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(Received for publication, May 4,
1995; and in revised form, October 4, 1995) From the
Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has
unique pharmacological properties, including sensitivity to the
coumarin drugs novobiocin and coumermycin, which are classical
inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit
gyrase by binding the GyrB subunit and thereby blocking the ATP-binding
site, they inhibit vaccinia topoisomerase by binding to the protein and
blocking the interaction of enzyme with DNA. Noncovalent DNA binding
and single-turnover DNA cleavage by topoisomerase are inhibited with K
DNA topoisomerases are targeted by a variety of antimicrobial
and antineoplastic drugs(1, 2, 3) . In
addition to their therapeutic value, these drugs provide indispensable
research tools. Insights into topoisomerase mechanism are provided by
inhibitors and poisons affecting specific steps in the catalytic cycle.
Studies of drug effects in vivo, supported by genetic analyses
of drug resistance, underscore the important role played by
topoisomerases in virtually all DNA transactions. Drug-target
relationships have been firmly established for bacterial DNA gyrase,
which is sensitive to the quinolone and coumarin
antibiotics(2, 3) ; for eukaryotic type II
topoisomerases, which are blocked by epipodophyllotoxins, acridines,
and quinolones(4, 5, 6, 7) ; and for
eukaryotic topoisomerase I, which is susceptible to
camptothecin(8, 9, 10, 11, 12) . Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, displays
unusual pharmacological properties. The vaccinia enzyme is resistant to
camptothecin(13) , the hallmark topoisomerase I poison, yet it
is sensitive to the coumarin drugs novobiocin and
coumermycin(14, 15) , which are classical inhibitors
of DNA gyrase. Coumarin action on gyrase has been studied in
detail(3) . The coumarins bind to the GyrB subunit and inhibit
its ATPase activity, which is required for enzyme-catalyzed DNA
supercoiling. A 24-kDa subdomain of GyrB is sufficient to bind the
coumarins(16) ; moreover, specific amino acids implicated in
drug binding are defined by drug-resistant gyrase mutants that map
within this subdomain(17) . The molecular basis for coumarin
action emerges from the crystal structure of the GyrB-novobiocin
complex, in which the bound drug impinges on the binding pocket for ATP (18, 19) ( Fogelsong and Bauer (14) first documented coumarin
inhibition of DNA relaxation by vaccinia topoisomerase using enzyme
purified from infectious virions. The reported potency of novobiocin
against vaccinia topoisomerase was comparable to its efficacy against
eukaryotic topoisomerase
II(14, 15, 20, 21, 22, 23) .
The identification of the gene encoding vaccinia topoisomerase (24) made it clear that the viral enzyme is structurally
homologous to other eukaryotic type I enzymes, with no discernible
similarity to DNA gyrase. Because the vaccinia topoisomerase, like
other eukaryotic type I enzymes, does not require ATP binding or ATP
hydrolysis for DNA relaxation, it is unclear how the coumarins might
inhibit the viral protein. We now show that the coumarins target the
vaccinia enzyme by a distinctive mechanism in which drug binding to the
topoisomerase prevents the binding of the enzyme to DNA.
Figure 2:
Coumarin effects on covalent complex
formation (DNA cleavage). The DNA substrate consisted of an 18-mer
CCCTT-containing oligonucleotide hybridized to a 24-mer
oligonucleotide, as shown. The scissile strand was radiolabeled using
[
For this analysis, the absorption maximum at 347 nm and the
entire fluorescence emission peak area were used to calculate the
fractional intensities, where I
In , K
To prevent irreversible covalent binding of the
32-mer(27, 32) , we included in the reaction an 8-mer
DNA strand, 5`-ATTCTCGC, which was complementary to the 5`-overhang
generated by release of the 4-mer. Reaction mixtures (0.15 ml) were
prepared containing 10 mM MES, pH 6.3, 8% Me
Figure 1:
Inhibition of DNA relaxation by
novobiocin and coumermycin. Relaxation assays were performed as
described under ``Experimental Procedures.'' In the series to
the left, novobiocin was included at 50, 100, 200, 500, 1000, and 2000
µM final concentrations (proceeding from left to right).
Control reactions were performed without novobiocin (lane
-) or without enzyme (lane C). In the series to the
right, coumermycin A
The drug effect on suicide cleavage was subject to a substantial
order-of-addition effect, as shown for coumermycin in Fig. 3. In
this set of experiments, preincubation of topoisomerase with
coumermycin for 5 min again elicited a concentration-dependent
inhibition of cleavage, with 50% inhibition at 25 µM (Fig. 3, closed circles). However, when drug and
DNA substrate were premixed and the reactions were initiated by
addition of enzyme, the inhibition profile was shifted significantly to
the right, with 50% inhibition at 125 µM coumermycin (Fig. 3, open circles). A simple view of the
order-of-addition phenomenon is that coumermycin binds to the
topoisomerase and dissociates relatively slowly (resulting in higher
drug potency by virtue of prebinding). Decreased efficacy of
coumermycin when copresented with DNA is consistent with a slower
on-rate for drug than for the DNA. (By adding the DNA substrate at
various times after exposure of enzyme to 60 µM coumermycin, we determined that the order-of-addition effect was
fully established within 30 s of preincubation (data not shown).)
Figure 3:
Order-of-drug addition effect on DNA
cleavage. Topoisomerase was preincubated for 5 min with coumermycin at
the concentrations indicated (closed circles); the cleavage
reaction was then initiated by addition of the labeled DNA substrate.
Alternatively, coumermycin was added to the reaction mixtures along
with the DNA, and the cleavage reaction was initiated by addition of
topoisomerase (open circles). The extent of covalent adduct
formation (expressed as the percent of the input DNA; average of three
separate experiments) is plotted as a function of drug
concentration.
Coumarin inhibition of suicide cleavage can be explained by either
of the following: (i) the drugs directly inhibit transesterification,
or (ii) the drugs block noncovalent binding of topoisomerase to the
DNA. We sought to address this issue by circumventing the DNA binding
step, i.e. by studying the ability of topoisomerase already
bound covalently to the suicide substrate to catalyze religation to an
acceptor DNA provided in trans (40, 41). The acceptor strand
was a 5`-OH-terminated 12-mer complementary to the 5`-tail of the
``donor'' complex. The religation product was a 24-mer that
was resolved electrophoretically from the input 18-mer strand. As noted
previously(32, 38, 39) , single-turnover
strand transfer was very efficient; 80% of the input substrate was
religated to the exogenous acceptor (Fig. 4). Treatment of the
covalently bound topoisomerase for 5 min with 2 mM novobiocin
or 200 µM coumermycin (concentrations that abrogated the
suicide cleavage reaction) prior to the addition of the
acceptor strand had no discernible effect on strand religation.
