©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Inhibition of Vaccinia DNA Topoisomerase by Novobiocin and Coumermycin (*)

(Received for publication, May 4, 1995; and in revised form, October 4, 1995)

JoAnn Sekiguchi (1) James T. Stivers (2)(§) Albert S. Mildvan (2) Stewart Shuman (1)(¶)

From the  (1)Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the (2)Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K values of 10-25 µM for coumermycin and 350 µM for novobiocin. Spectroscopic and fluorescence measurements of drug binding to enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (K = 27 ± 5 µM) and two classes of binding sites for novobiocin, one tight site (K(1) = 20 ± 5 µM) and several weak sites (K(2) = 513 ± 125 µM; n = 4.9 ± 0.7). Addition of a stoichiometric amount of DNA to a preformed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.


INTRODUCTION

DNA topoisomerases are targeted by a variety of antimicrobial and antineoplastic drugs(1, 2, 3) . In addition to their therapeutic value, these drugs provide indispensable research tools. Insights into topoisomerase mechanism are provided by inhibitors and poisons affecting specific steps in the catalytic cycle. Studies of drug effects in vivo, supported by genetic analyses of drug resistance, underscore the important role played by topoisomerases in virtually all DNA transactions. Drug-target relationships have been firmly established for bacterial DNA gyrase, which is sensitive to the quinolone and coumarin antibiotics(2, 3) ; for eukaryotic type II topoisomerases, which are blocked by epipodophyllotoxins, acridines, and quinolones(4, 5, 6, 7) ; and for eukaryotic topoisomerase I, which is susceptible to camptothecin(8, 9, 10, 11, 12) .

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, displays unusual pharmacological properties. The vaccinia enzyme is resistant to camptothecin(13) , the hallmark topoisomerase I poison, yet it is sensitive to the coumarin drugs novobiocin and coumermycin(14, 15) , which are classical inhibitors of DNA gyrase. Coumarin action on gyrase has been studied in detail(3) . The coumarins bind to the GyrB subunit and inhibit its ATPase activity, which is required for enzyme-catalyzed DNA supercoiling. A 24-kDa subdomain of GyrB is sufficient to bind the coumarins(16) ; moreover, specific amino acids implicated in drug binding are defined by drug-resistant gyrase mutants that map within this subdomain(17) . The molecular basis for coumarin action emerges from the crystal structure of the GyrB-novobiocin complex, in which the bound drug impinges on the binding pocket for ATP (18, 19) (^1)The ATP-dependent eukaryotic type II topoisomerases are also inhibited by novobiocin and coumermycin, albeit at much higher drug concentrations(20, 21, 22, 23) .

Fogelsong and Bauer (14) first documented coumarin inhibition of DNA relaxation by vaccinia topoisomerase using enzyme purified from infectious virions. The reported potency of novobiocin against vaccinia topoisomerase was comparable to its efficacy against eukaryotic topoisomerase II(14, 15, 20, 21, 22, 23) . The identification of the gene encoding vaccinia topoisomerase (24) made it clear that the viral enzyme is structurally homologous to other eukaryotic type I enzymes, with no discernible similarity to DNA gyrase. Because the vaccinia topoisomerase, like other eukaryotic type I enzymes, does not require ATP binding or ATP hydrolysis for DNA relaxation, it is unclear how the coumarins might inhibit the viral protein. We now show that the coumarins target the vaccinia enzyme by a distinctive mechanism in which drug binding to the topoisomerase prevents the binding of the enzyme to DNA.


EXPERIMENTAL PROCEDURES

Enzyme Preparation

Vaccinia topoisomerase was expressed in Escherichia coli and purified as described(13, 26) . The SP5PW preparation used for assay of DNA relaxation, DNA cleavage, DNA binding, and protease sensitivity was homogeneous with respect to the topoisomerase polypeptide, as determined by SDS-PAGE. (^2)Protein concentration of this enzyme fraction was determined using the Bio-Rad dye reagent with bovine serum albumin as a standard. For the equilibrium drug binding measurements, samples of purified topoisomerase were dialyzed extensively at 4 °C against 0.5 mM MES, pH 6.3, and then lyophilized to dryness and dissolved in deionized distilled water at a final concentration of 200-600 µM enzyme. These manipulations of the protein preparation did not affect its catalytic activity in relaxing supercoiled plasmid DNA. The molar concentration of the resuspended protein was determined spectrophotometrically using the relationship that a 1 mg/ml solution of topoisomerase gives an A of 1.08 cm(27) .

Coumarin Drugs

Novobiocin, purchased from Sigma, was stored as a 100 mM stock solution in water and diluted in water prior to each use. Coumermycin A(1) was purchased from Sigma and stored as a 10 mM solution in 100% Me(2)SO. Coumermycin was diluted in 100% Me(2)SO prior to each use and added to DNA relaxation, DNA cleavage, or DNA binding reaction mixtures such that the final Me(2)SO concentration was 10% (v/v).

DNA Relaxation Assay

Reaction mixtures (20 µl) contained 50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5 mM MgCl(2), 0.3 µg (170 fmol) of pUC19 plasmid DNA, and drug as indicated. The reaction was initiated by addition of 0.1 ng (3 fmol) of topoisomerase. After incubation for 10 min at 37 °C, the reactions were quenched by addition of a solution containing glycerol, xylene cyanol, bromphenol blue, and SDS (0.2% final concentration). The samples were analyzed by electrophoresis through a horizontal 1.0% agarose gel in Tris/glycine buffer (50 mM Tris base, 160 mM glycine). After staining for 15 min in 0.5 µg/ml ethidium bromide, the gel was soaked for 30 min in water and then photographed under short-wave UV illumination using Polaroid Type 57 film.

Assay of Covalent Complex Formation (DNA Cleavage)

Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 0.5 pmol of topoisomerase were preincubated in the presence of novobiocin or coumermycin (final drug concentrations as specified in the figure legends). The DNA cleavage reactions were initiated by addition of 1 pmol of 5`-P-labeled CCCTT-containing DNA (18-mer scissile strand hybridized to a 24-mer strand as shown in Fig. 2). After incubation for 5 min at 37 °C, the reactions were halted by addition of SDS to 1%. (Alternatively, the drug was added to the reaction mixtures along with the DNA, and the cleavage reaction was initiated by addition of topoisomerase.) Samples were analyzed by SDS-PAGE. Free DNA migrated with the bromphenol blue dye front. Covalent complex formation was revealed by transfer of radiolabeled DNA to the topoisomerase polypeptide(36) . The extent of adduct formation was quantitated by scanning the gel using a FUJIX BAS1000 Bio-Imaging Analyzer and was expressed as the percent of the input 5`-P-labeled oligonucleotide that was covalently transferred to protein.


