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• W2024632389 abstract "Vaccinia topoisomerase catalyzes DNA cleavage and rejoining via transesterification to pentapyrimidine recognition site 5′-(C/T)CCTT↓ in duplex DNA. The proposed reaction mechanism involves general-base catalysis of the attack by active site nucleophile Tyr-274 on the scissile phosphodiester and general-acid catalysis of the expulsion of the 5′-deoxyribose oxygen on the leaving DNA strand. The pKa values suggest histidine and cysteine side chains as candidates for the roles of proton acceptor and donor, respectively. To test this, we replaced each of the eight histidines and two cysteines of the vaccinia topoisomerase with alanine. Single mutants C100A and C211A and a double mutant C100A-C211A were fully active in DNA relaxation, indicating that a cysteine is not the general acid. Only one histidine mutation, H265A, affected enzyme activity. The rates of DNA relaxation, single-turnover strand cleavage, and single-turnover religation by H265A were 2 orders of magnitude lower than the wild-type rates. Yet the H265A mutation did not alter the dependence of the cleavage rate on pH, indicating that His-265 is not the general base. Replacing His-265 with glutamine or asparagine slowed DNA relaxation and single-turnover cleavage to about one-third of the wild-type rate. All three mutations, H265A, H265N, and H265Q, skewed the cleavage-religation equilibrium in favor of the covalently bound state. His-265 is strictly conserved in every member of the eukaryotic type I topoisomerase family. Vaccinia topoisomerase catalyzes DNA cleavage and rejoining via transesterification to pentapyrimidine recognition site 5′-(C/T)CCTT↓ in duplex DNA. The proposed reaction mechanism involves general-base catalysis of the attack by active site nucleophile Tyr-274 on the scissile phosphodiester and general-acid catalysis of the expulsion of the 5′-deoxyribose oxygen on the leaving DNA strand. The pKa values suggest histidine and cysteine side chains as candidates for the roles of proton acceptor and donor, respectively. To test this, we replaced each of the eight histidines and two cysteines of the vaccinia topoisomerase with alanine. Single mutants C100A and C211A and a double mutant C100A-C211A were fully active in DNA relaxation, indicating that a cysteine is not the general acid. Only one histidine mutation, H265A, affected enzyme activity. The rates of DNA relaxation, single-turnover strand cleavage, and single-turnover religation by H265A were 2 orders of magnitude lower than the wild-type rates. Yet the H265A mutation did not alter the dependence of the cleavage rate on pH, indicating that His-265 is not the general base. Replacing His-265 with glutamine or asparagine slowed DNA relaxation and single-turnover cleavage to about one-third of the wild-type rate. All three mutations, H265A, H265N, and H265Q, skewed the cleavage-religation equilibrium in favor of the covalently bound state. His-265 is strictly conserved in every member of the eukaryotic type I topoisomerase family. The eukaryotic type I DNA topoisomerase family includes the nuclear type I enzymes and the topoisomerases encoded by vaccinia and other poxviruses. These proteins relax supercoiled DNA via a common reaction mechanism, which entails 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 (1Gupta M. Fujimori A. Pommier Y. Biochim. Biophys. Acta. 1995; 1262: 1-14Crossref PubMed Scopus (284) Google Scholar). A shared structural basis for transesterification and strand passage is inferred from the considerable amino acid sequence conservation found by alignment of the cellular and virus-encoded enzymes (1Gupta M. Fujimori A. Pommier Y. Biochim. Biophys. Acta. 1995; 1262: 1-14Crossref PubMed Scopus (284) Google Scholar, 2Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (99) Google Scholar). Catalytically important residues have been identified via mutational analysis of the 314-amino acid vaccinia virus topoisomerase. Three strategies have been used: (i) random mutagenesis followed by in vivo genetic selection of mutations that adversely affect enzyme activity (3Morham S.G. Shuman S. Genes & Dev. 1990; 4: 515-524Crossref PubMed Scopus (27) Google Scholar, 4Morham S.G. Shuman S. J. Biol. Chem. 1992; 267: 15984-15992Abstract Full Text PDF PubMed Google Scholar); (ii) site-directed mutagenesis of specific regions of the enzyme (5Klemperer N. Traktman P. J. Biol. Chem. 1993; 268: 15887-15899Abstract Full Text PDF PubMed Google Scholar, 6Wittschieben J. Shuman S. J. Biol. Chem. 1994; 269: 29978-29983Abstract Full Text PDF PubMed Google Scholar, 7Petersen B.