rigorously dialyzed against buffer C before measurement. All of the spectra were recorded with a 0.2-nm step as follows. Buffer C only was added to a single chamber, and the spectrum was recorded. The protein sample then was added to the same chamber, and the spectrum was recorded. The protein-only spectrum then was obtained by subtracting the free protein spectrum from the mixture spectrum. The measurement results are reported as mean residue ellipticity (θ) (degrees per square centimeter per decimole). The ATPase activity, helicase activity, and protein concentration were determined before and after each measurement. ATPase Assay and Determination of Ki for mantATP—The ATPase activity was determined in an assay by measuring the radioactive 32Pi liberated during hydrolysis (25Dessinges M.N. Lionnet T. Xi X.G. Bensimon D. Croquette V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6439-6444Crossref PubMed Scopus (159) Google Scholar). The measurement was carried out at 37 °C in a reaction mixture containing 1.5 μm (nucleotide) of heat-denatured HindIII-cut pGEM-7Zf linear DNA (3 kb) or ssDNA (60-mer) at the indicated concentration of ATP. The reactions were initiated by the addition of RecQ helicase into 100 μl of reaction mixture and stopped by pipetting 80 μl of aliquots from the reaction mixture every 30 s into a hydrochloric solution of ammonium molybdate. The liberated radioactive 32Pi was extracted with a solution of 2-butanol-benzene-acetoneammonium molybdate (750:750:15:1) saturated with water. An aliquot of the organic phase was counted in 6 ml of Aquasol. The competitive inhibition constant, Ki, for mantATP was determined by measuring the ATPase rate as a function of mantATP concentration and fit to Equation 7, v=[ATP]kcat[ATP]+KmATP(1+[mantATP]Ki) (Eq. 7) where v is the initial ATPase rate, kcat is the catalytic constant, and KATPm is the Km for ATP. Helicase Assay—An unwinding assay was performed using the Beacon 2000 fluorescence polarization instrument (26Xu H.Q. Zhang A.H. Auclair C. Xi X.G. Nucleic Acids Res. 2003; 31: e70Crossref PubMed Scopus (41) Google Scholar). An appropriate quantity of fluorescein-labeled duplex oligonucleotide was added to the helicase-unwinding buffer (150 μl of total volumes) in a temperature-controlled cuvette. The anisotropy was measured successively until it stabilized. Helicase then was added. When the higher anisotropy value became stable, the unwinding reaction was initiated by the rapid addition of ATP solution to give a final ATP concentration of 1 mm. The decrease of the anisotropy was recorded every 8 s until it became stable. The unwinding buffer contained 25 mm Tris-HCl, pH 8, 30 mm sodium chloride, 3 mm magnesium acetate, and 0.1 mm DTT. The data were fit to the exponential equation: A = A0exp(–kobst), where A is the anisotropy amplitude at time t, A0 is a constant, and kobs is the observed rate constant. Size Exclusion Chromatography—Size exclusion chromatography was performed at 18 °C using an fast protein liquid chromatography system (ΔKTA, Amersham Biosciences) on a Superdex 200 (analytical grade) column equilibrated with elution buffer. Fractions of 0.5 ml were collected at a flow rate of 0.4 ml/min, and the absorbance was measured at 280 and 260 nm. The proteins used to prepare a calibration curve were as follows: thyroglobulin (bovine), 670 kDa; γ-globulin (bovine), 443 kDa; apoferritin, 158 kDa; bovine serum albumin, 66 kDa; ovalbumin, 44 kDa; and myoglobin, 17 kDa. Gel filtration chromatography was performed using a standard elution buffer (50 mm Tris-Cl at pH 7.5, 300 mm NaCl, 0.1 mm EDTA) with 1 mm ATP and 1 mm Mg(OAc)2.5 μm RecQ protein was incubated in the elution buffer with ATP and MgCl2 for 2 min prior to injection onto the column. Rationale for Site-directed Mutagenesis of the Zinc Finger Motif of RecQ Helicase—Site-directed mutagenesis was used to explore the functional significance of the putative zinc finger motif of the RecQ helicase. As shown in Fig. 1, six