Figure 2: Coumarin effects on covalent complex formation (DNA cleavage). The DNA substrate consisted of an 18-mer CCCTT-containing oligonucleotide hybridized to a 24-mer oligonucleotide, as shown. The scissile strand was radiolabeled using [-P]ATP and T4 polynucleotide kinase and then gel-purified and annealed to the unlabeled complementary strand. Topoisomerase was preincubated with coumermycin or novobiocin for 10 min at room temperature at the concentrations indicated and then mixed with the radiolabeled DNA as described under ``Experimental Procedures.'' The extent of covalent adduct formation (expressed as the percent of the input DNA) is plotted as a function of drug concentration. (Each data point is the average of three separate experiments.)



Assay of DNA Binding by Native Gel Electrophoresis

A 60-bp duplex DNA containing a single CCCTT motif was used for this analysis. The sequence of the DNA was reported previously(28) . The scissile strand was radiolabeled using [-P]ATP and T4 polynucleotide kinase and then gel-purified and annealed to an unlabeled 60-mer complementary strand by heating for 10 min at 65 °C in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.2 M NaCl and then cooling to room temperature over 1-2 h. DNA binding reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 1 pmol of enzyme were preincubated for 5 min at 37 °C in the presence of novobiocin or coumermycin (final drug concentrations as specified in the figure legends). The DNA binding reaction was initiated by addition of 1 pmol of 5`-P-labeled 60-bp duplex DNA. The mixtures were incubated for 5 min at 37 °C and then adjusted to 5% glycerol and electrophoresed through a native 6% polyacrylamide gel containing 0.25 times TBE (22.5 mM Tris borate, 0.6 mM EDTA) at 100 V for 2.5 h. Free 60-mer DNA and a topoisomerase-DNA complex of retarded electrophoretic mobility were visualized by autoradiographic exposure of the dried gel. The extent of binding was quantitated by scanning the gel using a phosphoimager and was expressed as the percent of the input 5`-P-labeled oligonucleotide shifted to the protein-DNA complex.

Spectroscopic Measurements

Ultraviolet absorbance and fluorescence measurements were made on a Perkin-Elmer Lambda-9 UV-visible spectrophotometer and a Perkin-Elmer 650-10S spectrofluorophotometer, respectively, using a 0.2-ml quartz cell with a 0.5-cm path length. For all experiments, the temperature of the cell compartment was maintained at 37 °C with a circulating water bath. The fluorescence excitation and emission slit widths were set to 4 nm for all measurements.

Reaction Conditions for Drug Binding

Because accurate binding measurements require concentrations of enzyme similar to the dissociation constant, the coumarin binding experiments employed much higher concentrations of enzyme than those used routinely for relaxation assays. It was necessary to optimize the pH and buffer conditions for the drug binding experiments to prevent enzyme aggregation that occurred upon addition of coumermycin (or novobiocin) to concentrated solutions of topoisomerase (>50 µM enzyme) at pH >7. Thus, the binding studies were performed in 10 mM MES, pH 6.3, in the presence or absence of 8% Me(2)SO. Control experiments revealed no discernible difference in the K(0.5) for inhibition of DNA relaxation by coumermycin or novobiocin at pH 6.3 compared with the standard assay conditions (data not shown).

Coumermycin Binding to Topoisomerase

Drug binding was quantitated by measuring the decrease in the fluorescence intensity and UV absorbance of coumermycin as it binds to topoisomerase. (It was not practical to measure changes in fluorescence of the topoisomerase because of the high UV absorbance by coumermycin in the excitation and emission wavelengths of the enzyme.) Binding reactions were performed at 37 °C in a solution containing 10 mM MES, pH 6.3, 8% Me(2)SO (v/v). The concentration of coumermycin was held constant (L = 30 µM), and the topoisomerase concentration was varied in the range 5-127 µM. Serial dilutions of enzyme were performed such that a solution of 127 µM topoisomerase and 30 µM coumermycin in a volume of 130 µl was diluted with a 30 µM solution of coumermycin in MES/Me(2)SO buffer. After each stepwise dilution of the enzyme, an absorbance spectrum in the range 300-400 nm and a fluorescence emission spectrum in the range 390-410 nm were measured. The isosbestic point at 327 nm in the absorbance spectrum was used as the fluorescence excitation wavelength. The pH of each sample was measured at the conclusion of the experiment; the variation between samples was <0.03 pH unit. Prior to data analysis, a background spectrum of topoisomerase in the absence of coumermycin was measured and subtracted from each sample absorption spectrum. The dissociation constant (K(D)) for coumermycin was determined from a nonlinear least-squares fit of the fractional absorbance or fluorescence intensities, (I(0) - I)/(I(0) - I), against total topoisomerase concentration (E) according to , which assumes one binding site.

For this analysis, the absorption maximum at 347 nm and the entire fluorescence emission peak area were used to calculate the fractional intensities, where I(0) and I are the absorbance or fluorescence intensities of coumermycin in the absence and presence of topoisomerase, respectively, and I is the intensity at saturation.

Novobiocin Binding to Topoisomerase

The quenching of topoisomerase fluorescence by novobiocin was determined as a function of drug concentration as follows. Aliquots (2-12 µl) from a concentrated novobiocin stock solution were added to a solution of 25 µM topoisomerase in 10 mM MES, pH 6.3, maintained at 37 °C. Prior to measuring the protein fluorescence, the samples were equilibrated in the cell holder for 5 min with the excitation shutter closed. The excitation wavelength was 275 nm; emission was measured at 340 nm. The observed fluorescence intensities were corrected for optical filtering effects caused by novobiocin absorption at 275 nm(29) , and the fractional fluorescence quenching was calculated as (I(0) - I)/(I(0) - I), where I(0) and I are the measured fluorescence intensities of the protein in the absence and presence of novobiocin, respectively, and I is the intensity at saturation. The equilibrium dissociation constant (K(D)) for novobiocin binding to topoisomerase (E) was determined from a nonlinear least-squares fit of the fractional fluorescence intensities against total novobiocin concentration (L) according to .