Ø. Wittschieben J. Shuman S. J. Mol. Biol. 1996; 263: 181-195Crossref PubMed Scopus (26) Google Scholar); and (iii) targeted mutagenesis of a specific class of amino acid residues irrespective of their location within the protein. The latter approach was used to identify Tyr-274 as the active site of the vaccinia enzyme, i.e. through systematic replacement of tyrosines by phenylalanines (8Shuman S. Kane E.M. Morham S.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9793-9797Crossref PubMed Scopus (52) Google Scholar). Physical mapping of the active site of yeast TOP1 to Tyr-727 (9Lynn R.M. Bjornsti M.-A. Caron P.R. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3559-3563Crossref PubMed Scopus (111) Google Scholar, 10Eng W. Pandit S.D. Sternglanz R. J. Biol. Chem. 1989; 264: 13373-13376Abstract Full Text PDF PubMed Google Scholar), supported by mutational analysis of the yeast, human, and vaccinia enzymes (8Shuman S. Kane E.M. Morham S.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9793-9797Crossref PubMed Scopus (52) Google Scholar, 9Lynn R.M. Bjornsti M.-A. Caron P.R. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3559-3563Crossref PubMed Scopus (111) Google Scholar, 10Eng W. Pandit S.D. Sternglanz R. J. Biol. Chem. 1989; 264: 13373-13376Abstract Full Text PDF PubMed Google Scholar, 11Madden K.R. Champoux J.J. Cancer Res. 1992; 52: 525-532PubMed Google Scholar, 12Jensen A.D. Svejstrup J.Q. Eur. J. Biochem. 1996; 236: 389-394Crossref PubMed Scopus (28) Google Scholar), localizes the active site tyrosines within a common sequence element, Ser-Lys-X-X-Tyr, situated near the carboxyl termini of all family members (2Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (99) Google Scholar). Additional residues that we and others have identified as essential or important for covalent catalysis by the vaccinia topoisomerase are conserved in the cellular counterparts (3Morham S.G. Shuman S. Genes & Dev. 1990; 4: 515-524Crossref PubMed Scopus (27) Google Scholar, 4Morham S.G. Shuman S. J. Biol. Chem. 1992; 267: 15984-15992Abstract Full Text PDF PubMed Google Scholar, 5Klemperer N. Traktman P. J. Biol. Chem. 1993; 268: 15887-15899Abstract Full Text PDF PubMed Google Scholar, 6Wittschieben J. Shuman S. J. Biol. Chem. 1994; 269: 29978-29983Abstract Full Text PDF PubMed Google Scholar, 7Petersen B.Ø. Wittschieben J. Shuman S. J. Mol. Biol. 1996; 263: 181-195Crossref PubMed Scopus (26) Google Scholar, 13Gupta M. Zhu C.-X. Tse-Dinh Y.-C. J. Biol. Chem. 1994; 269: 573-578Abstract Full Text PDF PubMed Google Scholar). Indeed, the effects of mutations at the corresponding positions in cellular type I topoisomerases are generally concordant with the findings for the vaccinia enzyme (12Jensen A.D. Svejstrup J.Q. Eur. J. Biochem. 1996; 236: 389-394Crossref PubMed Scopus (28) Google Scholar, 14Levin N.A. Bjornsti M. Fink G.R. Genetics. 1993; 133: 799-814Crossref PubMed Google Scholar, 15Tanizawa A. Bertrand R. Kohlhagen G. Tabuchi A. Jenkins J. Pommier Y. J. Biol. Chem. 1993; 268: 25463-25468Abstract Full Text PDF PubMed Google Scholar). This suggests a common structural basis for DNA strand cleavage by the vaccinia and cellular topoisomerases. A distinctive feature of the vaccinia topoisomerase is its specificity for cleaving duplex DNA at pentapyrimidine recognition site 5′-(C/T)CCTT↓ (16Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar, 17Shuman S. J. Biol. Chem. 1991; 266: 1796-1803Abstract Full Text PDF PubMed Google Scholar, 18Shuman S. J. Biol. Chem. 1991; 266: 11372-11379Abstract Full Text PDF PubMed Google Scholar). Using simple model substrates containing a single CCCTT cleavage site, Stivers et al. (19Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar) have determined the rate constants for the cleavage and religation reactions at 20°C and defined the rate-limiting steps under single-turnover and steady-state conditions. Analysis of the pH dependence of the rate constant for cleavage (kcl) and the internal equilibrium constant (Kcl) indicated the presence of two titratable groups on the enzyme (20Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 15449-15458Crossref PubMed