residues (Cys380, Arg381, Cys397, Cys400, Asp401, and Cys403) within this region are totally conserved among RecQ family members. Mutant RecQ enzymes were first engineered with single alanine or serine substitution at the position of each of the four cysteines. In addition, a careful analysis of the three-dimensional structure of the enzyme revealed that the conformation of the zinc finger motif is obviously stabilized through three hydrogen bonds among the highly conserved residues, Arg381, Asp401, and Phe374 (Fig. 1). These interactions may contribute to the relative positioning of the helices α16, α17, and α18 in the zinc finger motif (Fig. 1B). The residues Arg381 and Asp401 were thus replaced by asparagine and alanine, respectively. We found that all of these mutants displayed a high level of proteolysis and could not be purified to homogeneity, making functional studies impossible. To disturb mildly the zinc finger motif, we decided to simultaneously substitute C2 and C3 with asparagine. This residue was chosen, because its side chain could conceivably act as ligand to zinc ion (27Simpson R.J. Cram E.D. Czolij R. Matthews J.M. Crossley M. Mackay J.P. J. Biol. Chem. 2003; 278: 28011-28018Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Moreover, the single mutation with asparagine may have only a modest effect, whereas a double mutation should lead to a zinc finger motif that no longer binds to zinc ion. The expression analysis of these mutants shows that both single point mutants (C397N and C400N) became the inclusion body and could not be purified. However, ∼50% of the expressed double mutant RecQ helicase (C397N/C400N) is soluble and is purified to homogeneity. Based on Sypro Orangestained SDS-PAGE and electrospray mass spectrometry analyses, the purity of this modified RecQ helicase was determined to be >92%. The ability of the purified double mutant enzyme to bind zinc was determined using the PAR assay. Although 0.98 mol of zinc were bound to 1 mol of the wild-type protein, only 0.02 mol of zinc was bound to 1 mol of the double mutant enzyme (Table I). These results are consistent with both the prediction from the primary amino acid sequence and the study of the three-dimensional structure of RecQ helicase, indicating that the enzyme contains a functional zinc-binding domain.Table IComparison of the basic properties of wild-type, double mutant, and zinc-demetalated helicasesHelicase[Zn2+]/[protein]Stokes radiusCatalysisaDetermined from Fig. 4.DNA bindingbDetermined from Fig. 5B. ND, non detectable, i.e., no DNA binding detected up to 3 μm protein.ATP bindingcDetermined from Fig. 3D.kcat(ATPase)kcat(Helicase)Kd (ssDNA)Kd (dsDNA)Kd (mantATP)s-1nmμmWild type0.98 ± 0.1135.236.1 ± 1.12 ± 0.058 × 10-252 ± 1.281 ± 1.143.6 ± 1.1Mutant (C397N/C400N)0.02 ± 0.01535.600NDbDetermined from Fig. 5B. ND, non detectable, i.e., no DNA binding detected up to 3 μm protein.NDbDetermined from Fig. 5B. ND, non detectable, i.e., no DNA binding detected up to 3 μm protein.56.8 ± 1.3Zinc-demetalated0.28 ± 0.0534.534.6 ± 1.21.8 ± 0.089 × 10-265 ± 2.198 ± 1.347.6 ± 0.9a Determined from Fig. 4.b Determined from Fig. 5B. ND, non detectable, i.e., no DNA binding detected up to 3 μm protein.c Determined from Fig. 3D. Open table in a new tab To assess whether zinc ion is required for RecQ helicase function, zinc ion was extracted from wild-type enzyme by extensive dialysis against the dialysis solution (20 mm Tris-HCl at pH 7.9, 150 mm NaCl, 1 mm DTT, 5% glycerol) containing 15 mm EDTA. The obtained zinc-extracted wild-type helicase was termed zinc-demetalated RecQ helicase. Therefore, three preparations of enzymes were used for the following studies: the wild-type helicase; the double mutant helicase; and the zinc-demetalated helicases. Structural Characterization of the Mutant Protein—It is well established that metal ions have important effects on secondary structure formation. We were wondering whether the purified double mutant RecQ helicase (C397N/C400N) has