From the Molecular Biology Program, Sloan-Kettering Institute,
New York, New York 10021
Eukaryotic type IB topoisomerases catalyze the
cleavage and rejoining of DNA strands through a
DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid
vaccinia topoisomerase is the smallest member of this family and is
distinguished from its cellular counterparts by its specificity for
cleavage at the target sequence 5'-CCCTT
. Here we show that
Topo-(81-314), a truncated derivative that lacks the N-terminal
domain, performs the same repertoire of reactions as the full-sized
topoisomerase: relaxation of supercoiled DNA, site-specific DNA
transesterification, and DNA strand transfer. Elimination of the
N-terminal domain slows the rate of single-turnover DNA cleavage by
10
3.6, but has little effect on the rate of
single-turnover DNA religation. DNA relaxation and strand cleavage by
Topo-(81-314) are inhibited by salt and magnesium; these effects are
indicative of reduced affinity in noncovalent DNA binding. We report
that identical properties are displayed by a full-length mutant
protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within
the N-terminal domain that contact the DNA target site in the major
groove. We speculate that Topo-(81-314) is fully competent for
transesterification chemistry, but is compromised with respect to a
rate-limiting precleavage conformational step that is contingent on DNA
contacts made by Tyr-70 and Tyr-72.
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INTRODUCTION |
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
10
2 to 10
7 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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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).

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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.
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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.
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The rates of DNA relaxation by Topo-(81-314) and full-length wild type
(WT) topoisomerase were compared at 25 nM input protein (Fig. 3). Screening assays were performed
in the absence of added salt or magnesium (Fig. 3, Control).
The rate of DNA relaxation by WT topoisomerase under these conditions
was proportional to enzyme concentration from 8 nM to 100 nM (data not shown); higher concentrations were not tested.
25 nM WT topoisomerase relaxed 0.3 µg of supercoiled
pUC19 DNA to completion within 5-10 min. Reaction products of
intermediate superhelicity were not observed, suggesting that the WT
enzyme relaxed individual DNA molecules to completion before
dissociating and engaging a new DNA. 25 nM Topo-(81-314)
relaxed all input supercoiled DNA within 30 min, as evinced by the
decay of the supercoiled substrate. However, in contrast to WT, the
truncated enzyme accumulated partially relaxed intermediates
(e.g. at 10 and 20 min) and did not relax the DNA to
completion even after 60 min (Fig. 3). This suggested that
Topo-(81-314) acted distributively on supercoiled plasmid DNA.

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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.
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The DNA relaxation assays were also performed in the presence of 100 mM NaCl or 5 mM MgCl2. Either salt
or magnesium stimulated the relaxation rate of the WT enzyme
~20-fold, such that all supercoils were relaxed in 15-30 s (Fig. 3).
Salt and magnesium together have an additive effect on relaxation rate
(23); this is not apparent in Fig. 3, because the reaction was complete
with either solute alone at the earliest time analyzed. The response of
Topo-(81-314) to salt and magnesium was the exact opposite of the WT
protein. Relaxation by Topo-(81-314) was slowed severely by 100 mM NaCl; MgCl2 blocked activity almost
completely (Fig. 3). The combination of salt and magnesium was
similarly deleterious.
Prior studies indicated that product dissociation is rate-limiting
during relaxation by WT enzyme in the absence of salt or magnesium.
Salt and magnesium stimulate relaxation by enhancing product off-rate,
without affecting the rate of DNA cleavage by the WT topoisomerase (23,
24). In the presence of salt plus magnesium, the DNA cleavage step is
apparently rate-limiting. The paradoxical response of Topo-(81-314) to
salt and magnesium is strongly suggestive of reduced affinity of the
truncated protein for the DNA substrate. Strand cleavage is likely to
be rate-limiting in the absence of salt or magnesium. However, in the
presence of salt or magnesium, the DNA binding step becomes limiting
for Topo-(81-314). The relative rates of DNA cleavage can be gauged crudely by comparing the WT relaxation rate in the presence of salt and
magnesium to the relaxation rate of Topo-(81-314) in the absence of
added solutes. By this criteria, we estimate that the rate of DNA
cleavage by Topo-(1-314) was less than 1% of the WT rate.
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
(kcl) for the WT topoisomerase was 0.28 s
1 (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.

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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) = 100e kt.
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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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Topo-(81-314) cleaved the 18-mer/30-mer DNA very slowly (Fig. 4). An
end point of 87% label transfer was attained at 24 h. The
apparent cleavage rate constant (kcl) was 7 × 10
5 s
1. Thus, the rate of DNA cleavage
by Topo-(81-314) was slower than WT by a factor of
10
3.6. Because the observed kcl
did not increase when the Topo-(81-314) concentration was doubled, we
surmise that deletion of the N-terminal domain affects either
transesterification chemistry per se or a step in the
reaction pathway that occurs after initial binding, but prior to strand
scission.
The site specificity of cleavage of the 18-mer scissile strand by
Topo-(81-314) was initially compared with that of the WT enzyme by
treating the respective covalent adducts with proteinase K in the
presence of SDS, then resolving the digestion products by
electrophoresis through a 17% polyacrylamide gel containing 7 M urea. Digestion of the wild type covalent adduct yielded
a cluster of labeled species migrating faster than the 18-mer input strand, but slower than a free 12-mer strand (data not shown). These
digestion products consisted of the 12-mer oligonucleotide 5'-pCGTGTCGCCCTTp linked to short peptides of heterogeneous size. Digestion of the Topo-(81-314) covalent adduct yielded an identical cluster of 32P-labeled DNA-peptide adducts (data not
shown). Because any alteration in the site of covalent adduct formation
on the 32P-labeled scissile strand would result in an
easily detectable shift in the mobility of the proteinase K digestion
products (25), we surmise that Topo-(81-314) transesterified at the
same phosphodiester bond as the WT enzyme.
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 CCCTTp
A 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 Keq 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.

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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.
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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.

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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.
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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.

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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.
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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.
Here, we analyzed the kinetics of DNA relaxation by the Y70A/Y72A
double mutant under the same nonstringent conditions (no salt or
magnesium) and enzyme concentration used to detect relaxation by
Topo-(81-314). We found that the topoisomerase activity of Y70A/Y72A was comparable to that of Topo-(81-314) with respect to rate
and the accumulation of partially relaxed topoisomers (Fig.
9). Y70A/Y72A also displayed the same
profound inhibition of relaxation by 100 mM NaCl or 5 mM MgCl2 that was observed for Topo-(81-314)
(Fig. 9). Y70A/Y72A formed a covalent intermediate with the
18-mer/30-mer CCCTT-containing DNA under nonstringent conditions;
however, the rate of single turnover cleavage was extremely slow
(kcl = 3.6 × 10
5
s
1) (data not shown). The cleavage rate decrement
elicited by the double-alanine replacement was nearly identical to that
caused by removal of the entire N-terminal domain. The sensitivity of covalent adduct formation by Y70A/Y72A to inhibition by salt and magnesium paralleled that of Topo-(81-314) (Fig. 6).

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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.
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DISCUSSION |
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 10
2 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 (krel = 1.3 s
1), 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.