The Zinc Finger Motif of Escherichia coli RecQ Is Implicated in Both DNA Binding and Protein Folding*

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) 1 intermediate is essential to all aspects of DNA metabolism including DNA replication, recombination, and repair. The unwinding and separation of the individual strands of doublestranded DNA (dsDNA) is catalyzed by a class of specialized enzymes known as DNA helicases (1,2). 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 (3)(4)(5). These ubiquitous enzymes have been identified in all living organisms from virus to human. It appears that they evolved from a common ancestor (6).
The RecQ helicase family is critical to the maintenance of genomic stability in prokaryotes and eukaryotes (7). Mutations of RecQ genes can lead to genomic instability and several human diseases including the Bloom and Werner syndromes (8). 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 (9). The mutant BACH1 participates directly in breast and ovarian cancer development (9). The RecQ helicase from Escherichia coli is the prototype helicase of this family (10) and has been shown to initiate homologous recombination as well as suppress illegitimate recombination (11,12).
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 (13,14). 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 (15,16), and the RecQ C-terminal domain that contains a conserved CX n CX n CDXC 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 Cterminal 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 (17). The zinc atom is bound by four conserved cysteine residues located at a platform composed of ␣-helices17 and 18. The cysteine residue Cys 380 (labeled as C1) is located at the beginning of the ␣-helix 17. Cys 400 (labeled as C3) and Cys 403 (labeled as C4) are at the beginning and the middle of the ␣-helix 18, respectively, whereas Cys 397 (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 (Phe 374 ), arginine (Arg 381 ), and asparagine (Asp 401 ) (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 (18). 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 * This work was supported by the Centre National de la Recherche Scientifique (CNRS), the National Natural Science Foundation of China, and the Innovation Project of the Chinese Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. DNA binding and stabilization of the three-dimensional structure of RecQ molecules.
Expression of Wild-type Enzyme and Construction of the Zinc Finger Mutant of RecQ Helicase-The E. coli RecQ helicase containing a Nterminal His tag was expressed in E. coli and purified by nickelchelating and anion-exchange chromatography as described previously (20). Mutations were made by a two-step polymerase chain reaction method (21). The outside primers were QFN (5Ј-GGAATTCCATATG-GTGAATGTGGCGCAGGCGGAAGTGTTG-3Ј) and QRX (5Ј-CCGCTC-GAGCTACTCTTCGTCATCGCCATCAACATG-3Ј). The primers used to introduce mutations are QMF1 (5Ј-CAGGAGCCGAACGGGAACAAC-GATATCTGC-3Ј) and QMF2 (5Ј-GCAGATATCGTTGTTCCCGTTCG-GCTCCTG-3Ј). 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. (22). 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 PAR 2 ⅐Zn 2ϩ 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 l) 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 M. The absorbance at 500 nm was measured. The quantity of zinc ion was determined from a standard curve of ZnCl 2 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 ⅐Zn 2ϩ complex (⑀ 500 nm ϭ 6.6 ϫ 10 4 M Ϫ1 cm Ϫ1 ). 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 (17).
Fluorescence Measurements-Fluorescence spectra were determined using Fluoro Max-2 spectrofluorimeter (Jobin Yvon, Spex Instruments S.A., Inc.) as described by Levin et al. (23). Some results were further confirmed by using PiStar-180 spectrometer (Applied Photophysics). In a 10 ϫ 10 ϫ 40-mm 3 quartz cuvette, 0.5 M 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, F obs , was corrected for inner filter and sample dilution effect according to Equation 1, where F is the corrected fluorescence intensity, V 0 is the initial sample volume, V i is the total volume of titrant added, and A EX and A EM 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 (17). Therefore, the oneto-one binding model was used to establish the mantATP binding to RecQ. We have chosen the macroscopic interaction constant defined in Equation 2, where E f represents the concentration of free enzyme, D f represents the concentration of free mantATP, K d is the equilibrium interaction constant, and C is the concentration of the ATP-RecQ complex formed. The concentrations of total enzyme, E T , and total mantATP, D T , can be written in terms of E f and D f as shown in Equations 3 and 4.
