(Received for publication, September 9, 1996)
From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021
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 (1). 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 (1, 2).
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 (3, 4); (ii) site-directed mutagenesis of specific regions of
the enzyme (5-7); 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 (8).
Physical mapping of the active site of yeast TOP1 to Tyr-727 (9, 10), supported by mutational analysis of the yeast, human, and vaccinia enzymes (8-12), localizes the active site tyrosines within a common sequence element, Ser-Lys-X-X-Tyr, situated near the carboxyl termini of all family members (2). 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 (3-7, 13). Indeed, the effects of mutations at the corresponding positions in cellular type I topoisomerases are generally concordant with the findings for the vaccinia enzyme (12, 14, 15). 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
(16-18). Using simple model substrates containing a
single CCCTT cleavage site, Stivers et al. (19) 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 (20). 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 (20). 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 (21). Plasmid pA9topo (22) 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 (6, 7). All mutations were confirmed by dideoxy sequencing.
Topoisomerase Expression and PurificationpET11-based
plasmids were transformed into Escherichia coli BL21.
Topoisomerase expression was induced by infection with bacteriophage CE6 as described (22), except that the cultures were adjusted to 1 mM isopropyl-1-thio-
-D-galactopyranoside
immediately before inoculation with phage. Wild-type and mutant
topoisomerases were purified from soluble bacterial lysates by
phosphocellulose column chromatography (22). 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.
Suicide Cleavage AssayAn 18-mer CCCTT-containing DNA
oligonucleotide was 5 end-labeled by enzymatic phosphorylation in the
presence of [
-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.
Equilibrium Cleavage AssayA 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 (18). 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 (22). 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 (19, 23). 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.
H265A Is Defective in Relaxing Supercoiled Plasmid DNAThe
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.
H265A Affects the Rate of DNA CleavageA suicide substrate
containing a single CCCTT cleavage site was used to examine DNA
cleavage under single-turnover conditions (24). The substrate consisted
of an 18-mer scissile strand annealed to a 30-mer complementary strand
to produce an 18-bp1 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).
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 s1. 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 (19).
It was hypothesized previously
that a histidine might function as a general base during
transesterification (20). 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.
Substitution of His-265 with Asn and Gln
His-265 was replaced
with glutamine and asparagine, which are nearly isosteric with
histidine (30) 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 s1 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.
Effect of H265 Mutations on DNA Religation
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 (24, 25). 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 s1. 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 (19). 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 s1. 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. 16 and 24; 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 (16). 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 s1). 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 s1 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 (2). In the alignment of Caron and Wang (2), 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 (26)
and to the type I topoisomerases of Ustilago maydis (27),
Arabidopsis thaliana (2), and Plasmodium falciparum (28) (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 (2). 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 (29). 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 (6, 7). Alanine substitution mutagenesis of conserved acidic positions is currently under way.