INTRODUCTION

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The eukaryotic type IB DNA topoisomerase family includes topoisomerase I, a ubiquitous nuclear enzyme, and the topoisomerases encoded by vaccinia and other cytoplasmic poxviruses (1). These proteins relax supercoiled DNA via a common reaction pathway, which involves noncovalent binding of the topoisomerase to duplex DNA, cleavage of one DNA strand with concomitant formation of a covalent DNA-(3'-phosphotyrosyl)-protein intermediate, strand passage, and strand religation. Our aim is to understand the structural requirements for DNA recognition and transesterification chemistry. Toward that end, we have undertaken a structure-function analysis of the vaccinia topoisomerase.

The 314-amino acid vaccinia enzyme is the smallest topoisomerase known and thus affords a more tractable target for structure-function studies than the cellular type IB enzymes, which range from 765 to 1019 amino acids (2, 3). Another attractive feature of the vaccinia topoisomerase is its sequence specificity in transesterification; it forms a covalent adduct at sites containing the sequence 5'-(C/T)CCTT immediately 5' of the scissile phosphodiester (4). Thus far, we have identified key contacts on the DNA target site by enzymatic and chemical footprinting (5-7), probed the protein side of the protein-DNA interface by limited proteolysis of the topoisomerase in the free and DNA-bound states (8), mapped specific DNA contact points on the enzyme by UV photo-cross-linking (9), and performed targeted mutagenesis of 140 individual amino acid residues (10-18).

The vaccinia virus topoisomerase consists of three protease-resistant polypeptide fragments separated by two protease-sensitive interdomain segments, which we have referred to as the bridge and hinge (Fig. 1) (8). Specific functional groups identified through mutagenesis as being required for transesterification chemistry are situated near the hinge and within the C-terminal domain. These include the active site nucleophile (Tyr-274) and four other residues (Arg-130, Lys-167, Arg-223, and His-265) that are essential for the DNA cleavage and religation steps (11-15, 17, 19). Two other residues (Gly-132 and Tyr-136) are critical for the cleavage reaction, but not for religation (17). None of the essential residues appears to play a role in target site affinity, insofar as alanine substitutions that elicit from 10² to 10⁷ decrements in transesterification rate have no significant effect on the noncovalent binding of topoisomerase to CCCTT-containing duplex DNA (17).

The interdomain bridge is defined by trypsin-accessible sites at Arg-80, Lys-83, and Arg-84 (8, 20). Residues implicated in noncovalent DNA binding are situated within the bridge and in the N-terminal domain just proximal to the bridge. Tyr-70 and Tyr-72 were identified as the sites of UV cross-linking between topoisomerase and the +4 and +3 bromocytosine-substituted bases, respectively, of the CCCTT element (9). Alanine-scanning mutagenesis of the N-terminal domain suggests that Arg-67, Tyr-70, Tyr-72, and Arg-80 contribute to target site affinity (18). Mutational effects on DNA binding are evinced by inhibition of topoisomerase activity in the presence of magnesium and salt (16, 18).

These results have prompted the suggestion (9) that low affinity DNA binding and reaction chemistry are performed by the carboxyl two-thirds of the vaccinia enzyme, the sequence of which is similar to that of the cellular topoisomerases, whereas discrimination of the DNA sequence at the cleavage site is facilitated by the N-terminal domain, which is divergent in sequence and three-dimensional structure between the viral and cellular enzymes (20, 21). Here, we demonstrate that a deleted version of vaccinia topoisomerase, Topo-(81-314), that lacks the N-terminal 80-amino acid domain (Fig. 1), is active in relaxing supercoiled DNA, but is exquisitely sensitive to inhibition by salt or magnesium. The capacity of Topo-(81-314) to cleave a CCCTT-containing substrate under nonstringent conditions suggests that the catalytic domain per se can discriminate the target site. We propose a revised model for catalysis whereby the N-terminal domain enhances DNA binding and is required for a pre-cleavage conformational step.