Scopus (46) Google Scholar). A reaction mechanism was proposed involving general-base catalysis of the attack by Tyr-274 on the scissile phosphodiester and general-acid catalysis of the expulsion of the 5′-deoxyribose oxygen (20Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 15449-15458Crossref PubMed Scopus (46) Google Scholar). The pKa values point toward unperturbed histidine and cysteine side chains as candidates for the roles of proton acceptor and donor, respectively. In the present study, we test the importance of histidines and cysteines in topoisomerase reaction chemistry by replacing each of the eight histidines and two cysteines of the vaccinia topoisomerase with alanine. All Ala-substitution mutations except one had no discernible effect on topoisomerase activity in vitro Alanine substitution for His-265 slowed the overall rate of DNA relaxation by reducing the rates of the strand cleavage and the strand religation steps. Mutations were introduced into the vaccinia virus topoisomerase gene by using the two-stage polymerase chain reaction-based overlap extension method (21Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). Plasmid pA9topo (22Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Abstract Full Text PDF PubMed Google Scholar) was used as the template for the first-stage polymerase chain reaction. Gene fragments with overlapping ends obtained from the first-stage reactions were paired and used as template in the second-stage amplification. Products containing the entire topoisomerase gene were cloned into the T7-based expression vector pET11b (Novagen) as described (6Wittschieben J. Shuman S. J. Biol. Chem. 1994; 269: 29978-29983Abstract Full Text PDF PubMed Google Scholar, 7Petersen B.Ø. Wittschieben J. Shuman S. J. Mol. Biol. 1996; 263: 181-195Crossref PubMed Scopus (26) Google Scholar). All mutations were confirmed by dideoxy sequencing. pET11-based plasmids were transformed into Escherichia coli BL21. Topoisomerase expression was induced by infection with bacteriophage λCE6 as described (22Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Abstract Full Text PDF PubMed Google Scholar), except that the cultures were adjusted to 1 mM isopropyl-1-thio-β-D-galactopyranoside immediately before inoculation with phage. Wild-type and mutant topoisomerases were purified from soluble bacterial lysates by phosphocellulose column chromatography (22Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Abstract Full Text PDF PubMed Google Scholar). The protein concentrations of the phosphocellulose preparations were determined by using the dye-binding method (Bio-Rad) with bovine serum albumin as the standard. Reaction mixtures (20 μl) containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.3 μg of pUC19 plasmid DNA, either 2.5 mM EDTA or 5 mM MgCl2, and wild-type or mutant topoisomerases (12, 4, 1.3, 0.44, 0.15, 0.05, or 0.016 ng of the phosphocellulose enzyme preparations) were incubated at 37°C for 15 min. The reactions were quenched by the addition of a solution containing SDS (0.3% final concentration), glycerol, xylene cyanol, and bromphenol blue. Samples were analyzed by electrophoresis through a 1.2% horizontal agarose gel in TBE buffer (90 mM Tris borate and 2.5 mM EDTA). The gels were stained in 0.5 μg/ml ethidium bromide solution, destained in water, and photographed under short-wave UV illumination. An 18-mer CCCTT-containing DNA oligonucleotide was 5′ end-labeled by enzymatic phosphorylation in the presence of [γ-32P]ATP and T4 polynucleotide kinase and then gel-purified and hybridized to a complementary 30-mer strand (present at 4-fold molar excess). Reaction mixtures (20 μl) containing 50 mM Tris-HCl (pH 7.5), 0.3 pmol of 18-mer/30-mer DNA, and topoisomerase were incubated at 37°C. Covalent complexes were denatured by adding SDS to 1%. The denatured samples were electrophoresed though a 10% polyacrylamide gel containing 0.1% SDS. Free DNA migrated near the bromphenol blue dye front. Covalent complex formation was revealed by transfer of radiolabeled DNA to the topoisomerase polypeptide. The extent of covalent adduct formation (expressed as the percentage of the input 5′32P-labeled oligonucleotide that was covalently transferred to protein) was quantitated by scanning the dried gel using a FUJIX BAS1000 Bio-Imaging analyzer. Single-turnover cleavage assays were performed at 22°C with 50 mM of