Equilibrium Ultrafiltration

The affinity for novobiocin determined by the fluorescence method was higher than expected from the drug inhibition profile in the DNA relaxation assay. Consequently, the method of equilibrium ultrafiltration was employed to extend the measurements to higher novobiocin concentrations and thereby determine whether an additional weaker novobiocin-binding site (or sites) existed on the enzyme. Equilibrium ultrafiltration allows the direct determination of free novobiocin and is equivalent to equilibrium dialysis for measurement of ligand affinity(30) . MPS-1 micropartition ultrafiltration devices and membranes (14 mm; Diaflo YM-10) were obtained from Amicon, Inc.; the membranes were soaked for 1 h in distilled water and air-dried prior to use. Nine samples (0.15 ml) containing 200 µM topoisomerase and novobiocin in the range 31-1350 µM were equilibrated at 37 °C for 15 min in 10 mM MES, pH 6.3, 8% Me(2)SO. The samples were transferred to individual ultrafiltration devices, which had been prewarmed to 37 °C and placed in a Sorvall SS-34 rotor. The material was centrifuged for 4 min at 4000 rpm, and the A of the filtrate (which reflects free novobiocin) was determined. The concentrations of free and bound novobiocin were calculated as follows. First, the absorbance contributed by the small amount of topoisomerase leaking through the membrane (<0.1%; determined from control samples lacking novobiocin) was subtracted from the total filtrate absorbance. This correction was significant only at the two lowest drug concentrations, where novobiocin binding was nearly stoichiometric. Second, the fraction of free novobiocin was determined by dividing the corrected A of filtrates from topoisomerase-containing samples by the A of filtrates from control samples that contained identical concentrations of drug, but no enzyme. This method for determining the fraction of free novobiocin corrects for the background binding (<8%) of novobiocin to the membrane. Finally, the concentration of free novobiocin was calculated by multiplying the fraction of free drug by the total input drug concentration; the concentration of bound novobiocin was obtained by subtracting [novobiocin] from [novobiocin]. Binding data were analyzed by a nonlinear least-squares fit to , which describes ligand binding to multiple noninteracting sites.

In , K(D)(1) and K(D)(2) represent the dissociation constants, and C(1) = n(1)E and C(2) = n(2)E (that is, the concentration of tight and weak binding sites, respectively). Alternatively, the data were fit by Scatchard analysis using a graphical method that yielded identical results(31) .

Displacement of Bound Coumermycin by DNA

To examine whether coumermycin and DNA compete for a single binding site on the enzyme, a competition binding experiment was performed using the equilibrium ultrafiltration method (see above). The DNA competitor was a 32-mer self-complementary ``hairpin'' oligonucleotide as shown below (). Cleavage of the DNA by topoisomerase will liberate a 4-nucleotide leaving group, ATCC (underlined).

To prevent irreversible covalent binding of the 32-mer(27, 32) , we included in the reaction an 8-mer DNA strand, 5`-ATTCTCGC, which was complementary to the 5`-overhang generated by release of the 4-mer. Reaction mixtures (0.15 ml) were prepared containing 10 mM MES, pH 6.3, 8% Me(2)SO, 25 µM topoisomerase, and 25 µM coumermycin. The mixtures were then supplemented with a 0, 5, 10, 15, 20, 25, or 37.5 µM concentration of the hairpin DNA plus a 25 µM concentration of the 8-mer strand. The samples were incubated for 15 min at 37 °C before performing ultrafiltration as described above. An aliquot (25 µl) of the filtrate, which contained free coumermycin (as well as free 4-mer in the samples containing DNA), was diluted in 0.775 ml of water, and a UV absorption spectrum was taken in the range 240-370 nm. The absorbance at 310 nm, which was due to coumermycin and not free DNA, was used to quantitate the release of drug from the enzyme as the concentration of DNA competitor was increased.


RESULTS

Inhibition of DNA Relaxation by Novobiocin and Coumermycin

DNA relaxation by purified topoisomerase was assayed in the presence of 0.1 M NaCl and 5 mM MgCl(2) under conditions of DNA excess. Conversion of the input supercoiled plasmid DNA to relaxed circular DNA was essentially quantitative after 10 min of incubation at 37 °C (Fig. 1). Novobiocin inhibited relaxation in a concentration-dependent fashion, with a sharp decrement between 0.2 and 0.5 mM (Fig. 1). Coumermycin was more potent than novobiocin, inhibiting relaxation sharply in the range 50-200 µM (Fig. 1). (Note that because the control reaction in this experiment went to completion, the inhibitory effects of lower drug concentrations were probably obscured.) The effective inhibitory concentration ranges for novobiocin and coumermycin established in this assay for the recombinant enzyme were in agreement with earlier studies using topoisomerase isolated from vaccinia particles(14, 15) . Inhibition of relaxation by coumarins was not attributable to irreversible modification of the enzyme by drug insofar as topoisomerase that was preincubated with 1 mM coumermycin and then diluted 1000-fold in buffer without drug was just as active in relaxation of supercoiled DNA as enzyme that had not been exposed to coumermycin (data not shown).


Figure 1: Inhibition of DNA relaxation by novobiocin and coumermycin. Relaxation assays were performed as described under ``Experimental Procedures.'' In the series to the left, novobiocin was included at 50, 100, 200, 500, 1000, and 2000 µM final concentrations (proceeding from left to right). Control reactions were performed without novobiocin (lane -) or without enzyme (lane C). In the series to the right, coumermycin A(1) was included at 20, 50, 100, 200, 500, and 1000 µM final concentrations (proceeding from left to right). All reactions containing coumermycin included a 10% (v/v) final concentration of Me(2)SO. Control reactions contained topoisomerase and Me(2)SO, but no coumermycin (second lane -), or topoisomerase without coumermycin or Me(2)SO (first lane -).