Figure 4:
Coumarin effects on DNA strand transfer
(religation). Covalent complexes were formed in reaction mixtures
containing (per 20 µl) 50 mM Tris-HCl, pH 8.0, 1 pmol of
Figure 5:
Inhibition of DNA binding. A,
topoisomerase was preincubated with coumermycin at the concentrations
indicated and then mixed with the radiolabeled DNA ligand (60-mer) as
described under ``Experimental Procedures.'' The extent of
binding (expressed as the percent of the input DNA shifted to the
protein-DNA complex) is plotted as a function of drug concentration (closed circles). (Each data point is the average of three
separate experiments.) Alternatively, topoisomerase was incubated for 5
min at 37 °C with radiolabeled 60-mer DNA in the absence of drug
and then challenged with coumermycin (5-min incubation at 37 °C) at
the indicated concentrations (open circles) prior to native
gel electrophoresis. DNA binding (average of two separate experiments)
is plotted as a function of coumermycin concentration. B,
topoisomerase was preincubated with novobiocin and then mixed with the
radiolabeled 60-mer. DNA binding (closed circles; average of
two experiments) is plotted as a function of novobiocin concentration.
Alternatively, topoisomerase was incubated with the 60-mer DNA in the
absence of drug and then challenged with novobiocin (open
circles). DNA binding (average of two experiments) is plotted as a
function of novobiocin concentration.
Dramatically different effects were
observed when the order of addition was reversed such that
topoisomerase was incubated with the
Figure 6:
Stability of the binary complex to
competitor DNA is unaffected by coumermycin. Binding of topoisomerase
(1 pmol) to
These experiments
make it clear that coumarins inhibit noncovalent binding of
topoisomerase to duplex DNA. A simple hypothesis for this inhibition is
that the coumarins themselves bind to a site (or sites) on the
topoisomerase and that site occupancy by drug and DNA would be mutually
exclusive (by steric hindrance at the ligand-binding site). Failure of
the coumarins to dissociate the DNA-bound enzyme can be easily
accounted for on this basis (i.e. exclusion of drug binding to
the relatively stable topoisomerase-DNA complex). This would also
account for the failure of the drugs to inhibit strand religation by
preformed covalent complexes. Although these experiments do not exclude
the existence of a drug ternary complex (topoisomerase-DNA complex with
bound coumarin), we detected no effect of coumarins on the stability or
the strand transferase activity of the binary protein-DNA complex.
Given that reaction chemistry was not affected, at least not at the
level of religation, and that coumarin inhibition of binding
(predominantly noncovalent) to the 60-mer paralleled the inhibition of
suicide cleavage, it is likely that the inhibition of suicide cleavage
was caused by inhibition of precleavage binding. Taken together, these
data suggest that coumarin interference with noncovalent binding of
topoisomerase to DNA is sufficient to account for coumarin inhibition
of DNA relaxation. Further experiments to substantiate these points are
described below.
Figure 7:
Binding of coumermycin to topoisomerase.
Ultraviolet absorbance spectra (A) and fluorescence emission
spectra (
Figure 8:
Displacement of bound coumermycin by DNA.
Increasing concentrations of CCCTT-containing DNA were added to
solutions of topoisomerase (topo) and coumermycin as described
under ``Experimental Procedures.'' After incubation for 15
min at 37 °C, the samples were subjected to ultrafiltration, and
the absorbance of the filtrate at 310 nm was determined. The fractional
absorbance change is plotted as a function of the molar ratio of DNA to
topoisomerase (
Figure 9:
Equilibrium binding of novobiocin to
topoisomerase. A, the fluorescence of topoisomerase (25
µM) was measured as a function of novobiocin (NB)
concentration in the range 0-147 µM. The observed
fluorescence intensities were corrected for optical filtering effects
as described(29) . The fractional fluorescence intensity of
topoisomerase is plotted as a function of total novobiocin
concentration. The line describes the nonlinear least-squares
fit of the data to . The inset shows a
double-reciprocal plot of the data. B, equilibrium
ultrafiltration measurements of novobiocin binding were performed as
described under ``Experimental Procedures'' at 200 µM topoisomerase. Novobiocin concentration was varied in the range
0-1350 µM. Me
The affinity for novobiocin determined by the fluorescence method
was higher than expected based on the concentrations inhibitory for DNA
relaxation. Therefore, the binding measurements were extended to higher
concentrations of enzyme and novobiocin to ascertain whether additional
weaker novobiocin-binding sites existed on the enzyme. Because of large
optical filtering effects when high enzyme and ligand concentrations
are used, the fluorescence method is not generally suitable for
quantitation of weak binding sites. However, the method of equilibrium
ultrafiltration is not subject to these problems and was therefore used
to separate free and enzyme-bound novobiocin. This provided an
independent determination of the binding equilibria. In Fig. 9B, the data obtained by the ultrafiltration
method are shown as a plot of bound novobiocin, normalized to the total
concentration of enzyme present, against free novobiocin. The data show
two classes of binding sites. A nonlinear least-squares fit to (Fig. 9B, solid line) reveals a
single tight site (K
Figure 10:
Structure probing by proteolysis with
chymotrypsin. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 5 µg of topoisomerase were preincubated
at room temperature for 10 min without drug or with 1 mM novobiocin or 0.1 mM coumermycin. All reaction mixtures
containing coumermycin included 10% Me
Novobiocin (1 mM) and coumermycin (0.1 mM) both
rendered the topoisomerase resistant to chymotrypsin; a
10-20-fold higher level of chymotrypsin was required to achieve a
comparable extent of digestion of the drug-topoisomerase complex
compared with free topoisomerase. More important, however, was the
marked shift in the distribution of proteolytic fragments. Cleavage by
chymotrypsin in the presence of the coumarin drugs yielded a
polypeptide doublet at 18 kDa. Peptide sequencing after transfer of the
cleavage products to a polyvinylidene difluoride membrane showed that
this doublet consisted of a C-terminal fragment starting at Thr-147 and
a fragment derived from the N terminus of the topoisomerase. (Note that
the fragment from residues 1 to 146 has a predicted molecular mass of
17.4 kDa (Fig. 10).) Cleavage between Tyr-136 and Leu-137 within
the hinge was suppressed strongly, as reflected by the much lower
abundance of the 20- and 16-kDa chymotryptic fragments. (Note that the
drugs themselves display characteristic mobility during SDS-PAGE and
that they weakly take up Coomassie Blue dye. Hence, novobiocin appears
as a diffusely staining band at The shift in the protease
susceptibility of the topoisomerase depended on the concentration of
coumermycin or novobiocin included in the digests (Fig. 11).