each of the following reaction buffers: sodium acetate, pH 4.6; sodium 2-(N-morpholino)ethanesulfonic acid, pH 5.6 and 6.5; Tris-HCl, pH 7.5 and 8.5; and sodium 3-(cyclohexylamino)-1-propanesulfonic acid, pH 9.5. The wild-type and H265A topoisomerases were preincubated in a 50 mM solution of reaction buffer for 5 min. The cleavage reactions were initiated by mixing the enzyme solution with an equal volume of 50 mM reaction buffer containing the DNA substrate. (Final concentrations were 50 mM buffer, 1.9 μg/ml topoisomerase, and 15 nM DNA.) To determine the rate of cleavage by the H265A mutant, aliquots (20 μl) were withdrawn at 15 and 30 s; 1, 2, 5, 10, 20, and 30 min; and 1, 2, 4, 6, 8, and 12 h. (An additional 24-h time point was taken for rate determination at pH 4.6). The samples were quenched immediately by adding SDS. The protein-DNA adducts were resolved by SDS-polyacrylamide gel electrophoresis and quantitated by scanning the gels with a Bio-Imaging analyzer. A plot of the percentage of input DNA cleaved versus time-established end-point values for cleavage. The data were normalized to the end-point values, and kobs was determined by fitting the data to the equation (100 −%Clnorm) = 100e−kt. Aliquots were taken from wild-type topoisomerase cleavage reaction mixtures at 10, 20, and 30 s and 1, 2, 5, 10, 20, and 60 min. To better determine the initial rates of wild-type cleavage, additional sets of reaction mixtures were quenched at a single time point (5 s). The 5-s reactions were performed in triplicate at each pH; the average 5-s value was used to calculate the cleavage rate constant. Control experiments were performed to test whether the H265A protein was inactivated during a 24-h incubation at 22°C at pH 4.6, 5.6, 6.5, 7.5, 8.5, or 9.5. After this incubation, the protein was adjusted to pH 7.5 and assayed for suicide cleavage during a 5-min incubation at pH 7.5. We found that the H265A protein suffered no loss of activity during these incubations. A 60-mer oligonucleotide containing a centrally placed CCCTT element was 5′ end-labeled and then gel-purified and annealed to an unlabeled complementary 60-mer strand. Reaction mixtures (20 μl) containing 50 mM Tris-HCl (pH 7.5), 0.3 pmol of 60-mer DNA duplex, and topoisomerase were incubated for 10 min at 37°C. Covalent complexes were trapped by the addition of SDS to 1%. The denatured samples were digested for 60 min at 45°C with 10 μg of proteinase K. The volume was adjusted to 50 μl, and the digests were then extracted with an equal volume of phenol/chloroform. DNA was recovered from the aqueous phase by ethanol precipitation. The pelleted material was resuspended in formamide, and the samples were electrophoresed through a 17% polyacrylamide gel containing 7 M urea in TBE. The cleavage product, a 32P-labeled 30-mer bound to a short peptide, was well resolved from the input 60-mer substrate (18Shuman S. J. Biol. Chem. 1991; 266: 11372-11379Abstract Full Text PDF PubMed Google Scholar). The extent of strand cleavage was quantitated by scanning the wet gel using a Bio-Imaging analyzer. Single alanine substitutions were introduced at each of the eight histidines and two cysteines of the 314-amino acid vaccinia topoisomerase. A double mutant in which both cysteines were replaced by alanine was also included in the analysis. The wild-type and mutated proteins were expressed in E. coli using a T7 RNA polymerase-based expression system (22Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Abstract Full Text PDF PubMed Google Scholar). The recombinant proteins were purified from bacterial extracts by phosphocellulose column chromatography. The polypeptide compositions of the enzyme preparations were analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1). In every case, the 33-kDa topoisomerase polypeptide constituted the major species, and the extents of purification were essentially equivalent. To assess the impact of these mutations, all proteins were tested for their ability to relax supercoiled plasmid DNA in vitro Screening assays were performed in the absence of magnesium. (The rate-limiting step under these conditions is the dissociation of topoisomerase from the relaxed plasmid product.) Activity was quantitated by end-point dilution, beginning with 12 ng of the phosphocellulose topoisomerase preparation and decreasing by serial 3-fold decrements to 16 pg. We observed that