DNA Cleavage and Religation

The topoisomerase catalytic cycle entails multiple steps: (i) noncovalent binding of enzyme to duplex DNA, (ii) scission of one strand with concomitant formation of a covalent protein-DNA adduct, (iii) strand passage, and (iv) religation. Shaffer and Traktman (15) showed that covalent binding of vaccinia topoisomerase to nick-translated duplex DNA was inhibited partially by 200 µM novobiocin and completely by 200 µM coumermycin. It was shown subsequently that vaccinia topoisomerase displays considerable specificity in DNA cleavage(25) ; it binds and forms a covalent adduct at sites containing the sequence 5`-(C/T)CCTT. This feature of the vaccinia enzyme facilitates analysis of the partial reactions using model substrates containing a single CCCTT cleavage site. ``Suicide'' substrates have been especially useful for studying the cleavage reaction (first transesterification) under single-turnover conditions(27, 32) . An example of such a substrate is shown in Fig. 2. Covalent adduct formation is accompanied by spontaneous dissociation of the 3`-fragment of the cleaved strand from the protein-DNA complex, which leaves a 12-nucleotide single-strand tail on the noncleaved strand. With no readily available acceptor for religation, the topoisomerase is covalently trapped on the DNA. The suicide cleavage assay measures the yield of covalent adduct in a single-turnover reaction that is complete within 15 s at 37 °C (data not shown). The yield is proportional to input topoisomerase when DNA is in excess, and the reaction is near-quantitative at saturating enzyme(37) . Drug effects were evaluated at enzyme concentrations sufficient to cleave 35-45% of the input substrate (Fig. 2). In the experiment shown in Fig. 2, topoisomerase was exposed to drug prior to addition of the DNA cleavage substrate. The coumarins inhibited covalent adduct formation in a concentration-dependent manner. Cleavage was abolished completely at 200 µM coumermycin and 1 mM novobiocin; 50% inhibition occurred at 20 µM coumermycin and 0.35 mM novobiocin (Fig. 2).

The drug effect on suicide cleavage was subject to a substantial order-of-addition effect, as shown for coumermycin in Fig. 3. In this set of experiments, preincubation of topoisomerase with coumermycin for 5 min again elicited a concentration-dependent inhibition of cleavage, with 50% inhibition at 25 µM (Fig. 3, closed circles). However, when drug and DNA substrate were premixed and the reactions were initiated by addition of enzyme, the inhibition profile was shifted significantly to the right, with 50% inhibition at 125 µM coumermycin (Fig. 3, open circles). A simple view of the order-of-addition phenomenon is that coumermycin binds to the topoisomerase and dissociates relatively slowly (resulting in higher drug potency by virtue of prebinding). Decreased efficacy of coumermycin when copresented with DNA is consistent with a slower on-rate for drug than for the DNA. (By adding the DNA substrate at various times after exposure of enzyme to 60 µM coumermycin, we determined that the order-of-addition effect was fully established within 30 s of preincubation (data not shown).)


Figure 3: Order-of-drug addition effect on DNA cleavage. Topoisomerase was preincubated for 5 min with coumermycin at the concentrations indicated (closed circles); the cleavage reaction was then initiated by addition of the labeled DNA substrate. Alternatively, coumermycin was added to the reaction mixtures along with the DNA, and the cleavage reaction was initiated by addition of topoisomerase (open circles). The extent of covalent adduct formation (expressed as the percent of the input DNA; average of three separate experiments) is plotted as a function of drug concentration.



Coumarin inhibition of suicide cleavage can be explained by either of the following: (i) the drugs directly inhibit transesterification, or (ii) the drugs block noncovalent binding of topoisomerase to the DNA. We sought to address this issue by circumventing the DNA binding step, i.e. by studying the ability of topoisomerase already bound covalently to the suicide substrate to catalyze religation to an acceptor DNA provided in trans (40, 41). The acceptor strand was a 5`-OH-terminated 12-mer complementary to the 5`-tail of the ``donor'' complex. The religation product was a 24-mer that was resolved electrophoretically from the input 18-mer strand. As noted previously(32, 38, 39) , single-turnover strand transfer was very efficient; 80% of the input substrate was religated to the exogenous acceptor (Fig. 4). Treatment of the covalently bound topoisomerase for 5 min with 2 mM novobiocin or 200 µM coumermycin (concentrations that abrogated the suicide cleavage reaction) prior to the addition of the acceptor strand had no discernible effect on strand religation.


Figure 4: Coumarin effects on DNA strand transfer (religation). Covalent complexes were formed in reaction mixtures containing (per 20 µl) 50 mM Tris-HCl, pH 8.0, 1 pmol of P-labeled suicide substrate, and 2 pmol of topoisomerase. Aliquots (20 µl) were transferred to individual tubes and adjusted to either 2 mM novobiocin or 0.2 mM coumermycin as indicated. Control samples received no drug. The mixtures were incubated for 10 min at room temperature. Strand transfer was then induced by addition of 50 pmol of a 5`-OH 12-mer acceptor strand that was complementary to the 12-nucleotide single-strand tail of the covalent donor complex. (The structures of the donor complex and the acceptor strand are shown.) After incubation for 5 min at 37 °C, the samples were adjusted to 0.2 M NaCl. Formamide was added to 33% (v/v), and the samples were denatured for 5 min at 95 °C. Aliquots (7 µl) were analyzed by electrophoresis through a 12% polyacrylamide gel containing 7 M urea in TBE (90 mM Tris base, 90 mM boric acid, 2.5 mM EDTA). Religation of the labeled input strand to the acceptor was revealed by the appearance of a radiolabeled 24-mer strand. The extent of religation was quantitated by scanning the gel using a FUJIX BAS1000 Bio-Imaging Analyzer and is expressed as the percent of the input 5`-P-labeled 18-mer oligonucleotide that was converted to the 24-mer product.



Drug Inhibition of DNA Binding

We assayed the effect of the coumarins on the binding of vaccinia topoisomerase to a radiolabeled synthetic 60-bp duplex DNA containing a single centrally placed CCCTT recognition site(28) . Protein-DNA complex formation was detected as the formation of a discrete complex of retarded electrophoretic mobility during native gel electrophoresis(28, 37) . In contrast to the suicide substrate, for which all bound enzymes are trapped in the covalent state, the cleavage-religation equilibrium of vaccinia topoisomerase bound to the 60-mer DNA is strongly skewed toward religation, i.e. only 10-15% of the DNA molecules that are bound will be linked covalently to the protein(32) . Hence, the gel shift assay largely reflects the noncovalent binding of enzyme to the DNA ligand. Exposure of topoisomerase to coumermycin prior to addition of the DNA caused a concentration-dependent decrease in the extent of topoisomerase-DNA complex formation (Fig. 5A). DNA binding was inhibited almost completely at 80-100 µM coumermycin; 50% inhibition occurred at 10 µM coumermycin (Fig. 5A). Novobiocin also inhibited DNA binding in a concentration-dependent fashion, but was less potent than coumermycin; half-maximal inhibition occurred at 0.36 mM (Fig. 5B).