From Equations 2-4, the expression of C is obtained as shown in Equation 5.
The value of K d can be obtained by fitting the fluorescence intensity values to Equation 6, where F S is the starting fluorescence of the reaction mixture, f D is the fluorescence coefficient of free mantATP, and f C is the fluorescence coefficient of complex formed.
Limited Proteolytic Digestion-The wild-type, mutant, and Zn 2ϩextracted RecQ helicases at a concentration of 15 M and in a total volume of 30 l were digested with ␣-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 l corresponding to 1 g of protein) from the reaction were quenched with 15 l 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 (24). The reaction mixture (25 l) contained 20 mM Tris-HCl at pH 7.6, 1 mM DTT, 0.1 mg/ml bovine serum albumin, 2 mM MgCl 2 , 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 l 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 (24). An appropriate quantity of fluorescein-labeled ssDNA or dsDNA was added to a standard titration buffer (150 l 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 5Ј-fluorescein-labeled synthetic oligonucleotide (5Ј-F-AGACCC-TTTTAGTCAGTGTGGAAAATCTCTAGCAGT-3Ј). The dsDNA was generated with its complementary sequence (5Ј-ACTGCTA-GAGATTTTCCACACTGACTAAAAGGGTCT-3Ј).
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 () (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 K i for mantATP-The ATPase activity was determined in an assay by measuring the radioactive 32 P i liberated during hydrolysis (25). The measurement was carried out at 37°C in a reaction mixture containing 1.5 M (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 l of reaction mixture and stopped by pipetting 80 l of aliquots from the reaction mixture every 30 s into a hydrochloric solution of ammonium molybdate. The liberated radioactive 32 P i was extracted with a solution of 2-butanol-benzeneacetoneammonium 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, K i , for mantATP was determined by measuring the ATPase rate as a function of mantATP concentration and fit to Equation 7, where v is the initial ATPase rate, k cat is the catalytic constant, and K m ATP is the K m for ATP. Helicase Assay-An unwinding assay was performed using the Beacon 2000 fluorescence polarization instrument (26). An appropriate quantity of fluorescein-labeled duplex oligonucleotide was added to the helicase-unwinding buffer (150 l of total volumes) in a temperaturecontrolled 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: coli RecQ conserved regions and amino acid sequence alignment of the conserved putative C 4 -type zinc finger motif among the RecQ family helicases. The multiple alignments were performed with the program ClustalW and refined manually. The numbers at the beginning and end of each sequence correspond, respectively, to positions of the first and the last amino acid residues. Highly conserved amino acid residues are shadowed in gray. In boldface are the four conserved cysteine residues and the fully conserved arginine, aspartic acid, and aromatic residues involved in three very important hydrogen bonds. The consensus sequence was generated by ClustalW. The protein accession numbers used by the NCBI are as follows: RecQ, P15043 (E. coli); SgS1, P35187 (Saccharomyces cerevisiae); Rqh1, Q09811 (Schizosaccharomyces pombe); RecQL1, NP_002898/P46063 (Homo sapiens); WRN, NP_000544/Q14191 (H. sapiens); BLM, A57570/P54132 (H. sapiens); DmBLM, Q9VGI8 (H. sapiens); and RECQL5, NP_004250 (H. sapiens). Secondary structure elements of RecQ appear with boxes designing the ␣-helices. B, conserved residues in the zinc finger motif. The positions of the four conserved cysteine residues, Cys 380 (labeled as C1), Cys 397 (labeled as C2), Cys 400 (labeled as C3), and Cys 403 (labeled as C4), are drawn in gray as they appear in the structure of the E. coli RecQ core. The zinc atom is in dark gray. Labels for the secondary structures are also indicated. The conformation of the zinc finger motif is obviously stabilized through three hydrogen bonds between NH2 Arg 381 and OD2 Asp 401 , between NE Arg 381 and OD1 Asp 401 and between NH1 Arg 381 and the main chain atom O of Phe 374 , involving highly conserved residues in the RecQ family (NH2, OD2, OD1, NE, and NH1 represent the positions of the atoms in the molecule; NH, N heta; OD, O delta; NE, N epsilon). These three residues drawn in light gray could contribute to the relative positioning of the helices ␣16, ␣17, and ␣18. ropy amplitude at time t, A 0 is a constant, and k obs is the observed rate constant.