a normal structure. We first performed CD studies to check the effect of mutation on the secondary structure of the protein. The replacement of both cysteines with asparagines leads to a subtle modification in the secondary structure of RecQ helicase as judged from the CD spectra (Fig. 2A), suggesting that the double mutation induced slightly a local conformational change. In contrast, no significant CD spectra change was observed with the zinc-demetalated RecQ helicase. We next performed the limited proteolysis experiments on wild-type, mutant, and zinc-demetalated helicases under the same experimental conditions. Fig. 2B shows that both mutant and zinc-demetalated proteins display similar proteolysis-resistant patterns as the wild-type RecQ helicase, suggesting that the three proteins assume similar structures. These results have been further confirmed by size-exclusion chromatographic studies. Because both ultracentrifugation analyses and three-dimensional structure studies have shown that RecQ helicase takes a globular shape (17Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (212) Google Scholar, 20Xu 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), the apparent molecular masses of wild-type, double mutant, and zinc-demetalated helicases were used to estimate their Stokes radii, thus determining their overall spherical shapes. As shown in Table I, these helicases have similar Stokes radii. Taken together, these results indicate that these enzymes (wild-type, double mutant, and zinc-demetalated helicases) possess similar three-dimensional structures. Zinc Finger Motif Is Not Required for ATP Binding—We next studied the binding of ATP to the double mutant using a fluorescent nucleotide analogue (Fig. 3A, mantATP). The spectral properties of the mant fluorophore are ideally suited for monitoring nucleotide binding by FRET from the intrinsic tryptophan fluorescence of RecQ to the mant fluorophore bound at the ATP-binding site. The comparison between the fluorescence excitation and emission spectra of RecQ and those of mantATP showed that the emission spectrum of RecQ overlaps with the excitation spectrum of mantATP (Fig. 3B), indicating the possibility of FRET. Although the three-dimensional structure of RecQ helicase has shown that the enzyme has only one ATP-binding site, we want to first determine whether mantATP binds to the same ATP-binding site of RecQ. For this purpose, the competitive inhibition constant, Ki, for mantATP was determined by measuring the ATPase rate of the helicase as a function of mantATP concentration (Fig. 3C). The data were fit to a competitive inhibition equation with a Ki of 85 μm, indicating that mantATP binds competitively to the ATP-binding site. The apparent Kd values for the wild-type, double mutant, and zinc-demetalated helicases were measured using standard fluorimetric titration methods. From the titration curves as shown in Fig. 3D, the apparent Kd values determined are 43.6 μm for the wild-type helicase, 56.8 μm for the double mutant helicase, and 47.6 μm for the zinc-demetalated helicase, revealing that neither the zinc finger nor zinc ion is essential to ATP binding. This study sheds light not only on ATP binding but also on folding of the mutant protein. The fact that the double mutant protein binds ATP normally indicates that the overall three-dimensional structure of the double mutant was not altered. It is also interesting to no" @default.
• W2057808601 created "2016-06-24" @default.
• W2057808601 creator A5009837778 @default.
• W2057808601 creator A5015600384 @default.
• W2057808601 creator A5018344454 @default.
• W2057808601 creator A5035739001 @default.
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• W2057808601 date "2004-10-01" @default.
• W2057808601 modified "2023-09-29" @default.
• W2057808601 title "The Zinc Finger Motif of Escherichia coli RecQ Is Implicated in Both DNA Binding and Protein Folding" @default.
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