Acquisition of overall protease resistance (indicated by the amount of
intact topoisomerase polypeptide) and the enhancement of cleavage at
the secondary chymotryptic site (reflected by increased abundance of
the 18-kDa doublet) occurred in parallel between 20 and 60 µM coumermycin (Fig. 11, top panel) and between 0.4
and 0.8 mM novobiocin (bottom panel). The drug
concentrations for coumarin-dependent alteration of protease
sensitivity correlated well with those for drug binding and for
inhibition of DNA binding and DNA relaxation.
Figure 11:
Protection from proteolysis depends on
drug concentration. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, 5 µg of topoisomerase, and various
concentrations of coumermycin (top panel) or novobiocin (bottom panel) were preincubated at room temperature for 10
min. Chymotrypsin (0.5 µg) was added to each mixture. Proteolysis
proceeded for another 15 min at room temperature. The samples were then
denatured and analyzed by SDS-PAGE. The concentration of drug included
in each sample is indicated above the lanes. All reaction mixtures in
the coumermycin experiment (top panel) included 10%
Me
It is worth pointing
out that neither ATP nor AMP-PNP had any effect on the chymotrypsin
sensitivity of the topoisomerase, or on the distribution of the
proteolytic fragments, at nucleotide concentrations as high as 5 mM (data not shown). This is relevant because the coumarin drugs
block gyrase-catalyzed ATP hydrolysis, apparently by obstructing the
ATP-binding site(3) . Purified recombinant vaccinia
topoisomerase has no associated ATPase(35) . Although
nucleoside triphosphates at 5 mM can stimulate DNA relaxation
by the vaccinia topoisomerase, this effect is mediated by the
pyrophosphate moiety, not by the nucleoside(35) . Thus, there
is no indication that coumarin inhibition of vaccinia topoisomerase is
related mechanistically to coumarin action on DNA gyrase.
Figure 12:
Structure probing by proteolysis with
trypsin. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 5 µg of topoisomerase were preincubated
at room temperature for 10 min without drug or with 1 mM novobiocin or 0.1 mM coumermycin. All reaction mixtures
containing coumermycin included 10% Me
The results presented above indicate that the coumarin drugs
novobiocin and coumermycin inhibit the vaccinia type I topoisomerase
via a distinctive mechanism. Whereas coumarins inhibit DNA gyrase by
binding the GyrB subunit and thereby blocking the ATP-binding site on
GyrB, they inhibit vaccinia topoisomerase by binding to the protein and
blocking the interaction of enzyme with DNA. Coumermycin binding and
DNA binding to the enzyme are mutually exclusive, as judged by the
ability of added DNA to displace coumermycin from a preformed
drug-protein complex. The simplest interpretation of the data is that
the site of drug binding coincides or overlaps with the DNA-binding
site on the topoisomerase. These findings illuminate new properties of
the coumarins and provide additional insights into the ligand binding
properties of the vaccinia type I enzyme. An intriguing finding is
that the binding of the drugs to the topoisomerase results in
protection of the interdomain bridge and hinge regions from
proteolysis, the same effects observed upon binding of the enzyme to
duplex DNA. Models to account for the effects of drug binding on
proteolysis follow naturally from the two simple cases discussed
previously for DNA binding(34) , i.e. (i) that
protection by ligand from proteolysis stems from direct binding of
ligand to the protected region of the enzyme or (ii) that ligand
binding induces a conformational change in the topoisomerase that
affects the hinge and bridge. In the second case, no assumptions are
made about the location of the ligand-binding site. The bridge and
hinge are separated in the linear protein sequence, but their proximity
in three dimensions is not known. Although duplex DNA constitutes a
relatively large ligand (the ``minimal'' substrate for
covalent complex formation by vaccinia topoisomerase is an 11-bp
duplex, whereas stable noncovalent binding requires a 20-bp DNA
duplex(36, 37) ) compared with coumermycin (1110 kDa),
it is conceivable that the binding of a single coumermycin molecule at
or near the DNA site could elicit the same protective effects if the
bridge and hinge are relatively close to each other in the native
protein. The structures of novobiocin and coumermycin bear little
resemblance to DNA; therefore, it seems unlikely that they would make
the same spectrum of contacts with the enzyme that are made by duplex
DNA. A more plausible explanation for hindrance of DNA binding by
coumarins is that the drugs bind to the enzyme and overlap with the
DNA-binding site. This is similar to the gyrase case, where novobiocin
does not bind in the ATP site, but binds adjacent to it and sterically
hinders ATP binding. The binding data indicate that occupancy by one
coumermycin molecule at a high affinity site is sufficient to account
for inhibition of vaccinia topoisomerase, whereas novobiocin inhibition
entails binding to one or more weak sites. Coumermycin, which resembles
a dimer of novobiocin, may therefore extend from its tight binding site
into the DNA-binding site. The present data do not address whether
coumermycin and novobiocin bind to the same high affinity site on the
topoisomerase (with novobiocin also binding to additional weak sites).
Invoking a common site is nonetheless in keeping with the similar
chemical structures and the similar dissociation constants for a tight
binding site. The increased susceptibility of the
coumarin-topoisomerase complex to proteolysis at sites outside the
bridge and hinge segments is consistent with a ligand-induced
conformational change. The increased cleavage by chymotrypsin at
Leu-146 in the presence of coumermycin or novobiocin compared with free
enzyme is precisely what was observed for the topoisomerase-DNA
complex(34) . The induction of multiple novel tryptic cleavage
sites upon coumarin binding was more dramatic than the (primarily
protective) shifts in the tryptic pattern seen with the protein-DNA
complex(34) . To the extent that competitive inhibition of DNA
binding by coumermycin likely involves an overlapping ligand-binding
site, we view the exposure of protease-sensitive sites upon drug
binding as additional indirect evidence for a conformational change
upon binding of topoisomerase to DNA. Again, this does not rule out
direct contact between DNA or drugs and the hinge or bridge regions. Our understanding of the interaction of vaccinia topoisomerase with
DNA would be enhanced immeasurably by a crystal structure of the
enzyme, preferably in the DNA-bound state. The crystal structure of the
9-kDa N-terminal tryptic fragment of the enzyme (33) is not
informative in this regard because this fragment does not bind DNA by
itself. Efforts to crystallize the full sized protein alone or with DNA
have not yet been successful. The binary complex of topoisomerase and
coumermycin, with drug bound at or near the DNA-binding site, offers an
alternative target for crystallization.
Volume 271,
Number 4,
Issue of January 26, 1996 pp. 2313-2322
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
values of 10-25 µM for coumermycin and 350 µM for novobiocin.