Figure 5: Inhibition of DNA binding. A, topoisomerase was preincubated with coumermycin at the concentrations indicated and then mixed with the radiolabeled DNA ligand (60-mer) as described under ``Experimental Procedures.'' The extent of binding (expressed as the percent of the input DNA shifted to the protein-DNA complex) is plotted as a function of drug concentration (closed circles). (Each data point is the average of three separate experiments.) Alternatively, topoisomerase was incubated for 5 min at 37 °C with radiolabeled 60-mer DNA in the absence of drug and then challenged with coumermycin (5-min incubation at 37 °C) at the indicated concentrations (open circles) prior to native gel electrophoresis. DNA binding (average of two separate experiments) is plotted as a function of coumermycin concentration. B, topoisomerase was preincubated with novobiocin and then mixed with the radiolabeled 60-mer. DNA binding (closed circles; average of two experiments) is plotted as a function of novobiocin concentration. Alternatively, topoisomerase was incubated with the 60-mer DNA in the absence of drug and then challenged with novobiocin (open circles). DNA binding (average of two experiments) is plotted as a function of novobiocin concentration.



Dramatically different effects were observed when the order of addition was reversed such that topoisomerase was incubated with the P-labeled 60-mer DNA prior to addition of drug. Preformed topoisomerase-DNA complexes were refractory to coumermycin in the range 10-100 µM and to novobiocin in the range 0.1-1 mM (Fig. 5, A and B). Thus, the topoisomerase, once bound to DNA, was not induced to dissociate by the drugs. This finding was extended by order-of-addition competition experiments designed to provoke dissociation of the prebound protein by challenge with unlabeled DNA. A control assay established that addition of unlabeled 60-mer DNA to the binding reaction mixtures prior to addition of enzyme reduced the extent of DNA binding in accordance with the molar ratio of unlabeled competitor to labeled ligand (Fig. 6). When competitor was added after preincubation of topoisomerase with the labeled 60-mer, the protein-DNA complex was relatively resistant to competition, but could dissociate at higher ratios of unlabeled competitor to labeled probe (Fig. 6). Addition of 200 µM coumermycin to the preformed protein-DNA complexes had no apparent effect on enzyme-DNA dissociation by unlabeled competitor. Thus, coumermycin did not stabilize or destabilize the enzyme-DNA complex.


Figure 6: Stability of the binary complex to competitor DNA is unaffected by coumermycin. Binding of topoisomerase (1 pmol) to P-labeled CCCTT-containing 60-bp DNA (1 pmol) was assayed as described under ``Experimental Procedures.'' Unlabeled 60-mer DNA was added as a competitor in the amounts indicated. The order of addition of competitor relative to enzyme was varied as follows: open circles, competitor was included in the reaction mixtures along with labeled 60-mer, and the binding reaction was initiated by addition of topoisomerase; closed circles, topoisomerase was incubated for 5 min at 37 °C with labeled 60-mer in the absence of competitor and then challenged with unlabeled 60-mer (5-min incubation at 37 °C) prior to native gel electrophoresis; open squares, topoisomerase was incubated for 5 min at 37 °C with labeled 60-mer, then exposed to 200 µM coumermycin (for 5 min at 37 °C), and finally challenged with competitor DNA (5-min incubation at 37 °C) prior to native gel electrophoresis. The extent of binding to labeled 60-mer (percent of the input ligand shifted to the protein-DNA complex) is plotted as a function of unlabeled 60-mer added to the reaction. (Each data point is the average of three separate experiments.)



These experiments make it clear that coumarins inhibit noncovalent binding of topoisomerase to duplex DNA. A simple hypothesis for this inhibition is that the coumarins themselves bind to a site (or sites) on the topoisomerase and that site occupancy by drug and DNA would be mutually exclusive (by steric hindrance at the ligand-binding site). Failure of the coumarins to dissociate the DNA-bound enzyme can be easily accounted for on this basis (i.e. exclusion of drug binding to the relatively stable topoisomerase-DNA complex). This would also account for the failure of the drugs to inhibit strand religation by preformed covalent complexes. Although these experiments do not exclude the existence of a drug ternary complex (topoisomerase-DNA complex with bound coumarin), we detected no effect of coumarins on the stability or the strand transferase activity of the binary protein-DNA complex. Given that reaction chemistry was not affected, at least not at the level of religation, and that coumarin inhibition of binding (predominantly noncovalent) to the 60-mer paralleled the inhibition of suicide cleavage, it is likely that the inhibition of suicide cleavage was caused by inhibition of precleavage binding. Taken together, these data suggest that coumarin interference with noncovalent binding of topoisomerase to DNA is sufficient to account for coumarin inhibition of DNA relaxation. Further experiments to substantiate these points are described below.

Evidence for Coumermycin Binding to Topoisomerase

The binding of coumermycin to the enzyme was measured in solution in the absence of DNA. The assay was based on changes in the absorbance and fluorescence of the drug as a consequence of interaction with protein. The absorbance and fluorescence spectra during titration of coumermycin with increasing concentrations of topoisomerase are shown in Fig. 7(A and B, respectively). Both the absorbance maximum at 347 nm ( = 25,800 M cm) and the fluorescence emission spectrum ((max) = 360 nm) for coumermycin showed decreases upon binding to the enzyme. The maximal fluorescence decrease, extrapolated to infinite enzyme concentration, was 38%. Similarly, the maximal absorbance decrease was 0.106 absorbance unit, corresponding to a 27% decrease in . The absorption spectra also indicated an isosbestic point at 327 nm, which provides evidence for a simple two-state binding equilibrium between topoisomerase and coumermycin. (The error in the isosbestic point is caused by the combined errors introduced by subtracting the background absorbance of the enzyme from each titration spectrum and the small volumes used for the titration.) A plot of the fractional absorbance (closed circles) and fluorescence (open circles) intensities as a function of total topoisomerase concentration is shown in Fig. 7C along with a double-reciprocal plot of the data (Fig. 7C, inset). The absorbance and fluorescence data were both well fit to a one-site binding isotherm () with a best fit value for K(D) of 27 ± 5 µM (Fig. 7C). This value is similar to the K(I) of 40 µM reported previously for DNA relaxation by topoisomerase from virions (14) and to the coumermycin concentrations of 10-25 µM required for half-maximal inhibition of DNA binding and suicide cleavage noted above. These similarities provide evidence that the observed coumermycin-binding site is the same site that, when occupied by drug, inhibits DNA binding and relaxation. Two- or three-fold discrepancies between K(I) values measured at 10M enzyme (DNA relaxation assays) or 10M enzyme (DNA binding assays) and K(D) values measured at 10M enzyme (drug binding assays) are not unexpected.