the specific activity of every mutant protein except one (H265A) was equivalent to that of the wild-type topoisomerase (data not shown). The DNA relaxation assays were also performed in the presence of 5 mM magnesium. Magnesium stimulates the activity of the wild-type enzyme ∼9-fold under conditions of DNA excess by enhancing the product off-rate without affecting the rate of DNA cleavage (19Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar, 23Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 29760-29764Abstract Full Text PDF PubMed Google Scholar). The specific activities of all the mutant proteins (except H265A) were enhanced ∼9-fold by 5 mM magnesium and were again equivalent to that of the wild-type enzyme (data not shown). Hence, we conclude that Cys-100 and Cys-211 are dispensable for topoisomerase activity and that seven of the histidines (His-33, His-39, His-76, His-152, His-172, His-177, and His-307) are nonessential. The experiments that follow focus on the catalytic contributions of His-265. The effects of the H265A mutation were reflected in the kinetics of DNA relaxation (Fig. 2). 4 ng (110 fmol) of wild-type topoisomerase relaxed 0.3 μg of supercoiled pUC19 DNA (∼180 fmol) to completion within 1 min. In reactions containing 4 ng of the H265A protein, relaxed DNA accumulated slowly and steadily over 30 min, and relaxation was complete only after 60 min (Fig. 2). Hence, the H265A mutation reduced the rate of DNA relaxation to about one-sixtieth of that of the wild-type enzyme. Magnesium stimulated the rate of relaxation by wild-type topoisomerase such that the plasmid was relaxed to completion within 15 s (Fig. 2). In contrast, magnesium had no effect on the kinetics of relaxation by H265A (Fig. 2). DNA relaxation by H265A in the presence of magnesium was at least 2 orders of magnitude slower than that by the wild-type topoisomerase. There are two ways in which the H265A mutation can slow DNA relaxation: (i) by slowing the rate-limiting step, or (ii) by retarding another component step to the point that it becomes rate-limiting. The failure of H265A to be stimulated by magnesium suggested that the product off-rate was not rate-limiting for the mutant protein as it is for wild-type topoisomerase. This suggested that H265A directly affected reaction chemistry. A suicide substrate containing a single CCCTT↓ cleavage site was used to examine DNA cleavage under single-turnover conditions (24Shuman S. J. Biol. Chem. 1992; 267: 8620-8627Abstract Full Text PDF PubMed Google Scholar). The substrate consisted of an 18-mer scissile strand annealed to a 30-mer complementary strand to produce an 18-bp 1The abbreviation used is: bpbase pair(s). duplex with a 12-mer 5′ tail (Fig. 3). Upon formation of the covalent protein-DNA adduct, the distal cleavage product 5′-ATTCCC is released, and the topoisomerase becomes covalently trapped on the DNA (as illustrated in Fig. 3). The extent of cleavage by the wild-type topoisomerase during a 5-min reaction was proportional to added enzyme; 95% of the input DNA became covalently bound at saturation (Fig. 3A). The concentration dependence of the H265A activity profile was similar to that of the wild type, but only 35-38% of the input substrate was covalently bound in 5 min (Fig. 3A). base pair(s). Suicide cleavage by the wild-type topoisomerase was nearly complete within 10 s at 37°C (Fig. 3B). In contrast, H265A cleaved the DNA quite slowly. Covalent adduct accumulated steadily over 20 min; 84% of the input substrate was cleaved after 1 h (Fig. 3B). The H265A data fit well to a single exponential with an apparent cleavage rate constant (kobs) of 0.002 s−1. The extent of cleavage by wild-type enzyme at 5 s was 76% of the end-point value (±4%; average of five experiments). We used this datum to estimate a wild-type rate constant of 0.28 s−1. Thus, we observed that the H265A mutation slowed the rate of cleavage by 2 orders of magnitude. Note that kcl for wild-type topoisomerase at 37°C was higher than the value of 0.07 s−1 determined at 20°C with a different DNA substrate (19Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar). It was hypothesized previously that a histidine might function as a general base during transesterification (20Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 15449-15458Crossref PubMed Scopus (46) Google Scholar). According to this model, the imidazole ring