	EXPERIMENTAL PROCEDURES

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The segment of the vaccinia virus topoisomerase gene encoding amino acids 81-314 was polymerase chain reaction-amplified using a sense-strand oligonucleotide primer that introduced an internal NdeI restriction site (CATATG) with an in-frame methionine codon in lieu of the codon for Arg-80. An NdeI-BglII restriction fragment containing the truncated topoisomerase gene was cloned into the T7-based expression vector pET3c to yield pET-Topo-(81-314). The entire insert of this plasmid was sequenced to confirm that no unwanted mutations had been introduced during amplification or cloning. The pET-Topo-(81-314) plasmid was transformed into Escherichia coli BL21. Topo-(81-314) expression was induced by infection with bacteriophage CE6 (22). Topo-(81-314) was purified from soluble bacterial lysates by phosphocellulose column chromatography and glycerol gradient sedimentation. The elution and sedimentation profiles of the Topo-(81-314) polypeptide were monitored by SDS-PAGE¹ of the column and gradient fractions. Topo-(81-314) adsorbed to phosphocellulose in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1 mM DTT, 10% sucrose) containing 150 mM NaCl, remained bound during a wash with buffer A (50 mM Tris-HCl (pH 8.0), 2 mM DTT, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100) containing 0.5 M NaCl, and was recovered by step elution with buffer A containing 1.0 M NaCl. An aliquot of the phosphocellulose preparation (250 µg) was applied to a 4.8-ml 15-30% glycerol gradient in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.1% Triton X-100. The gradient was centrifuged for 30 h at 50,000 rpm in a Beckman SW50 rotor. Fractions (0.2 ml) were collected dropwise from the bottom of the tube.

Full-length wild type topoisomerase and the double alanine-substitution mutant Topo(Y70A/Y72A) were purified from soluble bacterial lysates by phosphocellulose column chromatography and glycerol gradient sedimentation as described above for Topo-(81-314). The protein concentrations of the topoisomerase preparations were determined by using the dye-binding method (Bio-Rad) with bovine serum albumin as the standard.

	RESULTS

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Relaxation of Supercoiled DNA by Topo-(81-314)-- Topo-(81-314) is a truncated version of vaccinia topoisomerase that lacks the N-terminal structural domain defined by limited proteolysis with trypsin (Fig. 1). Topo-(81-314) was expressed in bacteria and purified from soluble lysates by phosphocellulose chromatography and glycerol gradient sedimentation. The 27-kDa Topo-(81-314) polypeptide sedimented as a discrete peak (Fig. 2). The N-terminal sequence of this species was determined by automated Edman chemistry after transferring the 27-kDa polypeptide from an SDS-polyacrylamide gel to a polyvinylidene difluoride membrane (8). The amino acid sequence (MNAKRDRIF) confirmed that the protein we purified was indeed Topo-(81-314). A single peak of DNA supercoil relaxation activity cosedimented with the Topo-(81-314) protein (Fig. 2).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Domain structure of vaccinia topoisomerase. The tripartite structure of the 314-amino acid wild type vaccinia topoisomerase is illustrated. The protease-resistant segments are punctuated by protease-sensitive interdomain bridge and hinge segments. The active site Tyr-274 is situated within the C-terminal domain. A truncated version of the enzyme, Topo-(81-314), which lacks the 80-amino acid N-terminal domain, is shown below.

View larger version (81K): [in this window] [in a new window]   Fig. 2.   Purification of Topo-(81-314) by glycerol gradient sedimentation. The polypeptide compositions of the glycerol gradient fractions were analyzed by SDS-PAGE; 20-µl aliquots of odd numbered fractions were applied to the gel. The fraction number is indicated above each lane. A photograph of the Coomassie Blue-stained gel is shown in the bottom panel. The positions and sizes (in kDa) of coelectrophoresed marker proteins are indicated on the left. Top panel, topoisomerase assay. Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 0.3 µg of pUC19 DNA, and 0.2 µl of the indicated glycerol gradient fractions were incubated for 60 min at 37 °C. The reactions were quenched by adding a solution containing SDS (0.3% final concentration), glycerol, xylene cyanol, and bromphenol blue. Reaction products were analyzed by electrophoresis through a 1% horizontal agarose gel in TG buffer (50 mM Tris, 150 mM glycine). The gels were stained in a 0.5 µg/ml ethidium bromide solution, destained in water, and photographed under short wave UV illumination.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Kinetics of DNA relaxation by WT topoisomerase and Topo-(81-314). Control reaction mixtures containing (per 20 µl) 50 mM Tris-HCl (pH 7.5), 0.3 µg (0.17 pmol) of pUC19 DNA and 0.5 pmol of WT topoisomerase or Topo-(81-314) were incubated at 37 °C. Other reaction mixtures were supplemented with 0.1 M NaCl (+NaCl), 5 mM MgCl2 (+Mg), or both 0.1 M NaCl and 5 mM MgCl2 (+NaCl +Mg), as indicated. The reactions were initiated by adding enzyme. Aliquots (20 µl) were withdrawn at the times indicated and quenched immediately with SDS. The "time 0" samples were taken prior to addition of enzyme. Reaction products were analyzed by agarose gel electrophoresis. A photograph of the ethidium bromide-stained gel is shown.