Figure 7: Binding of coumermycin to topoisomerase. Ultraviolet absorbance spectra (A) and fluorescence emission spectra ( = 327 nm) (B) of 30 µM coumermycin were determined as a function of topoisomerase concentration in the range 0-127 µM. C shows a plot of the fractional absorbance (closed circles) and fluorescence (open circles) intensities of coumermycin as a function of total topoisomerase (topo) concentration. For this plot, the absorbance intensities at 347 nm and the entire fluorescence emission peak areas were used. The line describes the nonlinear least-squares fit of the data to . A double-reciprocal plot of the data is shown in the inset. arb. units, arbitrary units.



Displacement of Bound Coumermycin by DNA

To directly test whether DNA binding and coumermycin binding are mutually exclusive, i.e. that there is no ternary complex of enzyme, drug, and DNA, we investigated whether protein-bound coumermycin would be displaced by added DNA. In this experiment, a coumermycin-topoisomerase complex was challenged with increasing concentrations of a 32-mer DNA substrate containing a single CCCTT recognition site. Because the concentration of topoisomerase used in the binding reactions (25 µM) was 500-fold greater than the K(D) value of 50 nM for DNA substrates of this size (27) and because the binding affinity of topoisomerase for DNA is 500-fold greater than for coumermycin (based on the results of Fig. 7), it is expected that the added DNA should displace the coumermycin from the protein if the drug and DNA bind to the same site on the enzyme. The method of equilibrium ultrafiltration was used to separate free and enzyme-bound coumermycin. Release of coumermycin into the filtrate was determined by measuring the absorbance at 310 nm (see ``Experimental Procedures''). A plot of the fractional increase in absorbance of the filtrate as a function of the molar ratio of DNA to enzyme is shown in Fig. 8. As expected for tight competitive binding, the DNA displaced the drug from the enzyme with a linear concentration dependence and saturated at a 1:1 stoichiometry of DNA to enzyme. Thus, binding of DNA and binding of coumermycin are mutually exclusive.


Figure 8: Displacement of bound coumermycin by DNA. Increasing concentrations of CCCTT-containing DNA were added to solutions of topoisomerase (topo) and coumermycin as described under ``Experimental Procedures.'' After incubation for 15 min at 37 °C, the samples were subjected to ultrafiltration, and the absorbance of the filtrate at 310 nm was determined. The fractional absorbance change is plotted as a function of the molar ratio of DNA to topoisomerase (DeltaA(max) = 0.01). The dashed line shows the expected curve for fractional binding of one DNA molecule to topoisomerase based on the reported Kvalue of 50 nM(32) .



Binding of Novobiocin to Topoisomerase

The binding of novobiocin to the vaccinia enzyme was studied by following the quenching of the intrinsic fluorescence of the enzyme upon binding of novobiocin. (Note that this method was unsuitable for studying coumermycin binding because of the large absorbance of coumermycin at the excitation wavelengths for enzyme fluorescence assays.) The enzyme has two tryptophan residues and 14 tyrosine residues that contribute to its intrinsic fluorescence. A plot of fractional fluorescence intensity of topoisomerase at the emission wavelength of 340 nm ( = 275 nm) as a function of total novobiocin concentration is shown in Fig. 9A. Extrapolating to saturating novobiocin, the topoisomerase fluorescence was quenched by 94 ± 4%. A K(D) value for novobiocin of 55 ± 15 µM was obtained from a nonlinear least-squares fit of the fluorescence data to , which assumes one binding site.


Figure 9: Equilibrium binding of novobiocin to topoisomerase. A, the fluorescence of topoisomerase (25 µM) was measured as a function of novobiocin (NB) concentration in the range 0-147 µM. The observed fluorescence intensities were corrected for optical filtering effects as described(29) . The fractional fluorescence intensity of topoisomerase is plotted as a function of total novobiocin concentration. The line describes the nonlinear least-squares fit of the data to . The inset shows a double-reciprocal plot of the data. B, equilibrium ultrafiltration measurements of novobiocin binding were performed as described under ``Experimental Procedures'' at 200 µM topoisomerase. Novobiocin concentration was varied in the range 0-1350 µM. Me(2)SO (8%, v/v) was included in the reactions to prevent aggregation of the drug-topoisomerase complex. The ratio of bound novobiocin to total topoisomerase (topo) is plotted as a function of free novobiocin. The solid line is the nonlinear least-squares fit of the data to . The dashed lines show the individual curves for the two classes of binding sites. C, shown is the Scatchard analysis of the binding data obtained by equilibrium ultrafiltration. The lines correspond to those shown in B. The stoichiometries for the tight and weak binding sites are 1 ± 0.1 and 4.9 ± 0.7, respectively.



The affinity for novobiocin determined by the fluorescence method was higher than expected based on the concentrations inhibitory for DNA relaxation. Therefore, the binding measurements were extended to higher concentrations of enzyme and novobiocin to ascertain whether additional weaker novobiocin-binding sites existed on the enzyme. Because of large optical filtering effects when high enzyme and ligand concentrations are used, the fluorescence method is not generally suitable for quantitation of weak binding sites. However, the method of equilibrium ultrafiltration is not subject to these problems and was therefore used to separate free and enzyme-bound novobiocin. This provided an independent determination of the binding equilibria. In Fig. 9B, the data obtained by the ultrafiltration method are shown as a plot of bound novobiocin, normalized to the total concentration of enzyme present, against free novobiocin. The data show two classes of binding sites. A nonlinear least-squares fit to (Fig. 9B, solid line) reveals a single tight site (K(D)(1) = 20 ± 5 µM; n = 1 ± 0.1) and a class of weak sites (K(D)(2) = 513 ± 125 µM; n = 4.9 ± 0.7). For illustration, the dashed lines in Fig. 9B show the individual binding curves for the two classes of sites. Scatchard analysis of the data gave similar results (Fig. 9C). Both the stoichiometry and affinity for the tight site are consistent with the values obtained by the fluorescence method. The K(D) value for the weak binding sites is similar to the concentrations of drug that inhibit DNA relaxation and DNA binding, suggesting that binding of novobiocin to one or more weak sites is necessary for enzyme inhibition.