nitrogen would accept a proton from the hydroxyl of the active site tyrosine (Tyr-274), thereby facilitating nucleophilic attack by the phenolic oxygen on the scissile phosphate. If His-265 plays such a role, we would expect the H265A mutant to display an altered pH-rate profile in single-turnover cleavage. We therefore measured the rate of suicide cleavage by H265A as a function of pH in the range of pH 4.6-9.5. A plot of log kobs versus pH is shown in Fig. 4. The shape of the H265A pH-rate profile was similar to that of wild-type topoisomerase. This argued that His-265 is not the general base in topoisomerase-mediated strand cleavage. His-265 was replaced with glutamine and asparagine, which are nearly isosteric with histidine (30Plapp B.V. Methods Enzymol. 1995; 249: 91-119Crossref PubMed Scopus (58) Google Scholar) but cannot be protonated like histidine. The H265N and H265Q proteins were expressed in bacteria and purified by phosphocellulose chromatography. The polypeptide compositions of these enzyme preparations were similar to that of the wild type depicted in Fig. 1 (data not shown). The rates of relaxation of supercoiled plasmid DNA by H265N and H265Q in the absence of a divalent cation were about one-half to one-fourth of the wild-type rate (Fig. 5). Relaxation by H265N and H265Q was stimulated 2-fold by 5 mM magnesium (Fig. 5). Suicide cleavage by H265N and H265Q in a 5-min reaction was proportional to added enzyme; 93% of the input DNA was covalently bound at saturation (Fig. 6A). Cleavage by H265N and H265Q was slowed compared to that by wild-type topoisomerase (Fig. 6B). Apparent cleavage rate constants of 0.08 and 0.06 s−1 for H265N and H265Q were estimated from the extents of cleavage at 10 s (Fig. 6B). The mild effects of the H265N and H265Q mutations on the rate of single-turnover DNA cleavage contrasted with the severe rate decrement observed for the H265A mutant. Religation of the cleaved strand occurs by attack of a 5′-OH-terminated polynucleotide on the 3′ phosphodiester bond between Tyr-274 and the DNA. This transesterification step can be studied independent of strand cleavage by assaying the ability of a preformed topoisomerase-DNA complex to religate the covalently held 5′32P-labeled strand to a heterologous acceptor strand (24Shuman S. J. Biol. Chem. 1992; 267: 8620-8627Abstract Full Text PDF PubMed Google Scholar, 25Shuman S. J. Biol. Chem. 1992; 267: 16755-16758Abstract Full Text PDF PubMed Google Scholar). The wild-type, H265N, H265Q, and H265A proteins were incubated with the suicide cleavage substrate for 60 min to attain near-equivalent levels of the covalent intermediate. We then added a 100-fold molar excess of an 18-mer acceptor strand complementary to the 5′ tail of the covalent donor complex (Fig. 7) while simultaneously increasing the ionic strength to 0.3 M NaCl. (Addition of NaCl during the religation phase promotes dissociation of the topoisomerase after strand closure and prevents recleavage of the strand transfer product.) Religation to the 18-mer yielded the 32P-labeled 30-mer depicted in Fig. 7. The strand transfer product was resolved from the input 32P-labeled 18-mer strand by denaturing gel electrophoresis. The wild-type enzyme transferred 96% of the input CCCTT-containing strand to the exogenous acceptor (Fig. 7). The extent of religation at the earliest time point analyzed (10 s) was 95% of the end-point value. Similarly, H265N and H265Q transferred >90% of the input DNA to the acceptor, with ∼80% of the end-point value attained in 10 s. Thus, the Asn and Gln substitutions caused a relatively mild slowing of the strand transfer reaction. In contrast, strand transfer by H265A was much slower. The religated 30-mer accumulated steadily over 10 min; 74% of the input substrate was religated after 20 min. The observed religation rate constant (krel) was 0.004 s−1. Thus, the H265A mutation slowed the rate of religation by at least 2 orders of magnitude relative to the wild-type religation rate. We used a 60-bp DNA duplex containing a centrally placed cleavage site with 30 bp upstream and 30 bp downstream of the scissile bond to study topoisomerase cleavage under true equilibrium conditions. Cleavage of the 60-mer duplex by the wild-type topoisomerase was linear up to 20 ng of protein and plateaued at 38-152 ng (Fig. 8). At saturation, 17% of the substrate was cleaved. The cleavage equilibrium