Spectroscopic and fluorescence measurements of drug binding to enzyme
indicate a single binding site on vaccinia topoisomerase for
coumermycin (K
= 27 ± 5
µM) and two classes of binding sites for novobiocin, one
tight site (K
= 20
± 5 µM) and several weak sites (K
= 513 ± 125
µM; n = 4.9 ± 0.7). Addition of a
stoichiometric amount of DNA to a preformed coumermycin-topoisomerase
complex quantitatively displaces the drug, indicating that coumermycin
binding and DNA binding to topoisomerase are mutually exclusive. A
simple interpretation is that the site of drug binding coincides or
overlaps with the DNA-binding site on the topoisomerase. Both
novobiocin and coumermycin alter the susceptibility of vaccinia
topoisomerase to proteolysis with either chymotrypsin or trypsin;
similar effects occur when topoisomerase binds to duplex DNA.
)The ATP-dependent eukaryotic
type II topoisomerases are also inhibited by novobiocin and
coumermycin, albeit at much higher drug
concentrations(20, 21, 22, 23) .
Enzyme Preparation
Vaccinia topoisomerase was
expressed in Escherichia coli and purified as
described(13, 26) . The SP5PW preparation used for
assay of DNA relaxation, DNA cleavage, DNA binding, and protease
sensitivity was homogeneous with respect to the topoisomerase
polypeptide, as determined by SDS-PAGE. ()Protein
concentration of this enzyme fraction was determined using the Bio-Rad
dye reagent with bovine serum albumin as a standard. For the
equilibrium drug binding measurements, samples of purified
topoisomerase were dialyzed extensively at 4 °C against 0.5 mM MES, pH 6.3, and then lyophilized to dryness and dissolved in
deionized distilled water at a final concentration of 200-600
µM enzyme. These manipulations of the protein preparation
did not affect its catalytic activity in relaxing supercoiled plasmid
DNA. The molar concentration of the resuspended protein was determined
spectrophotometrically using the relationship that a 1 mg/ml solution
of topoisomerase gives an A of 1.08
cm
(27) .
Coumarin Drugs
Novobiocin, purchased from Sigma,
was stored as a 100 mM stock solution in water and diluted in
water prior to each use. Coumermycin A was purchased from
Sigma and stored as a 10 mM solution in 100% MeSO.
Coumermycin was diluted in 100% MeSO prior to each use and
added to DNA relaxation, DNA cleavage, or DNA binding reaction mixtures
such that the final MeSO concentration was 10% (v/v).DNA Relaxation Assay
Reaction mixtures (20 µl)
contained 50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5
mM MgCl, 0.3 µg (170 fmol) of pUC19 plasmid
DNA, and drug as indicated. The reaction was initiated by addition of
0.1 ng (3 fmol) of topoisomerase. After incubation for 10 min at 37
°C, the reactions were quenched by addition of a solution
containing glycerol, xylene cyanol, bromphenol blue, and SDS (0.2%
final concentration). The samples were analyzed by electrophoresis
through a horizontal 1.0% agarose gel in Tris/glycine buffer (50 mM Tris base, 160 mM glycine). After staining for 15 min in
0.5 µg/ml ethidium bromide, the gel was soaked for 30 min in water
and then photographed under short-wave UV illumination using Polaroid
Type 57 film.Assay of Covalent Complex Formation (DNA
Cleavage)
Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 0.5 pmol of topoisomerase were preincubated
in the presence of novobiocin or coumermycin (final drug concentrations
as specified in the figure legends). The DNA cleavage reactions were
initiated by addition of 1 pmol of 5`-P-labeled
CCCTT-containing DNA (18-mer scissile strand hybridized to a 24-mer
strand as shown in Fig. 2). After incubation for 5 min at 37
°C, the reactions were halted by addition of SDS to 1%.
(Alternatively, the drug was added to the reaction mixtures along with
the DNA, and the cleavage reaction was initiated by addition of
topoisomerase.) Samples were analyzed by SDS-PAGE. Free DNA migrated
with the bromphenol blue dye front. Covalent complex formation was
revealed by transfer of radiolabeled DNA to the topoisomerase
polypeptide(36) . The extent of adduct formation was
quantitated by scanning the gel using a FUJIX BAS1000 Bio-Imaging
Analyzer and was expressed as the percent of the input
5`-
P-labeled oligonucleotide that was covalently
transferred to protein.
-
P]ATP and T4 polynucleotide kinase and
then gel-purified and annealed to the unlabeled complementary strand.
Topoisomerase was preincubated with coumermycin or novobiocin for 10
min at room temperature at the concentrations indicated and then mixed
with the radiolabeled DNA as described under ``Experimental
Procedures.'' The extent of covalent adduct formation (expressed
as the percent of the input DNA) is plotted as a function of drug
concentration. (Each data point is the average of three separate
experiments.)
Assay of DNA Binding by Native Gel
Electrophoresis
A 60-bp duplex DNA containing a single CCCTT
motif was used for this analysis. The sequence of the DNA was reported
previously(28) . The scissile strand was radiolabeled using
[-
P]ATP and T4 polynucleotide kinase and
then gel-purified and annealed to an unlabeled 60-mer complementary
strand by heating for 10 min at 65 °C in 10 mM Tris-HCl,
pH 8.0, 1 mM EDTA, and 0.2 M NaCl and then cooling to
room temperature over 1-2 h. DNA binding reaction mixtures (20
µl) containing 50 mM Tris-HCl, pH 8.0, and 1 pmol of
enzyme were preincubated for 5 min at 37 °C in the presence of
novobiocin or coumermycin (final drug concentrations as specified in
the figure legends). The DNA binding reaction was initiated by addition
of 1 pmol of 5`-
P-labeled 60-bp duplex DNA. The mixtures
were incubated for 5 min at 37 °C and then adjusted to 5% glycerol
and electrophoresed through a native 6% polyacrylamide gel containing
0.25
TBE (22.5 mM Tris borate, 0.6 mM EDTA)
at 100 V for 2.5 h. Free 60-mer DNA and a topoisomerase-DNA complex of
retarded electrophoretic mobility were visualized by autoradiographic
exposure of the dried gel. The extent of binding was quantitated by
scanning the gel using a phosphoimager and was expressed as the percent
of the input 5`-P-labeled oligonucleotide shifted to the
protein-DNA complex.
Spectroscopic Measurements
Ultraviolet absorbance
and fluorescence measurements were made on a Perkin-Elmer Lambda-9
UV-visible spectrophotometer and a Perkin-Elmer 650-10S
spectrofluorophotometer, respectively, using a 0.2-ml quartz cell with
a 0.5-cm path length. For all experiments, the temperature of the cell
compartment was maintained at 37 °C with a circulating water bath.