Probing the Structure of the Coumarin-Topoisomerase Complex by Partial Proteolysis

Structural domains of the 314-amino acid vaccinia topoisomerase are demarcated by two interdomain regions that are susceptible to proteolysis(33, 34) . The ``hinge'' region, from residues 135 to 142, is defined by accessibility to chymotrypsin, trypsin, and V8 proteases(34) . Chymotrypsin cleaves the purified topoisomerase at a single site in the hinge, between Tyr-136 and Leu-137, to generate a 16-kDa N-terminal fragment and a 20-kDa C-terminal fragment starting at residue 137(34) . This is seen clearly in Fig. 10as the topoisomerase was digested with increasing concentrations of chymotrypsin, and the products were analyzed by SDS-PAGE. The 20-kDa carboxyl-terminal species was largely resistant to digestion by chymotrypsin added in excess over the level sufficient to cleave all the input topoisomerase. Some breakdown of the 20-kDa species to an 18-kDa polypeptide was evident at the highest levels of protease (Fig. 10). This 18-kDa species arises via a secondary chymotryptic cleavage event between Leu-146 and Thr-147(34) . The 16-kDa N-terminal fragment was degraded at the higher levels of chymotrypsin to which the carboxyl-terminal domain was stable (Fig. 10). (Note that the 14-kDa polypeptide seen in Fig. 10at high levels of input protease corresponds to chymotrypsin, not a fragment derived from the topoisomerase.)


Figure 10: Structure probing by proteolysis with chymotrypsin. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 5 µg of topoisomerase were preincubated at room temperature for 10 min without drug or with 1 mM novobiocin or 0.1 mM coumermycin. All reaction mixtures containing coumermycin included 10% Me(2)SO. Increasing amounts of chymotrypsin were added (50, 100, 500, and 1000 ng, proceeding from left to right within each series), and the samples were digested for 15 min at room temperature. Control reactions were incubated without chymotrypsin (lanes -). The samples were then denatured by addition of SDS to 1% and analyzed by electrophoresis through a 15% polyacrylamide gel containing 0.1% SDS. A Coomassie Blue-stained gel is shown. The positions of 31- and 14-kDa marker proteins are indicated to the left. The identities of the predominant chymotryptic fragments are denoted in schematic form to the left and right. A polypeptide contributed by the chymotrypsin preparation (Chymo) is indicated by the arrow to right.



Novobiocin (1 mM) and coumermycin (0.1 mM) both rendered the topoisomerase resistant to chymotrypsin; a 10-20-fold higher level of chymotrypsin was required to achieve a comparable extent of digestion of the drug-topoisomerase complex compared with free topoisomerase. More important, however, was the marked shift in the distribution of proteolytic fragments. Cleavage by chymotrypsin in the presence of the coumarin drugs yielded a polypeptide doublet at 18 kDa. Peptide sequencing after transfer of the cleavage products to a polyvinylidene difluoride membrane showed that this doublet consisted of a C-terminal fragment starting at Thr-147 and a fragment derived from the N terminus of the topoisomerase. (Note that the fragment from residues 1 to 146 has a predicted molecular mass of 17.4 kDa (Fig. 10).) Cleavage between Tyr-136 and Leu-137 within the hinge was suppressed strongly, as reflected by the much lower abundance of the 20- and 16-kDa chymotryptic fragments. (Note that the drugs themselves display characteristic mobility during SDS-PAGE and that they weakly take up Coomassie Blue dye. Hence, novobiocin appears as a diffusely staining band at 14 kDa in Fig. 10, whereas coumermycin, which structurally resembles a dimer of novobiocin, appears as a diffuse band at 27 kDa.) A second key finding was that the actual amount of cleavage by chymotrypsin at Leu-146 in the presence of the coumarins (reflected by the abundance of the 18-kDa C-terminal species) was increased compared with the free enzyme. These changes in proteolysis in the drug-bound state suggest either that topoisomerase undergoes a conformational change upon ligand binding or that the coumarins bind directly to the protease-sensitive hinge region of the protein. We showed previously that identical changes in the chymotrypsin sensitivity of the topoisomerase are elicited when the enzyme binds to duplex DNA(34) .

The shift in the protease susceptibility of the topoisomerase depended on the concentration of coumermycin or novobiocin included in the digests (Fig. 11). Acquisition of overall protease resistance (indicated by the amount of intact topoisomerase polypeptide) and the enhancement of cleavage at the secondary chymotryptic site (reflected by increased abundance of the 18-kDa doublet) occurred in parallel between 20 and 60 µM coumermycin (Fig. 11, top panel) and between 0.4 and 0.8 mM novobiocin (bottom panel). The drug concentrations for coumarin-dependent alteration of protease sensitivity correlated well with those for drug binding and for inhibition of DNA binding and DNA relaxation.


Figure 11: Protection from proteolysis depends on drug concentration. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, 5 µg of topoisomerase, and various concentrations of coumermycin (top panel) or novobiocin (bottom panel) were preincubated at room temperature for 10 min. Chymotrypsin (0.5 µg) was added to each mixture. Proteolysis proceeded for another 15 min at room temperature. The samples were then denatured and analyzed by SDS-PAGE. The concentration of drug included in each sample is indicated above the lanes. All reaction mixtures in the coumermycin experiment (top panel) included 10% Me(2)SO. The position of the full-length topoisomerase polypeptide (Topo) is indicated to the right. The identities of the predominant chymotryptic fragments are denoted in schematic form to the left and right.



It is worth pointing out that neither ATP nor AMP-PNP had any effect on the chymotrypsin sensitivity of the topoisomerase, or on the distribution of the proteolytic fragments, at nucleotide concentrations as high as 5 mM (data not shown). This is relevant because the coumarin drugs block gyrase-catalyzed ATP hydrolysis, apparently by obstructing the ATP-binding site(3) . Purified recombinant vaccinia topoisomerase has no associated ATPase(35) . Although nucleoside triphosphates at 5 mM can stimulate DNA relaxation by the vaccinia topoisomerase, this effect is mediated by the pyrophosphate moiety, not by the nucleoside(35) . Thus, there is no indication that coumarin inhibition of vaccinia topoisomerase is related mechanistically to coumarin action on DNA gyrase.