Size Exclusion Chromatography-Size exclusion chromatography was performed at 18°C using an fast protein liquid chromatography system (⌬KTA, 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; ␥-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 M RecQ protein was incubated in the elution buffer with ATP and MgCl 2 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 (Cys 380 , Arg 381 , Cys 397 , Cys 400 , Asp 401 , and Cys 403 ) 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, Arg 381 , Asp 401 , and Phe 374 (Fig. 1). These interactions may contribute to the relative positioning of the helices ␣ 16 , ␣ 17 , and ␣ 18 in the zinc finger motif (Fig. 1B). The residues Arg 381 and Asp 401 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 (27). 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, ϳ50% 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 Ͼ92%.
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.
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 zincdemetalated 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 zincdemetalated 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-dimen-sional structure studies have shown that RecQ helicase takes a globular shape (17,20), 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 zincdemetalated 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 ATPbinding site of RecQ. For this purpose, the competitive inhibition constant, K i , 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 K i of 85 M, indicating that mantATP binds competitively to the ATP-binding site. The apparent K d 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 K d values determined are 43.6 M for the wild-type helicase, 56.8 M for the double mutant helicase, and 47.6 M 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 note that the apparent mantATP binding constant determined in this study (K d ϭ 43.6 M) is lower than that of ATP determined from bulk ATPase (250 M, Fig. 4A) and helicase assays (200 M) (28). However, the constant is close to the value determined from a single molecule assay (50 M). 2 The discrepancy among these values may be due to different experimental approaches and different experimental conditions. Regardless, the FRET method used in this study for determining the ATP binding constant is a more direct approach compared with that through the measurement of enzymatic activities.
The Zinc Finger Motif Is Important for DNA-dependent ATPase and Helicase Activities-Previous studies have shown that the ATPase activity of wild-type RecQ is greatly stimulated by ssDNA and, to a less extent, by dsDNA (10). To test the role of the zinc finger motif in ATP hydrolysis, the ATPase activities of the double mutant RecQ and zinc-demetalated helicases were compared with the wild-type enzyme in the presence of ssDNA (60-mer). The mutant protein was severely compromised in DNA-dependent ATPase activities, exhibiting no detectable ATPase activities when compared with wild-type and zinc-demetalated helicases ( Fig. 4A and Table I). Similar results were obtained with 3-kb linear plasmid DNA (results not shown).
The above observations demonstrate that the integrity of the zinc finger motif of RecQ is important for ATPase activity. We reasoned that the helicase activity should also be affected when the zinc binding motif is altered. As expected, although the wild-type enzyme unwinds the duplex DNA substrate completely within 5 min, no helicase activity was detectable under the same conditions for the double mutant protein (Fig. 4B and Table I). These results demonstrate that the zinc finger motif is needed for both ATPase and helicase activities.
The Zinc Finger Motif Is Required for Stable DNA Binding-To further understand the molecular basis of the observed decrease in ATPase and helicase activities for the double mutant protein, the effect of the mutation on DNA binding was first investigated using the gel mobility shift assay under the best experimental condition that we determined previously (25). Whereas a shift of 3 kb of DNA was observed as wild-type and zinc-demetalated helicase concentrations increases (Fig.  5A, lanes 2-5), no protein-DNA complexes were observed under the same condition for mutant enzyme (Fig. 5A, lanes 6 and 7), suggesting that the DNA binding ability of mutant protein is completely compromised. However, it is still possible that the mutant protein displays weak DNA binding activities that cannot be detected by the electrophoretic mobility shift assay method due to the physical separation of free DNA from the protein. Therefore, we measured the DNA binding activities of both wild-type and mutant protein under equilibrium conditions using fluorescence anisotropy assays as performed previously (24). Fig. 5B shows that both wild-type and zinc-demetalated helicases display a high affinity for the 5Ј-fluoresceinlabeled ssDNA. The apparent K d values of both wild-type and zinc-demetalated helicases determined from this experiment are very close to each other (Table I). In contrast, no DNA binding activity is detectable for the mutant helicase, even at a high protein concentration (3 M). Similar results were obtained with dsDNA (Fig. 5B, inset, and Table I). Together, these results demonstrate that the zinc finger motif plays an essential role in DNA binding.