The fluorescence excitation and emission slit widths were set to 4 nm
for all measurements.Reaction Conditions for Drug Binding
Because
accurate binding measurements require concentrations of enzyme similar
to the dissociation constant, the coumarin binding experiments employed
much higher concentrations of enzyme than those used routinely for
relaxation assays. It was necessary to optimize the pH and buffer
conditions for the drug binding experiments to prevent enzyme
aggregation that occurred upon addition of coumermycin (or novobiocin)
to concentrated solutions of topoisomerase (>50 µM enzyme) at pH >7. Thus, the binding studies were performed in
10 mM MES, pH 6.3, in the presence or absence of 8%
MeSO. Control experiments revealed no discernible
difference in the K
for inhibition of DNA
relaxation by coumermycin or novobiocin at pH 6.3 compared with the
standard assay conditions (data not shown).Coumermycin Binding to Topoisomerase
Drug binding
was quantitated by measuring the decrease in the fluorescence intensity
and UV absorbance of coumermycin as it binds to topoisomerase. (It was
not practical to measure changes in fluorescence of the topoisomerase
because of the high UV absorbance by coumermycin in the excitation and
emission wavelengths of the enzyme.) Binding reactions were performed
at 37 °C in a solution containing 10 mM MES, pH 6.3, 8%
MeSO (v/v). The concentration of coumermycin was held
constant (L = 30 µM), and the
topoisomerase concentration was varied in the range 5-127
µM. Serial dilutions of enzyme were performed such that a
solution of 127 µM topoisomerase and 30 µM coumermycin in a volume of 130 µl was diluted with a 30
µM solution of coumermycin in MES/Me
SO buffer.
After each stepwise dilution of the enzyme, an absorbance spectrum in
the range 300-400 nm and a fluorescence emission spectrum in the
range 390-410 nm were measured. The isosbestic point at 327 nm in
the absorbance spectrum was used as the fluorescence excitation
wavelength. The pH of each sample was measured at the conclusion of the
experiment; the variation between samples was <0.03 pH unit. Prior
to data analysis, a background spectrum of topoisomerase in the absence
of coumermycin was measured and subtracted from each sample absorption
spectrum. The dissociation constant (K
) for
coumermycin was determined from a nonlinear least-squares fit of the
fractional absorbance or fluorescence intensities, (I
- I)/(I - I), against total topoisomerase
concentration (E
) according to ,
which assumes one binding site.
and I are
the absorbance or fluorescence intensities of coumermycin in the
absence and presence of topoisomerase, respectively, and I is the intensity at saturation.
Novobiocin Binding to Topoisomerase
The quenching
of topoisomerase fluorescence by novobiocin was determined as a
function of drug concentration as follows. Aliquots (2-12 µl)
from a concentrated novobiocin stock solution were added to a solution
of 25 µM topoisomerase in 10 mM MES, pH 6.3,
maintained at 37 °C. Prior to measuring the protein fluorescence,
the samples were equilibrated in the cell holder for 5 min with the
excitation shutter closed. The excitation wavelength was 275 nm;
emission was measured at 340 nm. The observed fluorescence intensities
were corrected for optical filtering effects caused by novobiocin
absorption at 275 nm(29) , and the fractional fluorescence
quenching was calculated as (I - I)/(I - I), where I
and I are the measured fluorescence intensities of the protein in the
absence and presence of novobiocin, respectively, and I is the intensity at saturation. The
equilibrium dissociation constant (K
) for
novobiocin binding to topoisomerase (E) was determined from a
nonlinear least-squares fit of the fractional fluorescence intensities
against total novobiocin concentration (L) according to .
Equilibrium Ultrafiltration
The affinity for
novobiocin determined by the fluorescence method was higher than
expected from the drug inhibition profile in the DNA relaxation assay.
Consequently, the method of equilibrium ultrafiltration was employed to
extend the measurements to higher novobiocin concentrations and thereby
determine whether an additional weaker novobiocin-binding site (or
sites) existed on the enzyme. Equilibrium ultrafiltration allows the
direct determination of free novobiocin and is equivalent to
equilibrium dialysis for measurement of ligand affinity(30) .
MPS-1 micropartition ultrafiltration devices and membranes (14 mm;
Diaflo YM-10) were obtained from Amicon, Inc.; the membranes were
soaked for 1 h in distilled water and air-dried prior to use. Nine
samples (0.15 ml) containing 200 µM topoisomerase and
novobiocin in the range 31-1350 µM were equilibrated
at 37 °C for 15 min in 10 mM MES, pH 6.3, 8%
MeSO. The samples were transferred to individual
ultrafiltration devices, which had been prewarmed to 37 °C and
placed in a Sorvall SS-34 rotor. The material was centrifuged for 4 min
at 4000 rpm, and the A of the filtrate (which
reflects free novobiocin) was determined. The concentrations of free
and bound novobiocin were calculated as follows. First, the absorbance
contributed by the small amount of topoisomerase leaking through the
membrane (<0.1%; determined from control samples lacking novobiocin)
was subtracted from the total filtrate absorbance. This correction was
significant only at the two lowest drug concentrations, where
novobiocin binding was nearly stoichiometric. Second, the fraction of
free novobiocin was determined by dividing the corrected A
of filtrates from topoisomerase-containing
samples by the A
of filtrates from control
samples that contained identical concentrations of drug, but no enzyme.
This method for determining the fraction of free novobiocin corrects
for the background binding (<8%) of novobiocin to the membrane.
Finally, the concentration of free novobiocin was calculated by
multiplying the fraction of free drug by the total input drug
concentration; the concentration of bound novobiocin was obtained by
subtracting [novobiocin]
from
[novobiocin]
. Binding data were analyzed by a
nonlinear least-squares fit to , which describes ligand
binding to multiple noninteracting
sites.
and K represent the dissociation
constants, and C = nE and C
= nE (that is,
the concentration of tight and weak binding sites, respectively).
Alternatively, the data were fit by Scatchard analysis using a
graphical method that yielded identical results(31) .
Displacement of Bound Coumermycin by DNA
To
examine whether coumermycin and DNA compete for a single binding site
on the enzyme, a competition binding experiment was performed using the
equilibrium ultrafiltration method (see above). The DNA competitor was
a 32-mer self-complementary ``hairpin'' oligonucleotide as
shown below (). Cleavage of the DNA by topoisomerase will
liberate a 4-nucleotide leaving group, ATCC
(underlined).
SO, 25
µM topoisomerase, and 25 µM coumermycin. The
mixtures were then supplemented with a 0, 5, 10, 15, 20, 25, or 37.5
µM concentration of the hairpin DNA plus a 25 µM concentration of the 8-mer strand. The samples were incubated for
15 min at 37 °C before performing ultrafiltration as described
above. An aliquot (25 µl) of the filtrate, which contained free
coumermycin (as well as free 4-mer in the samples containing DNA), was
diluted in 0.775 ml of water, and a UV absorption spectrum was taken in
the range 240-370 nm. The absorbance at 310 nm, which was due to
coumermycin and not free DNA, was used to quantitate the release of
drug from the enzyme as the concentration of DNA competitor was
increased.