Digestion of the Coumarin-Topoisomerase Complex with Trypsin

Trypsin digestion defines three structural domains within the topoisomerase(33, 34) . Initial attack at limiting trypsin concentration occurs between Arg-80 and Asn-81 (we have referred to this site as the interdomain ``bridge'') to yield a large carboxyl-terminal fragment of 27 kDa and an N-terminal peptide of 9 kDa (Fig. 12). The 27-kDa species is converted to a 20-kDa polypeptide at intermediate levels of trypsin via cleavage between Lys-135 and Tyr-136 in the hinge. A difference peptide with Asn-81 at the amino terminus comigrates with the 9-kDa N-terminal fragment. In the presence of novobiocin (1 mM) or coumermycin (0.1 mM), the topoisomerase became resistant to trypsin digestion (Fig. 12). A 10-20-fold higher level of trypsin was required to cleave the drug-topoisomerase complex compared with free topoisomerase (this estimate was based on the amount of undigested topoisomerase present after incubation with trypsin). The specific domain fragments arising from cleavage within the bridge and hinge regions were dramatically reduced in the presence of the drugs, suggesting that the interdomain regions are shielded from trypsin in the coumarin-topoisomerase complex. Protection of the bridge and hinge from trypsin also occurs upon binding of topoisomerase to duplex DNA (34) . A striking aspect of the experiment shown in Fig. 12is the appearance of a novel array of tryptic fragments in the presence of the drug that was not detected during digestion of the free topoisomerase. It is obvious (even without knowing the N-terminal sequences of these many new tryptic fragments) that coumarin binding exposes multiple sites on the topoisomerase to protease digestion, a finding that is consistent with a ligand-induced conformational change.


Figure 12: Structure probing by proteolysis with trypsin. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 5 µg of topoisomerase were preincubated at room temperature for 10 min without drug or with 1 mM novobiocin or 0.1 mM coumermycin. All reaction mixtures containing coumermycin included 10% Me(2)SO. Increasing amounts of trypsin were added (1, 5, 10, 50, and 100 ng, proceeding from left to right within each series), and the samples were digested for 15 min at room temperature. Control reactions were incubated without trypsin (lanes -). The samples were then denatured and analyzed by SDS-PAGE. A Coomassie Blue-stained gel is shown. The positions of 31- and 14-kDa marker proteins are indicated to the left. The identities of the predominant tryptic fragments are denoted in schematic form to the left.




DISCUSSION

The results presented above indicate that the coumarin drugs novobiocin and coumermycin inhibit the vaccinia type I topoisomerase via a distinctive mechanism. Whereas coumarins inhibit DNA gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site on GyrB, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Coumermycin binding and DNA binding to the enzyme are mutually exclusive, as judged by the ability of added DNA to displace coumermycin from a preformed drug-protein complex. The simplest interpretation of the data is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. These findings illuminate new properties of the coumarins and provide additional insights into the ligand binding properties of the vaccinia type I enzyme.

An intriguing finding is that the binding of the drugs to the topoisomerase results in protection of the interdomain bridge and hinge regions from proteolysis, the same effects observed upon binding of the enzyme to duplex DNA. Models to account for the effects of drug binding on proteolysis follow naturally from the two simple cases discussed previously for DNA binding(34) , i.e. (i) that protection by ligand from proteolysis stems from direct binding of ligand to the protected region of the enzyme or (ii) that ligand binding induces a conformational change in the topoisomerase that affects the hinge and bridge. In the second case, no assumptions are made about the location of the ligand-binding site. The bridge and hinge are separated in the linear protein sequence, but their proximity in three dimensions is not known. Although duplex DNA constitutes a relatively large ligand (the ``minimal'' substrate for covalent complex formation by vaccinia topoisomerase is an 11-bp duplex, whereas stable noncovalent binding requires a 20-bp DNA duplex(36, 37) ) compared with coumermycin (1110 kDa), it is conceivable that the binding of a single coumermycin molecule at or near the DNA site could elicit the same protective effects if the bridge and hinge are relatively close to each other in the native protein.

The structures of novobiocin and coumermycin bear little resemblance to DNA; therefore, it seems unlikely that they would make the same spectrum of contacts with the enzyme that are made by duplex DNA. A more plausible explanation for hindrance of DNA binding by coumarins is that the drugs bind to the enzyme and overlap with the DNA-binding site. This is similar to the gyrase case, where novobiocin does not bind in the ATP site, but binds adjacent to it and sterically hinders ATP binding. The binding data indicate that occupancy by one coumermycin molecule at a high affinity site is sufficient to account for inhibition of vaccinia topoisomerase, whereas novobiocin inhibition entails binding to one or more weak sites. Coumermycin, which resembles a dimer of novobiocin, may therefore extend from its tight binding site into the DNA-binding site. The present data do not address whether coumermycin and novobiocin bind to the same high affinity site on the topoisomerase (with novobiocin also binding to additional weak sites). Invoking a common site is nonetheless in keeping with the similar chemical structures and the similar dissociation constants for a tight binding site.

The increased susceptibility of the coumarin-topoisomerase complex to proteolysis at sites outside the bridge and hinge segments is consistent with a ligand-induced conformational change. The increased cleavage by chymotrypsin at Leu-146 in the presence of coumermycin or novobiocin compared with free enzyme is precisely what was observed for the topoisomerase-DNA complex(34) . The induction of multiple novel tryptic cleavage sites upon coumarin binding was more dramatic than the (primarily protective) shifts in the tryptic pattern seen with the protein-DNA complex(34) . To the extent that competitive inhibition of DNA binding by coumermycin likely involves an overlapping ligand-binding site, we view the exposure of protease-sensitive sites upon drug binding as additional indirect evidence for a conformational change upon binding of topoisomerase to DNA. Again, this does not rule out direct contact between DNA or drugs and the hinge or bridge regions.

Our understanding of the interaction of vaccinia topoisomerase with DNA would be enhanced immeasurably by a crystal structure of the enzyme, preferably in the DNA-bound state. The crystal structure of the 9-kDa N-terminal tryptic fragment of the enzyme (33) is not informative in this regard because this fragment does not bind DNA by itself. Efforts to crystallize the full sized protein alone or with DNA have not yet been successful. The binary complex of topoisomerase and coumermycin, with drug bound at or near the DNA-binding site, offers an alternative target for crystallization.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM46330 (to S. S.) and DK28616 (to A. S. M.) and American Cancer Society Grant FRA-432 (to S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an American Cancer Society postdoctoral fellowship.

To whom correspondence should be addressed.

(^1)
D. Wigley, personal communication.

(^2)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; bp, base pair; AMP-PNP, adenosine 5`-(beta,-imino)triphosphate.


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