Role of the Zinc Atom-The zinc ion may play roles both in FIG. 4. A, ATPase activity of RecQ helicase as a function of ATP concentration. Experiments were performed at 37°C with 10 nM protein for each helicase preparation and 6 M ssDNA (nucleotide, 60-mer oligonucleotide). B, kinetics of RecQ helicase-mediated DNA unwinding. 50 nM RecQ helicases (wild-type (wt), mutant, and zinc-demetalated protein) were incubated with 2 nM DNA substrate (36 bases in duplex length). DNA unwinding was initiated by the addition of 1 mM ATP at 25°C. The time courses from the wild-type (open circles), zincdemetalated (closed circles), and double mutant (triangle) RecQ helicases were fit to the exponential equation: A ϭ A 0 exp(Ϫk obs t). The fraction of unwound DNA substrate is shown in the inset. structure and in enzymatic catalysis of RecQ. To better define the role of zinc atom, we performed three kinds of experiments. We first compared the properties of both wild-type and zincdemetalated RecQ helicases. For this purpose, the zinc-demetalated RecQ helicase was obtained by EDTA extraction protocol (17) and analyzed in parallel with wild-type and mutant enzymes. As can be seen from Figs. 2-5 and Table I, the zincdemetalated RecQ helicase displays very similar properties with wild-type helicase in terms of the full tertiary structure of holoenzyme, DNA binding, ATPase, and helicase activities. We next investigated the effect of the zinc atom on ATPase activity of both wild-type and zinc-demetalated RecQ helicases with increasing zinc concentrations. The results revealed that the zinc atom does not significantly influence the activities of both wild-type and zinc-demetalated RecQ helicases (Fig. 6A). Finally, the stabilities of the wild-type and zinc-demetalated helicases were assessed by measuring ATPase activity at different temperatures ranging from 25 to 54°C. Fig. 6B shows that both enzymes display similar thermostability except at 54°C where the zinc-demetalated enzyme displays a modest decrease in K cat and an increase in K m . The similar thermosta-bilities of both enzymes indicate that zinc ion is not essential to protein stability. Taken together, these observations indicate that the zinc atom is not absolutely required for enzymatic catalysis, DNA binding, or stabilization of the protein conformation.

DISCUSSION
The three-dimensional structure of the E. coli RecQ helicase (17) reveals the existence of a zinc binding motif. In this study, the importance of this motif to RecQ helicase function was highlighted by the loss-of-function mutations at highly conserved residues within the zinc finger motif by site-directed mutagenesis. Consistent with the notion that zinc fingers are structural modules of a major ubiquitous class of DNA binding motifs (29), our data illustrated that a mutation at the zinc finger, which drastically reduces zinc binding, abrogates DNA binding to the helicase and leads to decreased ATPase and helicases activities. Furthermore, this motif is also crucial for the integrity of the whole protein.