Inhibition of DNA Relaxation by Novobiocin and
Coumermycin
DNA relaxation by purified topoisomerase was assayed
in the presence of 0.1 M NaCl and 5 mM MgCl under conditions of DNA excess. Conversion of the input
supercoiled plasmid DNA to relaxed circular DNA was essentially
quantitative after 10 min of incubation at 37 °C (Fig. 1).
Novobiocin inhibited relaxation in a concentration-dependent fashion,
with a sharp decrement between 0.2 and 0.5 mM (Fig. 1).
Coumermycin was more potent than novobiocin, inhibiting relaxation
sharply in the range 50-200 µM (Fig. 1).
(Note that because the control reaction in this experiment went to
completion, the inhibitory effects of lower drug concentrations were
probably obscured.) The effective inhibitory concentration ranges for
novobiocin and coumermycin established in this assay for the
recombinant enzyme were in agreement with earlier studies using
topoisomerase isolated from vaccinia
particles(14, 15) . Inhibition of relaxation by
coumarins was not attributable to irreversible modification of the
enzyme by drug insofar as topoisomerase that was preincubated with 1
mM coumermycin and then diluted 1000-fold in buffer without
drug was just as active in relaxation of supercoiled DNA as enzyme that
had not been exposed to coumermycin (data not shown).
was included at 20, 50, 100, 200, 500,
and 1000 µM final concentrations (proceeding from left to
right). All reactions containing coumermycin included a 10% (v/v) final
concentration of MeSO. Control reactions contained
topoisomerase and MeSO, but no coumermycin (second lane
-), or topoisomerase without coumermycin or MeSO (first lane -).
DNA Cleavage and Religation
The topoisomerase
catalytic cycle entails multiple steps: (i) noncovalent binding of
enzyme to duplex DNA, (ii) scission of one strand with concomitant
formation of a covalent protein-DNA adduct, (iii) strand passage, and
(iv) religation. Shaffer and Traktman (15) showed that covalent
binding of vaccinia topoisomerase to nick-translated duplex DNA was
inhibited partially by 200 µM novobiocin and completely by
200 µM coumermycin. It was shown subsequently that
vaccinia topoisomerase displays considerable specificity in DNA
cleavage(25) ; it binds and forms a covalent adduct at sites
containing the sequence 5`-(C/T)CCTT. This feature of the
vaccinia enzyme facilitates analysis of the partial reactions using
model substrates containing a single CCCTT cleavage site.
``Suicide'' substrates have been especially useful for
studying the cleavage reaction (first transesterification) under
single-turnover conditions(27, 32) . An example of
such a substrate is shown in Fig. 2. Covalent adduct formation
is accompanied by spontaneous dissociation of the 3`-fragment of the
cleaved strand from the protein-DNA complex, which leaves a
12-nucleotide single-strand tail on the noncleaved strand. With no
readily available acceptor for religation, the topoisomerase is
covalently trapped on the DNA. The suicide cleavage assay measures the yield of covalent adduct in a single-turnover reaction that is
complete within 15 s at 37 °C (data not shown). The yield is
proportional to input topoisomerase when DNA is in excess, and the
reaction is near-quantitative at saturating enzyme(37) . Drug
effects were evaluated at enzyme concentrations sufficient to cleave
35-45% of the input substrate (Fig. 2). In the experiment
shown in Fig. 2, topoisomerase was exposed to drug prior to
addition of the DNA cleavage substrate. The coumarins inhibited
covalent adduct formation in a concentration-dependent manner. Cleavage
was abolished completely at 200 µM coumermycin and 1
mM novobiocin; 50% inhibition occurred at
20 µM coumermycin and 0.35 mM novobiocin (Fig. 2).
P-labeled suicide substrate, and 2 pmol of topoisomerase.
Aliquots (20 µl) were transferred to individual tubes and adjusted
to either 2 mM novobiocin or 0.2 mM coumermycin as
indicated. Control samples received no drug. The mixtures were
incubated for 10 min at room temperature. Strand transfer was then
induced by addition of 50 pmol of a 5`-OH 12-mer acceptor strand that
was complementary to the 12-nucleotide single-strand tail of the
covalent donor complex. (The structures of the donor complex and the
acceptor strand are shown.) After incubation for 5 min at 37 °C,
the samples were adjusted to 0.2 M NaCl. Formamide was added
to 33% (v/v), and the samples were denatured for 5 min at 95 °C.
Aliquots (7 µl) were analyzed by electrophoresis through a 12%
polyacrylamide gel containing 7 M urea in TBE (90 mM Tris base, 90 mM boric acid, 2.5 mM EDTA).
Religation of the labeled input strand to the acceptor was revealed by
the appearance of a radiolabeled 24-mer strand. The extent of
religation was quantitated by scanning the gel using a FUJIX BAS1000
Bio-Imaging Analyzer and is expressed as the percent of the input
5`-
P-labeled 18-mer oligonucleotide that was converted to
the 24-mer product.
Drug Inhibition of DNA Binding
We assayed the
effect of the coumarins on the binding of vaccinia topoisomerase to a
radiolabeled synthetic 60-bp duplex DNA containing a single centrally
placed CCCTT recognition site(28) . Protein-DNA complex
formation was detected as the formation of a discrete complex of
retarded electrophoretic mobility during native gel
electrophoresis(28, 37) . In contrast to the suicide
substrate, for which all bound enzymes are trapped in the covalent
state, the cleavage-religation equilibrium of vaccinia topoisomerase
bound to the 60-mer DNA is strongly skewed toward religation, i.e. only 10-15% of the DNA molecules that are bound will be
linked covalently to the protein(32) . Hence, the gel shift
assay largely reflects the noncovalent binding of enzyme to the DNA
ligand. Exposure of topoisomerase to coumermycin prior to addition of
the DNA caused a concentration-dependent decrease in the extent of
topoisomerase-DNA complex formation (Fig. 5A). DNA
binding was inhibited almost completely at 80-100 µM coumermycin; 50% inhibition occurred at
10 µM coumermycin (Fig. 5A). Novobiocin also inhibited
DNA binding in a concentration-dependent fashion, but was less potent
than coumermycin; half-maximal inhibition occurred at 0.36 mM (Fig. 5B).