Topo-(81-314) Forms a Covalent Adduct on CCCTT-containing DNA-- A DNA substrate containing a single CCCTT site was used to examine DNA cleavage under single-turnover conditions. The substrate consisted of a 5' ³²P-labeled 18-mer scissile strand 5'-pCGTGTCGCCCTTATTCCC annealed to a 30-mer strand 3'-GCACAGCGGGAATAAGGCTATCACTGATGT to produce an 18-mer duplex with a 12-mer 5' tail (Fig. 4). Upon formation of the covalent protein-DNA adduct, the distal cleavage product 5'-ATTCCC is released and the enzyme remains bound to the ³²P-labeled 12-mer strand pCGTGTCGCCCTTp. The labeled protein-DNA adduct formed by WT topoisomerase migrated as a discrete species of ~40 kDa during SDS-PAGE (Fig. 5, left panel). In reactions containing 25 nM 18-mer/30-mer DNA and 125 nM WT topoisomerase, 95% of the input DNA became covalently bound to protein and the reaction was nearly complete within 10 s at 37 °C. The observed cleavage rate constant (k_cl) for the WT topoisomerase was 0.28 s¹ (14, 15). We found that Topo-(81-314) cleaved the 18-mer strand to form a labeled protein-DNA adduct that migrated at ~32 kDa during SDS-PAGE (Fig. 5, left panel, N). The faster electrophoretic mobility of the Topo-(81-314)-DNA adduct was consistent with the smaller size of the Topo-(81-314) polypeptide.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Kinetics of covalent DNA-enzyme adduct formation by Topo-(81-314). The structure of the 18-mer/30-mer CCCTT-containing substrate is depicted at the top of the figure; the cleavage site is indicated by the arrow. The CCCTT-containing scissile strand is 5' 32P-labeled. Cleavage reaction mixtures containing (per 20 µl) 50 mM Tris-HCl (pH 7.5), 0.5 pmol of 18-mer/30-mer DNA, and 2.5 pmol of Topo-(81-314) were incubated at 37 °C. The reactions were initiated by the addition of enzyme. Aliquots (20 µl) were withdrawn at the times indicated and quenched immediately by adding SDS to 1%. The denatured samples were electrophoresed through a 12% polyacrylamide gel containing 0.1% SDS. Covalent complex formation was revealed by transfer of radiolabeled DNA to the topoisomerase polypeptide. The extent of covalent adduct formation (expressed as the percent of the input 5' 32P-labeled oligonucleotide that was transferred to protein) was quantitated by scanning the dried gel using a FUJIX BAS1000 Bio-Imaging Analyzer and is plotted as a function of reaction time. The data were normalized to the end point value of 87% label transfer, and kcl was determined by fitting the data to the equation (100  %Clnorm) = 100ekt.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   DNA cleavage and religation by WT topoisomerase and Topo-(81-314). Reaction mixtures containing (per 20 µl) 50 mM Tris-HCl (pH 7.5), 0.5 pmol of 18-mer/30-mer DNA, and 2.5 pmol of WT topoisomerase or Topo-(81-314) were incubated at 37 °C for either 5 min (WT) or 6 h (Topo(81-314)). At this point (religation time 0), duplicate aliquots (20 µl) were withdrawn from each mixture and quenched either with SDS-PAGE sample buffer or with formamide stop buffer (1% SDS, 95% formamide, 20 mM EDTA). The cleavage reaction products formed by WT topoisomerase and Topo-(81-314) (N) in SDS-sample buffer were analyzed by SDS-PAGE. An autoradiograph of the dried gel is shown in the left panel (Cleavage). The positions and sizes (in kDa) of coelectrophoresed prestained marker proteins are indicated. Religation was initiated by adding a 5'-hydroxyl terminated 18-mer strand (5'-ATTCCGATAGTGACTACA) to the reaction mixtures at a concentration of 25 pmol/20 µl and simultaneously adjusting the mixtures to 0.3 M NaCl. Aliquots (20 µl) were withdrawn after 10, 30, and 60 s and quenched immediately in formamide stop buffer. The samples were electrophoresed through a 15% polyacrylamide gel containing 7 M urea in TBE (90 mM Tris borate, 2.5 mM EDTA). A control sample containing the input DNA substrate, but lacking topoisomerase, was analyzed in parallel (lane ). An autoradiogram of the gel is shown in the right panel. The positions of the input 18-mer scissile strand and the 30-mer strand transfer product are indicated.