constant (Kcl = covalent complex/noncovalent complex) was 0.2, which was slightly higher than the Kcl of 0.13 determined at 20°C (19Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar). Covalent complex formation by H265N, H265Q, and H265A increased with enzyme up to 40 ng and saturated thereafter. Remarkably, 48-51% of the input 60-mer was cleaved by the three His-265 mutants at saturation; hence Kcl was about 5-fold higher than that of the wild-type topoisomerase. We conclude that the H265 mutations skew the cleavage-religation equilibrium in favor of the covalently bound state. These findings were confirmed by a kinetic analysis of cleavage of the 60-mer duplex by the H265N, H265Q, and H265A proteins (Fig. 9A). H265N and H265Q achieved end points of 51% cleavage (Kcl = 1.0) and 48% cleavage (Kcl = 0.92), respectively. Equilibrium cleavage by H265N and H265Q was virtually complete within 10-20 s. The H265A mutant displayed a slow approach to equilibrium over 5 min (Fig. 9A). 48% of the input 60-mer was covalently bound at equilibrium (Kcl = 0.92). The rate constant kobs for approach to equilibrium by H265A was 0.01 s−1. Knowing that Kcl = 0.96 and that kobs = kcl+ krel, we calculated that kcl = 0.005 s−1 and krel = 0.005 s−1. The rate constant for cleavage by H265A of the 60-mer DNA (0.005 s−1) was fairly close to the observed rate constant for single-turnover cleavage of the 18-mer/30-mer suicide substrate (0.002 s−1). We measured single-turnover religation on the 60-mer substrate by allowing the cleavage reaction to reach equilibrium and then adjusting the reaction mixtures to 0.3 M NaCl. This concentration of salt blocks both equilibrium cleavage and single-turnover cleavage by interfering with DNA binding (Refs. 16Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar and 24Shuman S. J. Biol. Chem. 1992; 267: 8620-8627Abstract Full Text PDF PubMed Google Scholar; data not shown). Topoisomerase prebound to an equilibrium cleavage substrate at low ionic strength is dissociated when the salt concentration is raised to >0.25 M (16Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar). Hence, topoisomerase molecules that have catalyzed strand closure on the 60-mer DNA will be dissociated from the DNA by salt and will be unable to rebind and recleave. The decrease in covalent complex as a function of time after the addition of NaCl is plotted in Fig. 9B The extent of cleavage by H265N and H265Q plummeted rapidly from 48-50% to 2%. The closure reaction was virtually complete at the earliest time point (10 s). In contrast, the level of H265A covalent complex declined slowly over 10 min (krel = 0.004 s−1). Note that the observed rate constant for H265A in single-turnover religation agreed with the value calculated from the rate of approach to equilibrium. In continuing our mutational analysis of the vaccinia DNA topoisomerase, we focused on cysteine and histidine side chains as potential catalysts during transesterification. Alanine scanning mutagenesis revealed that neither of the two cysteines in the protein was important for enzyme activity. Of the eight histidines, only His-265 was essential for catalysis. An essential amino acid side chain was defined operationally as one whose removal, e.g. by alanine replacement, results in drastic (100-fold) loss of function. The H265A mutation profoundly slowed the rates of single-turnover cleavage and single-turnover religation. We estimate that the rates of both chemical steps were reduced by about 2 orders of magnitude compared to the wild-type protein. These effects on reaction chemistry are probably sufficient to explain the effect of the H265A mutation on the rate of DNA relaxation. A noteworthy effect of the H265A mutation was to bias the cleavage-religation equilibrium toward covalent binding. Kcl of H265A (0.92) was nearly 5-fold higher than that of the wild-type topoisomerase (0.2) on the 60-bp CCCTT-containing DNA. Because Kcl = kcl/krel, the H265A mutation must have slowed the rate of religation of the 60-bp DNA to a greater extent (relative to the wild-type rate) than it slowed the rate of cleavage. The apparent rate constants for single-turnover cleavage on a suicide substrate and single-turnover religation on the suicide cleavage intermediate were 0.002 s−1 and 0.004 s−1, respectively. The equilibrium constant of 0.5 calculated from the ratio of the single-turnover rate