P-labeled 60-mer DNA
prior to addition of drug. Preformed topoisomerase-DNA complexes were
refractory to coumermycin in the range 10-100 µM and
to novobiocin in the range 0.1-1 mM (Fig. 5, A and B). Thus, the topoisomerase, once bound to DNA,
was not induced to dissociate by the drugs. This finding was extended
by order-of-addition competition experiments designed to provoke
dissociation of the prebound protein by challenge with unlabeled DNA. A
control assay established that addition of unlabeled 60-mer DNA to the
binding reaction mixtures prior to addition of enzyme reduced the
extent of DNA binding in accordance with the molar ratio of unlabeled
competitor to labeled ligand (Fig. 6). When competitor was added
after preincubation of topoisomerase with the labeled 60-mer, the
protein-DNA complex was relatively resistant to competition, but could
dissociate at higher ratios of unlabeled competitor to labeled probe (Fig. 6). Addition of 200 µM coumermycin to the
preformed protein-DNA complexes had no apparent effect on enzyme-DNA
dissociation by unlabeled competitor. Thus, coumermycin did not
stabilize or destabilize the enzyme-DNA complex.
P-labeled CCCTT-containing 60-bp DNA (1 pmol)
was assayed as described under ``Experimental Procedures.''
Unlabeled 60-mer DNA was added as a competitor in the amounts
indicated. The order of addition of competitor relative to enzyme was
varied as follows: open circles, competitor was included in
the reaction mixtures along with labeled 60-mer, and the binding
reaction was initiated by addition of topoisomerase; closed
circles, topoisomerase was incubated for 5 min at 37 °C with
labeled 60-mer in the absence of competitor and then challenged with
unlabeled 60-mer (5-min incubation at 37 °C) prior to native gel
electrophoresis; open squares, topoisomerase was incubated for
5 min at 37 °C with labeled 60-mer, then exposed to 200 µM coumermycin (for 5 min at 37 °C), and finally challenged with
competitor DNA (5-min incubation at 37 °C) prior to native gel
electrophoresis. The extent of binding to labeled 60-mer (percent of
the input ligand shifted to the protein-DNA complex) is plotted as a
function of unlabeled 60-mer added to the reaction. (Each data point is
the average of three separate experiments.)
Evidence for Coumermycin Binding to
Topoisomerase
The binding of coumermycin to the enzyme was
measured in solution in the absence of DNA. The assay was based on
changes in the absorbance and fluorescence of the drug as a consequence
of interaction with protein. The absorbance and fluorescence spectra
during titration of coumermycin with increasing concentrations of
topoisomerase are shown in Fig. 7(A and B,
respectively). Both the absorbance maximum at 347 nm ( =
25,800 M
cm
) and the
fluorescence emission spectrum (
= 360 nm)
for coumermycin showed decreases upon binding to the enzyme. The
maximal fluorescence decrease, extrapolated to infinite enzyme
concentration, was 38%. Similarly, the maximal absorbance decrease was
0.106 absorbance unit, corresponding to a 27% decrease in . The
absorption spectra also indicated an isosbestic point at
327 nm,
which provides evidence for a simple two-state binding equilibrium
between topoisomerase and coumermycin. (The error in the isosbestic
point is caused by the combined errors introduced by subtracting the
background absorbance of the enzyme from each titration spectrum and
the small volumes used for the titration.) A plot of the fractional
absorbance (closed circles) and fluorescence (open
circles) intensities as a function of total topoisomerase
concentration is shown in Fig. 7C along with a
double-reciprocal plot of the data (Fig. 7C, inset). The absorbance and fluorescence data were both well
fit to a one-site binding isotherm () with a best fit value
for K
of 27 ± 5 µM (Fig. 7C). This value is similar to the K
of 40 µM reported previously for
DNA relaxation by topoisomerase from virions (14) and to the
coumermycin concentrations of 10-25 µM required
for half-maximal inhibition of DNA binding and suicide cleavage noted
above. These similarities provide evidence that the observed
coumermycin-binding site is the same site that, when occupied by drug,
inhibits DNA binding and relaxation. Two- or three-fold discrepancies
between K
values measured at 10M enzyme (DNA relaxation assays) or 10
M enzyme (DNA binding assays) and K
values measured at 10M enzyme (drug
binding assays) are not unexpected.
= 327 nm) (B) of 30
µM coumermycin were determined as a function of
topoisomerase concentration in the range 0-127 µM. C shows a plot of the fractional absorbance (closed
circles) and fluorescence (open circles) intensities of
coumermycin as a function of total topoisomerase (topo)
concentration. For this plot, the absorbance intensities at 347 nm and
the entire fluorescence emission peak areas were used. The line describes the nonlinear least-squares fit of the data to . A double-reciprocal plot of the data is shown in the inset. arb. units, arbitrary
units.
Displacement of Bound Coumermycin by DNA
To
directly test whether DNA binding and coumermycin binding are mutually
exclusive, i.e. that there is no ternary complex of enzyme,
drug, and DNA, we investigated whether protein-bound coumermycin would
be displaced by added DNA. In this experiment, a
coumermycin-topoisomerase complex was challenged with increasing
concentrations of a 32-mer DNA substrate containing a single CCCTT
recognition site. Because the concentration of topoisomerase used in
the binding reactions (25 µM) was 500-fold greater
than the K
value of 50 nM for DNA
substrates of this size (27) and because the binding affinity
of topoisomerase for DNA is 500-fold greater than for coumermycin
(based on the results of Fig. 7), it is expected that the added
DNA should displace the coumermycin from the protein if the drug and
DNA bind to the same site on the enzyme. The method of equilibrium
ultrafiltration was used to separate free and enzyme-bound coumermycin.
Release of coumermycin into the filtrate was determined by measuring
the absorbance at 310 nm (see ``Experimental Procedures''). A
plot of the fractional increase in absorbance of the filtrate as a
function of the molar ratio of DNA to enzyme is shown in Fig. 8.
As expected for tight competitive binding, the DNA displaced the drug
from the enzyme with a linear concentration dependence and saturated at
a 1:1 stoichiometry of DNA to enzyme. Thus, binding of DNA and binding
of coumermycin are mutually exclusive.
A = 0.01). The dashed line shows the expected curve for fractional binding of
one DNA molecule to topoisomerase based on the reported Kvalue of 50
nM(32) .
Binding of Novobiocin to Topoisomerase
The binding
of novobiocin to the vaccinia enzyme was studied by following the
quenching of the intrinsic fluorescence of the enzyme upon binding of
novobiocin. (Note that this method was unsuitable for studying
coumermycin binding because of the large absorbance of coumermycin at
the excitation wavelengths for enzyme fluorescence assays.) The enzyme
has two tryptophan residues and 14 tyrosine residues that contribute to
its intrinsic fluorescence. A plot of fractional fluorescence intensity
of topoisomerase at the emission wavelength of 340 nm ( = 275 nm) as a function of total novobiocin concentration
is shown in Fig. 9A. Extrapolating to saturating
novobiocin, the topoisomerase fluorescence was quenched by 94 ±
4%. A K
value for novobiocin of 55 ± 15
µM was obtained from a nonlinear least-squares fit of the
fluorescence data to , which assumes one binding site.