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3.6

DNA Religation by the Covalent Topo-(81-314)-DNA Intermediate-- The religation reaction was studied under single-turnover conditions by assaying the ability of the covalent intermediate to transfer the covalently held 5' ³²P-labeled 12-mer strand to a 5'-hydroxyl-terminated 18-mer strand to form a 30-mer product (26). WT topoisomerase and Topo-(81-314) were first preincubated with the 18-mer/30-mer substrate. DNA analysis by denaturing gel electrophoresis established that virtually all of the input ³²P-labeled 18-mer strand had reacted with the WT topoisomerase (as gauged by disappearance of the 18-mer scissile strand; note that the covalent protein-DNA adduct does not enter the gel), whereas Topo-(81-314) reacted with ~70% of the substrate (Fig. 5, right panel, time 0 lanes). The religation reaction was initiated by adding a 50-fold molar excess of an 18-mer acceptor strand complementary to the 5' tail of the covalent intermediate. The reaction mixture was adjusted simultaneously to 0.3 M NaCl. Addition of NaCl during the religation phase promotes dissociation of the topoisomerase after strand closure and completely prevents recleavage of the strand transfer product. Religation by WT topoisomerase to form the 30-mer strand transfer product was complete within 10 s (Fig. 5, right panel). Remarkably, so was the religation reaction catalyzed by Topo-(81-314). This was in stark contrast to the severe rate defect evinced by Topo-(81-314) in the forward cleavage reaction. The religation results show that Topo-(81-314) catalyzed transesterification reaction chemistry at a near-wild type rate. The fact that the strand transfer product generated by Topo-(81-314) was identical in size to that formed by WT topoisomerase provides additional proof that the truncated enzyme cleaved the scissile strand at the CCCTTpA phosphodiester (Fig. 5). A profound and selective decrement in the rate of single-turnover strand cleavage versus religation should result in a drastic decrease in K_eq when Topo-(81-314) reacts with CCCTT-containing DNA under equilibrium conditions (17). WT topoisomerase attains a cleavage-religation equilibrium on a 60-bp duplex containing a centrally placed CCCTT element, such that ~20% of the substrate is covalently bound to the enzyme (17). Cleavage of the 60-bp equilibrium substrate by Topo-(81-314) was barely detectable; <0.1% of the scissile strand became covalently bound (data not shown).

Effects of Salt and Magnesium on DNA Cleavage by Topo-(81-314)-- Single turnover cleavage reactions are routinely performed at low ionic strength in the absence of a divalent cation. The findings that DNA relaxation by Topo-(81-314) was inhibited by 0.1 M NaCl and 5 mM MgCl₂ suggested that pre-cleavage binding might be rate-limiting under those more stringent conditions. To address this issue, we examined the effects of salt and magnesium on cleavage of the 18-mer/30-mer substrate. The amounts of covalent adduct formed in the presence of 50, 100, 150, and 200 mM NaCl or 1, 2, 4, 6, and 8 mM MgCl₂ were measured and normalized to the extent of cleavage in unsupplemented control reactions. WT topoisomerase cleavage reactions were quenched after 10 s, whereas Topo-(81-314) reactions were terminated after 6 h. The reaction times were chosen to attain comparable sensitivity for the effects of solution parameters on the cleavage reaction. The salt effects are shown in Fig. 6A; magnesium effects are shown in Fig. 6B. We observed that the WT topoisomerase was unaffected by up to 150 mM NaCl, but was inhibited by 26% at 200 mM NaCl. In contrast, covalent adduct formation by Topo-(81-314) was salt-sensitive; Topo-(81-314) was inhibited 29% and 85%, respectively, by 50 and 100 mM NaCl. Topo-(81-314) cleavage activity was abolished at 150 mM and 200 mM NaCl (Fig. 5A). WT topoisomerase was unaffected by magnesium up to 8 mM, whereas Topo-(81-314) was inhibited progressively by 1-8 mM MgCl₂. 52% inhibition occurred at 1 mM MgCl₂ and 88% inhibition was observed at 6 mM MgCl₂ (Fig. 5B). Susceptibility to salt and magnesium inhibition indicates that deletion of the N-terminal domain results in decreased affinity for the CCCTT-containing DNA substrate.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Inhibition of covalent adduct formation by salt and magnesium. Reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 0.5 pmol of 18-mer/30-mer DNA substrate, and 2.5 pmol of WT topoisomerase, Topo-(81-314), or Y70A/Y72A were supplemented with NaCl (A) or MgCl2 (B) as indicated. Reactions were initiated by adding protein and quenched after incubation at 37 °C for either 10 s (WT) or 6 h (Topo(81-314) and Y70A-Y72A). The reaction products were analyzed by SDS-PAGE. The extents of covalent complex formation were normalized to that of the unsupplemented control reaction (defined as 100%) and then plotted as a function of salt or magnesium concentration.