constants agreed reasonably well with the value of 0.92 for equilibrium cleavage of the 60-mer. His-265, which is essential for topoisomerase activity, is strictly conserved in every known eukaryotic type I topoisomerase (Fig. 10). None of the other seven histidine residues of the vaccinia topoisomerase is strictly conserved (2Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (99) Google Scholar). In the alignment of Caron and Wang (2Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (99) Google Scholar), His-265 is situated within a 9-amino acid motif that includes 3 other conserved residues: Lys-257 (which is Lys or Arg in all family members); Ala-260 (an invariant residue); and Val-262 (an aliphatic residue in most cases) (Fig. 10). Within this segment, the vaccinia topoisomerase is most closely related to the topoisomerases encoded by its poxvirus cousins Shope fibroma virus and orf virus (26Klemperer N. Lyttle D.J. Tauzin D. Traktman P. Robinson A.J. Virology. 1995; 206: 203-215Crossref PubMed Scopus (27) Google Scholar) and to the type I topoisomerases of Ustilago maydis (27Gerhold D. Thiyagarjan M. Kmiec E.B. Nucleic Acids Res. 1994; 22: 3773-3778Crossref PubMed Scopus (11) Google Scholar), Arabidopsis thaliana (2Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (99) Google Scholar), and Plasmodium falciparum (28Tosh K. Kilbey B. Gene (Amst.). 1995; 163: 151-154Crossref PubMed Scopus (27) Google Scholar) (Fig. 10). We predict that the conserved histidine plays an essential role in the chemistry of strand cleavage and religation by cellular type I topoisomerases. What is the role of His-265 during covalent catalysis? It apparently does not function as a general base during single-turnover cleavage because the shape of the pH-rate profile of H265A was similar to that of wild-type topoisomerase and because replacements by Asn and Gln were well tolerated. His-265 may thus engage in hydrogen-bonding interactions that are critical for topoisomerase activity; this hydrogen bonding potential is largely retained after substitution by Gln or Asn. Still, the H265N and H265Q proteins are slightly slowed with respect to their cleavage rates, and their equilibrium cleavage constants are higher than that of wild-type protein. The increase in Kcl was similar to that seen with H265A. The implication is that even subtle alterations at position 265 affect strand religation more than strand cleavage. His-265 is situated only 9 amino acids away from the active site Tyr in the linear sequence of the vaccinia topoisomerase. We speculate that His-265 is part of the active site in the native folded protein. The interval between the homologous histidines of the cellular topoisomerases and their respective active site tyrosines is interrupted by a linker region of variable length, e.g. 90 amino acids in the human topoisomerase and 167 amino acids in the yeast enzyme (2Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (99) Google Scholar). The linker has no counterpart in the poxvirus topoisomerases, is not well conserved even among the cellular topoisomerases, and is apparently dispensable for topoisomerase activity (29Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1996; 271: 7602-7608Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). We suspect therefore that the His-265 and Tyr-274 equivalents of the cellular enzymes are likely to be disposed in three dimensions as they are in the vaccinia topoisomerase. Definitive assessment of predictions regarding the action of His-265 and its location at the active site await the determination of the crystal structure of topoisomerase bound covalently to DNA. In conclusion, our results establish the importance of conserved residue His-265 but exclude all histidines and cysteines as essential acid-base catalysts in DNA cleavage. Aspartate and glutamate side chains emerge as the next likely candidates for the role of general base. We have already shown that 5 of the 35 acidic residues in vaccinia topoisomerase are nonessential (6Wittschieben J. Shuman S. J. Biol. Chem. 1994; 269: 29978-29983Abstract Full Text PDF PubMed Google Scholar, 7Petersen B.Ø. Wittschieben J. Shuman S. J. Mol. Biol. 1996; 263: 181-195Crossref PubMed Scopus (26) Google Scholar). Alanine substitution mutagenesis of conserved acidic positions is currently under way." @default.
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