The Zinc Finger Motif Is Essential to DNA Binding-The most striking observation in this report is that alterations of  (36-mer) were determined by measuring the changes in fluorescence anisotropy as described under "Materials and Methods." Varying amounts of the proteins were added to a binding assay buffer containing 1 nM DNA. Fluorescence anisotropy data were fit to a hyperbola using KaleidaGraph software. Given in the inset are the titration curves of the different RecQ helicases with 36-mer dsDNA. The association constant values obtained from the best fit are summarized in Table I. the zinc finger motif by site-directed mutagenesis lead to a complete loss of RecQ-DNA binding ability. In view of the fact that RecQ helicase is a DNA-stimulated ATPase and an ATPdependent helicase, the impairment of DNA binding should be the primary cause of the observed defects in ATPase and helicase activities. These observations indicate that the zinc finger motif is essential to DNA binding. Alternatively, the replacement of amino acids in the zinc finger motif leads to an overall conformational change of the helicase, which resulted in the observed defects in helicase functions. Consistent with this possibility, all of the single-point mutations were rapidly degraded, making functional analysis impossible. However, a careful characterization of the double mutant helicase indicated that the protein undergoes a subtle conformational change rather than a radical modification of the overall protein architecture. First, both wild-type and mutant enzymes displayed very similar limited proteolysis pattern. Second, the size-exclusion chromatography analysis indicated that both wild-type and mutant helicases have similar Stokes radii, suggesting that these proteins fold in overall similar fashions. Third, the fact that the mutant protein possesses almost the same affinity for mantATP binding as wild-type helicase suggests that the tertiary structure of the mutant protein is not dramatically altered. These results clearly demonstrate that a local subtle conformational change in the zinc finger motif abrogates DNA binding capability, and as a consequence, the ATPase and helicase activities were abolished. This interpretation gains support from CD experiments where the small reduction in ␣-helix may be attributed to the altered structure of the zinc finger motif because of the replacement of two cysteine amino acids with asparagines. The zinc finger motif thus has a dominant effect in DNA binding. We hypothesize that some conserved positively charged residues (Arg 382 and Lys 408 ) and solvent-exposed hydrophobic residues (Phe 374 and Phe 389 ) harbored by this zinc motif could be directly involved in the recognition and binding of DNA.
For other helicases without zinc finger motif within the molecules, such as PrcA, Rep, UvrD, and HCV (hepatitis C virus) helicases, their DNA binding surfaces are composed primarily of motifs Ia, IV, and V. Several lines of experimental evidence suggest that the same situation may be held in the RecQ family of helicases (13). It is also interesting to note that most RecQ family of helicases harbor a HRDC domain, which possesses DNA binding activities (16). Because the double mutant that we studied appears to keep its overall three-dimensional structure and because the helicase domain and HRDC domain could still bind DNA, the observation that DNA binding activity is completely compromised is not expected. How can we reconcile the observation that alterations of the zinc finger motif lead to the RecQ helicase losing its DNA binding activity almost completely? One of the answers to this question lies in the fact that, for RecQ helicase, the zinc finger motif plays an essential role in DNA binding, whereas both the helicase and HRDC domains may function as auxiliary DNA-binding domains. Without zinc finger motif, both helicase and HRDC domains display very low affinities for DNA binding. Indeed, in work to be published elsewhere, 3 it has been shown that the isolated helicase domain and HRDC domain fragment proteins display very low affinity for ssDNA and dsDNA. It is likely that when DNA is bound to zinc finger motif, the helicase domain plays an essential role in unwinding activity, whereas HRDC domain may direct DNA binding specificity. Thus, it is tempting to speculate that the coordination among the zinc finger motif, the helicase domain, and HRDC will determine the DNA substrate specificity, the kinetics, and the processivity of the helicase. Here again, these analyses further enforce the notion that the zinc finger motif plays an essential role in DNA binding.