Noncovalent DNA Binding by Topo-(81-314)-- The noncovalent binding of Topo-(81-314) to a ³²P-labeled 60-bp DNA containing a single centrally placed CCCTT site (17) was assessed by native gel electrophoresis (27). The full-sized active site mutant protein Topo(Phe-274) was analyzed in parallel (Fig. 7). The binding reaction mixtures contained no added salt or magnesium. The Phe-274 protein bound to the 60-mer ligand to form a single discrete complex of retarded electrophoretic mobility (indicated by the asterisk in Fig. 7). The extent of complex formation was proportional to input Phe-274 topoisomerase and was near quantitative at a 2:1 molar ratio of protein to DNA. Increasing the concentration of Phe-274 to attain 5:1 and 10:1 molar ratios of protein to DNA resulted in the appearance of at least two more slowly migrating complexes (Fig. 7). We presume that this reflects the binding of one or two more topoisomerase monomers to the 60-bp DNA. We showed previously that Phe-274 topoisomerase binds at nonspecific sites on duplex DNA with 7-10-fold lower affinity than to CCCTT sites (27). The same is true of noncovalent binding by WT topoisomerase (27). Incubation of Topo-(81-314) with the 60-mer ligand resulted in the formation of a somewhat diffuse smear of slowly migrating material (Fig. 7). We estimate from the protein concentrations required to attain a comparable decrease in the residual unbound DNA that Topo-(81-314) bound the 60-mer with one-fifth the affinity of the full-sized enzyme.

View larger version (85K): [in this window] [in a new window]   Fig. 7.   Assay of DNA binding by native gel electrophoresis. Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 0.5 pmol of 60-bp DNA 5' 32P-labeled on the CCCTT-containing strand (17), and increasing amounts of either Phe-274 topoisomerase or Topo-(81-314) as indicated (0.25, 0.5, 1, 2.5, or 5 pmol, proceeding from left to right within each titration series) were incubated at 37 °C for 5 min. A control reaction contained no topoisomerase (lane ). Glycerol was added to 5% and the samples were electrophoresed through a 6% native polyacrylamide gel in 0.25× TBE (22.5 mM Tris borate, 0.6 mM EDTA) at 100 V for 3 h. Free DNA and a topoisomerase-DNA complexes of retarded mobility were visualized by autoradiographic exposure of the dried gel.

Limited Proteolysis of Topo-(81-314) with Chymotrypsin-- To address the possibility that the altered biochemical properties of recombinant Topo-(81-314) were caused by aberrant protein folding, we probed the structure of Topo-(81-314) by digestion with increasing concentrations of chymotrypsin. At limiting protease concentrations, Topo-(81-314) was cleaved to yield a predominant polypeptide fragment of 20 kDa and a minor fragment of 18 kDa (Fig. 8). The 20-kDa carboxyl species was resistant to digestion by a level of chymotrypsin sufficient to cleave all the input Topo-(81-314). The 18-kDa polypeptide increased in abundance at the higher levels of protease. Sequencing of these polypeptides by automated Edman chemistry after transfer to a polyvinylidene difluoride membrane revealed that the 20-kDa species arose via cleavage between amino acids Tyr-136 and Leu-137 and the 18-kDa species arose via cleavage between residues Leu-146 and Thr-147. These two sites of chymotrypsin cleavage of Topo-(81-314) correspond precisely to the chymotrypsin-accessible sites in the hinge region of the WT topoisomerase (8). This result suggests that recombinant Topo-(81-314) was folded properly.