SO (8%, v/v) was included
in the reactions to prevent aggregation of the drug-topoisomerase
complex. The ratio of bound novobiocin to total topoisomerase (topo) is plotted as a function of free novobiocin. The solid line is the nonlinear least-squares fit of the data to . The dashed lines show the individual curves for
the two classes of binding sites. C, shown is the Scatchard
analysis of the binding data obtained by equilibrium ultrafiltration.
The lines correspond to those shown in B. The
stoichiometries for the tight and weak binding sites are 1 ± 0.1
and 4.9 ± 0.7, respectively.
= 20
± 5 µM; n = 1 ± 0.1) and a
class of weak sites (K = 513
± 125 µM; n = 4.9 ± 0.7).
For illustration, the dashed lines in Fig. 9B show the individual binding curves for the two classes of sites.
Scatchard analysis of the data gave similar results (Fig. 9C). Both the stoichiometry and affinity for the
tight site are consistent with the values obtained by the fluorescence
method. The K value for the weak binding sites is
similar to the concentrations of drug that inhibit DNA relaxation and
DNA binding, suggesting that binding of novobiocin to one or more weak
sites is necessary for enzyme inhibition.Probing the Structure of the Coumarin-Topoisomerase
Complex by Partial Proteolysis
Structural domains of the
314-amino acid vaccinia topoisomerase are demarcated by two interdomain
regions that are susceptible to proteolysis(33, 34) .
The ``hinge'' region, from residues 135 to 142, is defined by
accessibility to chymotrypsin, trypsin, and V8 proteases(34) .
Chymotrypsin cleaves the purified topoisomerase at a single site in the
hinge, between Tyr-136 and Leu-137, to generate a 16-kDa N-terminal
fragment and a 20-kDa C-terminal fragment starting at residue
137(34) . This is seen clearly in Fig. 10as the
topoisomerase was digested with increasing concentrations of
chymotrypsin, and the products were analyzed by SDS-PAGE. The 20-kDa
carboxyl-terminal species was largely resistant to digestion by
chymotrypsin added in excess over the level sufficient to cleave all
the input topoisomerase. Some breakdown of the 20-kDa species to an
18-kDa polypeptide was evident at the highest levels of protease (Fig. 10). This 18-kDa species arises via a secondary
chymotryptic cleavage event between Leu-146 and Thr-147(34) .
The 16-kDa N-terminal fragment was degraded at the higher levels of
chymotrypsin to which the carboxyl-terminal domain was stable (Fig. 10). (Note that the 14-kDa polypeptide seen in Fig. 10at high levels of input protease corresponds to
chymotrypsin, not a fragment derived from the topoisomerase.)
SO. Increasing
amounts of chymotrypsin were added (50, 100, 500, and 1000 ng,
proceeding from left to right within each series), and the samples were
digested for 15 min at room temperature. Control reactions were
incubated without chymotrypsin (lanes -). The samples
were then denatured by addition of SDS to 1% and analyzed by
electrophoresis through a 15% polyacrylamide gel containing 0.1% SDS. A
Coomassie Blue-stained gel is shown. The positions of 31- and 14-kDa
marker proteins are indicated to the left. The identities of the
predominant chymotryptic fragments are denoted in schematic form to the
left and right. A polypeptide contributed by the chymotrypsin
preparation (Chymo) is indicated by the arrow to
right.
14 kDa in Fig. 10, whereas
coumermycin, which structurally resembles a dimer of novobiocin,
appears as a diffuse band at
27 kDa.) A second key finding was
that the actual amount of cleavage by chymotrypsin at Leu-146 in the
presence of the coumarins (reflected by the abundance of the 18-kDa
C-terminal species) was increased compared with the free enzyme. These
changes in proteolysis in the drug-bound state suggest either that
topoisomerase undergoes a conformational change upon ligand binding or
that the coumarins bind directly to the protease-sensitive hinge region
of the protein. We showed previously that identical changes in the
chymotrypsin sensitivity of the topoisomerase are elicited when the
enzyme binds to duplex DNA(34) .
SO. The position of the full-length topoisomerase
polypeptide (Topo) is indicated to the right. The identities
of the predominant chymotryptic fragments are denoted in schematic form
to the left and right.
Digestion of the Coumarin-Topoisomerase Complex with
Trypsin
Trypsin digestion defines three structural domains
within the topoisomerase(33, 34) . Initial attack at
limiting trypsin concentration occurs between Arg-80 and Asn-81 (we
have referred to this site as the interdomain ``bridge'') to
yield a large carboxyl-terminal fragment of 27 kDa and an N-terminal
peptide of 9 kDa (Fig. 12). The 27-kDa species is converted
to a 20-kDa polypeptide at intermediate levels of trypsin via cleavage
between Lys-135 and Tyr-136 in the hinge. A difference peptide with
Asn-81 at the amino terminus comigrates with the 9-kDa N-terminal
fragment. In the presence of novobiocin (1 mM) or coumermycin
(0.1 mM), the topoisomerase became resistant to trypsin
digestion (Fig. 12). A 10-20-fold higher level of trypsin
was required to cleave the drug-topoisomerase complex compared with
free topoisomerase (this estimate was based on the amount of undigested
topoisomerase present after incubation with trypsin). The specific
domain fragments arising from cleavage within the bridge and hinge
regions were dramatically reduced in the presence of the drugs,
suggesting that the interdomain regions are shielded from trypsin in
the coumarin-topoisomerase complex. Protection of the bridge and hinge
from trypsin also occurs upon binding of topoisomerase to duplex DNA (34) . A striking aspect of the experiment shown in Fig. 12is the appearance of a novel array of tryptic fragments
in the presence of the drug that was not detected during digestion of
the free topoisomerase. It is obvious (even without knowing the
N-terminal sequences of these many new tryptic fragments) that coumarin
binding exposes multiple sites on the topoisomerase to protease
digestion, a finding that is consistent with a ligand-induced
conformational change.
SO. Increasing
amounts of trypsin were added (1, 5, 10, 50, and 100 ng, proceeding
from left to right within each series), and the samples were digested
for 15 min at room temperature. Control reactions were incubated
without trypsin (lanes -). The samples were then
denatured and analyzed by SDS-PAGE. A Coomassie Blue-stained gel is
shown. The positions of 31- and 14-kDa marker proteins are indicated to
the left. The identities of the predominant tryptic fragments are
denoted in schematic form to the left.
))
,-imino)triphosphate.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