The Zinc Finger Motif Plays an Important Role in the Tertiary Structural Stability-The results from this study and the three-dimensional structural analysis reveal that the zinc finger motif is stabilized by four cysteine residues. It is important to note that altering one of the four conserved cysteine residues leads to highly unstable proteins. These results showed that the zinc finger motif is unexpectedly involved in the integrity of the whole enzyme. It is possible that, in addition to its DNA binding function, the zinc finger motif also functions in the folding cascade, linking the domains to each other to stabilize the protein tertiary structure. In addition to the four highly conserved cysteine residues, the zinc finger motif could be further stabilized through very important interactions among ␣-helices 16, 17, and 18. It appears that the hydrogen bonds between Phe 374 and Arg 381 and between Arg 381 and Asp 401 play an important role in stabilizing the zinc finger motif and the whole protein structure. Consistent with this postulation, the mutation of Arg 381 or Asp 401 leads to the enzyme becoming very sensible to protease degradation. Similar results were observed with the Bloom syndrome protein (30). The replacement of Asp 1064 (which is located at a position equivalent to that of Asp 401 of RecQ helicase) with Ala substantially reduced DNA binding and helicase activities, whereas the protein displayed essentially the same activities as the wild-type enzyme when Asp 1064 was replaced by Asn. This phenomenon is probably the result of the properties of the side chain of the replacing residues. Asn probably could still possibly establish hydrogen bond with Arg 1037 , thus stabilizing the protein structure, whereas 3 J. L. Liu and X. G. Xi, unpublished observation. Ala cannot. Thus, both the cysteines, C1 and C4, and the hydrogen bonds between Phe 374 and Arg 381 and between Arg 381 and Asp 401 contribute to the stabilization of the zinc finger motif and increase the stability of the tertiary structure of the protein.
The observation that the wild-type and zinc-demetalated forms of RecQ are very similar to each other by virtue of their enzymatic activities suggests that the zinc atom is not directly implicated in DNA binding, ATPase, and helicase activities. It appears that the zinc ion is not essential for stabilizing the overall conformation of the protein, because both the wild-type and zinc-demetalated helicases display very similar thermostabilities between 25 and 54°C. Nevertheless, the zinc ion may have a quite relevant role in determining the full development of secondary structure of the zinc finger motif during the protein folding. Once the zinc finger motif has folded correctly in the presence of zinc ion, its structure remains stable even in the absence of zinc ion; thus, DNA can bind to the residues involved in base-specific contacts. There are several examples in which metal ions play an important role in the formation of secondary structural elements in metalloproteins, whereas demetalated proteins still have well defined tertiary structures (31).
Biological Relevance of the Cysteine Cluster to the RecQ Helicase Family-Amino acid sequence alignment of several RecQ helicases revealed that the cysteine residues involved in the formation of zinc finger in E. coli helicases are conserved among the RecQ family of helicases (Fig. 1). Available evidence indicates that mutations of the cysteine residues in the zinc finger motif in most RecQ family of helicases can have dramatic effects on the enzymatic activities. Two disease-causing Bloom missense mutations map to Cys 1036 and Cys 1055 , respectively (32,33), among four conserved cysteine residues in the zinc finger motif. In vitro analysis shows that these mutations abolish BLM ATPase and helicase activities (34). In vitro studies of the RecQ core of the Bloom syndrome protein have shown that when three of the four cysteines in the zinc finger motif were mutated, respectively, the resulting mutants were very unstable. Furthermore, even mutations of residues near the conserved cysteine residues such as R1038A and D1064A prevented the modified enzymes from binding DNA and caused them to lose ATPase and helicase activities (29). It has also been shown that this region is essential to in vitro function of the yeast RecQ homologue Sgs1 (35,36).
Sequence comparisons among the DNA helicases suggest that the zinc finger motif appears to be unique to the RecQ helicase family. Both the sequence analysis and three-dimensional structural studies showed that other DNA helicases, such as T7 gene 4 helicase (37), Rep (38), PcrA (39), and hepatitis C virus NS3 helicases (40), do not use the zinc finger motif for DNA binding. Whether the use of zinc finger motif for DNA binding is a unique feature of RecQ family of helicases is currently being investigated in our laboratory.
In summary, the present study reveals that the zinc finger motif in the RecQ helicase plays important roles both in DNA binding and folding of the whole enzyme. Thus, evolution has selected a complex mechanism to link all of the functions of the enzyme to this small subdomain. Although much remains to be discovered, RecQ enzymes without perfect folding of the zinc finger motif would indeed be unable to ensure the helicase functions.