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Chymotrypsin digestion of Topo-(81-314). Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 8.0), and 5 µg of Topo-(81-314) were incubated at 22 °C for 15 min with 25, 100, 250, or 500 ng of chymotrypsin. A control reaction was incubated without protease (lane ). Digestion products were resolved by SDS-PAGE and stained with Coomassie Blue. The positions and sizes (in kDa) of marker proteins are indicated at the left. The structures of the 20-kDa and 18-kDa chymotryptic fragments are depicted on the right.

Substitution of Tyr-70 and Tyr-72 by Alanine Mimics the Effects of Deleting the N-terminal Domain-- In light of prior findings that two specific residues within the N-terminal domain (Tyr-70 and Tyr-72) make major groove contacts with the nucleotide bases of the CCCTT target site (9), we considered the hypothesis that loss of these contacts might account for the effects of N-terminal domain removal on DNA binding and cleavage. To address this point, we purified and characterized a mutated version of the full-length vaccinia topoisomerase, Topo(Y70A/Y72A), in which Tyr-70 and Tyr-72 were both replaced by alanine. Previously, we reported the effects of single alanine-substitutions at Tyr-70 and Tyr-72 (18). The Y70A mutation had no effect on the kinetics of DNA relaxation in the presence of 0.1 M NaCl. However, Y70A mutant displayed an aberrant response to magnesium, whereby its rate of relaxation was lower by a factor of 5 in the presence of 5 mM magnesium plus 0.1 M NaCl compared with 0.1 M NaCl alone. The Y72A mutation slowed the rate of DNA relaxation in the presence of 0.1 M NaCl by a factor of 8 (compared with the WT rate), and the combination of NaCl and magnesium nearly abolished DNA relaxation by Y72A.

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View larger version (70K): [in this window] [in a new window]   Fig. 9.   DNA relaxation by Topo(Y70A/Y72A). Reaction mixtures contained (per 20 µl) 0.5 pmol of Topo(Y70A/Y72A). Relaxation assays were performed as described in the legend to Fig. 3.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The results presented in this study of vaccinia topoisomerase enhance our understanding of the eukaryotic type IB enzyme family as follows. (i) They define a catalytically active domain that is conserved between the cellular and poxvirus enzymes; (ii) they illuminate a clear distinction between structural elements required for transesterification chemistry in general and those required specifically for the cleavage reaction; (iii) they demonstrate that the catalytic domain per se is capable of recognizing the target sequence for transesterification.

The 234-amino acid catalytic domain, Topo-(81-314), performed the same repertoire of reactions as the full-sized vaccinia topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Our attempts to isolate a more extensively truncated, enzymatically active N-terminal deletion mutant of vaccinia topoisomerase were unsuccessful.² Therefore, we suggest that the vaccinia catalytic domain defines (for now) the minimum functional unit of a type IB topoisomerase. The catalytic domain includes the active site Tyr-274 and the four other conserved residues (Arg-130, Lys-167, Arg-223, and His-265) that we have shown are essential for transesterification reaction chemistry. Essential residues are defined as those at which side chain removal causes at least a 10² rate decrement in single-turnover religation.

We regard mutational effects on the rate of the religation reaction as the simplest measure of the contribution of any protein moiety to reaction chemistry. This is because single-turnover religation entails no site-recognition step, i.e. the topoisomerase is already bound covalently to the DNA. Although loss of the N terminus is not without consequences, it appears that the N-terminal domain is not directly involved in covalent catalysis, i.e. the rate of religation by Topo-(81-314) was similar to that of WT topoisomerase. Note that, because the rate of DNA religation to the 18-mer acceptor oligonucleotide is too fast to measure accurately by manual assay methods (k_rel = 1.3 s¹), we can only construe from the finding that the reactions were complete at 10 s that the rate of transesterification by Topo-(81-314) was within a factor of 10 of the WT rate. These results indicate that the N-terminal domain is nonessential for transesterification chemistry. Previously, we had mutated 22 individual residues of the 80-amino acid N-terminal domain to alanine and found that none of these single substitutions had a significant effect on transesterification rate (14, 16, 18).

The severe and biased effects of deleting the N-terminal domain on the rate of the forward cleavage reaction suggest that a post-binding, pre-chemical step applies during the cleavage reaction, which is not pertinent during religation. Although the precise nature of the pre-cleavage step remains unclear, the available evidence that residues within the N-terminal domain make major groove contacts with the CCCTT target site (9) suggests a post-binding alteration of the protein-DNA interface. Elimination of the two tyrosine side chains responsible for these major groove interactions (Tyr-70 and Tyr-72) elicits the same effects on relaxation and cleavage as does removal of the entire N-terminal domain. We propose that major groove contacts made by the two tyrosines induce a conformational change in the catalytic domain that activates the cleavage step.

The hinge segment of the catalytic domain is a likely mediator of this proposed conformational step. The hinge is highly susceptible to proteolysis when topoisomerase is free in solution and becomes protected from proteolysis when the enzyme is bound to DNA (8). Residues adjacent to the hinge become more accessible to proteolysis in the DNA-bound state (8). The alteration in the protease sensitivity of the hinge occurs prior to transesterification. Moreover, single alanine substitutions at residues Gly-132 and Tyr-136 within the hinge cause a reduction by more than 2 orders of magnitude in the rate of DNA cleavage, but only a modest effect on religation (17). These effects are strikingly similar to those reported here for deletion of the N terminus. We speculate that N-terminal domain and hinge dynamics may activate cleavage by properly orienting the catalytically essential residues with respect to the scissile phosphate.

The scissile phosphate and six other phosphates that contact the topoisomerase are located on the minor groove of the CCCTT target site (7). Our observation that the catalytic domain retains specificity for cleavage at CCCTT implies that vaccinia topoisomerase (i) interacts with the bases in the minor groove, (ii) senses the target site through indirect readout of backbone conformation, or (iii) recognizes the major groove through contacts intrinsic to the catalytic domain per se. Loss of contacts made by the N-terminal domain resulted in decreased affinity for the target site, which was manifest by salt and magnesium inhibition of single-turnover cleavage and DNA relaxation by Topo-(81-314). The salt and magnesium inhibition curves were essentially identical for Topo-(81-314) and Y70A/Y72A. We surmise that the two tyrosine side chains are largely responsible for the contribution made by the N-terminal domain to target site affinity. Studies of the effects of conservative replacement for Tyr-70 or Tyr-72 point to the aromatic character of these side chains as being important for enhancing DNA binding (18).

The present findings suggest that the distinctive target site specificities of the poxvirus and cellular type IB topoisomerases are not simply a function of structural differences between the domains N-terminal to the conserved catalytic core (20, 21). For the vaccinia enzyme, the specificity of cleavage is an intrinsic property of the core domain. This may also be the case for the cellular enzyme, to the extent that a core domain has been defined. Stewart et al. (28) have shown that active human topoisomerase can be reconstituted from a 58-kDa fragment derived from the central portion of the protein (this fragment contains all the residues of the vaccinia enzyme essential for transesterification except the active site tyrosine) plus a C-terminal 6.3-kDa fragment that includes the active site tyrosine. The fragment-reconstituted human enzyme cleaved DNA with the same sequence specificity as the full-sized protein. However, the cleavage-religation equilibrium of the reconstituted enzyme was skewed toward religation, DNA binding affinity was reduced, and the enzyme relaxed distributively (28). These properties of fragment-reconstituted human topoisomerase I vis à vis the intact enzyme are broadly similar to what we observe for the vaccinia catalytic domain. Nonetheless, the reconstituted human fragments together are still more than twice the size of the catalytic domain of the vaccinia enzyme.

Although mutational analysis of the vaccinia topoisomerase has implicated individual amino acid residues in general transesterification chemistry, in the cleavage reaction specifically, and in noncovalent DNA binding, a complete interpretation of these results will ultimately hinge on the availability of a crystal structure of the enzyme in both the free and DNA-bound states. Efforts in this and other laboratories to crystallize the full-sized vaccinia topoisomerase have been unsuccessful. We suspect this is due to flexibility of the protein at the interdomain bridge and hinge segments. We recently achieved success in crystallizing the catalytically active domain characterized in this paper.² The structure of the domain is under refinement and will be reported separately.