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J Biol Chem, Vol. 274, Issue 50, 35927-35932, December 10, 1999
From the Departments of Quinolones are the most active oral
antibacterials in clinical use and act by increasing DNA cleavage
mediated by prokaryotic type II topoisomerases. Although topoisomerase
IV appears to be the primary cytotoxic target for most quinolones in
Gram-positive bacteria, interactions between the enzyme and these drugs
are poorly understood. Therefore, the effects of ciprofloxacin on the
DNA cleavage and religation reactions of Staphylococcus
aureus topoisomerase IV were characterized. Ciprofloxacin doubled
DNA scission at 150 nM drug and increased cleavage
~9-fold at 5 µM. Furthermore, it dramatically inhibited
rates of DNA religation mediated by S. aureus topoisomerase
IV. This inhibition of religation is in marked contrast to the effects
of antineoplastic quinolones on eukaryotic topoisomerase II, and
suggests that the mechanistic basis for quinolone action against type
II topoisomerases has not been maintained across evolutionary
boundaries. The apparent change in quinolone mechanism was not caused
by an overt difference in the drug interaction domain on
topoisomerase IV. Therefore, we propose that the mechanistic basis for
quinolone action is regulated by subtle changes in drug orientation
within the enzyme·drug·DNA ternary complex rather than gross
differences in the site of drug binding.
Quinolone antibacterials were first synthesized over 30 years ago
(1-4). Although the founding member of this drug class, nalidixic
acid, had limited medical applications, its discovery spawned
tremendous interest in the clinical potential of these compounds. Since
that initial breakthrough, the use of quinolone-based drugs to treat
bacterial infections in humans has grown considerably (2-6). In fact,
this class is now the most active and broad spectrum family of oral
antibacterial agents in clinical use (2-5).
Quinolones are targeted to the prokaryotic type II topoisomerases, DNA
gyrase and topoisomerase IV (6-20). These enzymes are essential to all
bacterial species and play fundamental roles in most DNA processes
(20-22). DNA gyrase, the only known topoisomerase that can actively
underwind nucleic acids (23), is required for the maintenance of
superhelical density in the bacterial chromosome and relieves torsional
stress that accumulates in front of replication forks (20, 22, 24-27).
Topoisomerase IV is responsible for unlinking newly replicated daughter
chromosomes and resolving knots that result from recombination events
(9, 20, 28, 29). Although these enzymes play different roles in the
cell, they both alter the topological state of nucleic acids by passing a double helix through a transient break that they generate in a
separate DNA segment (19, 20, 22, 26, 27).
Quinolones do not kill bacterial cells by blocking the essential
functions of type II topoisomerases. Rather, they increase the cellular
concentration of covalent topoisomerase-cleaved DNA complexes that are
intermediates in the DNA strand passage reactions of these enzymes (4,
5, 10, 19, 20, 24, 26, 30-32). This action generates high levels of
double-stranded breaks in the chromosomes of treated bacteria, triggers
the SOS response, and ultimately induces cell death (4, 20, 26, 30,
32).
Before topoisomerase IV was discovered in 1990, DNA gyrase was believed
to be the only significant target for quinolones in bacterial cells. To
a great extent, this has proven true for Gram-negative species (4-8,
20). Although topoisomerase IV is a secondary target for these
compounds in Escherichia coli, the cellular consequences of
its interaction with quinolones are revealed only in the presence of
drug-resistant DNA gyrase mutants (4, 6, 12, 20). However, in
Gram-positive species, topoisomerase IV appears to be the primary
cytotoxic target for most quinolones (4-6, 11, 14-17, 20, 33). This
change in target reflects the increased sensitivity of Gram-positive
topoisomerase IV to quinolone-based compounds coupled with the
naturally drug-resistant form of gyrase found in these bacteria (4, 20,
34).
Quinolones targeted to topoisomerase IV, for the first time, have
opened many Gram-positive species to this family of potent antibacterial drugs. This has allowed improved treatment of many infections (especially those of the respiratory tract) that were resistant to other antibiotics (4-6, 35). Although Gram-positive topoisomerase IV has become an important new target for antibacterial drug discovery, its interactions with quinolones are poorly understood.
Therefore, the present study characterized the effects of quinolones on
the DNA cleavage/religation activity of topoisomerase IV from
Staphylococcus aureus. In marked contrast to the actions of
antineoplastic quinolones against eukaryotic type II topoisomerases (which increase levels of DNA breakage by stimulating the forward scission reaction), quinolones act primarily by inhibiting the ability
of topoisomerase IV to religate cleaved DNA molecules. Even though the
functional drug interaction domain on type II topoisomerases has been
maintained from bacterial to eukaryotic species, it appears that the
mechanistic basis for quinolone action has not been conserved across
evolutionary boundaries.
Topoisomerase IV was cloned from S. aureus 4220, overexpressed as the separate subunits (GrlA and GrlB) in E. coli, and purified by a modification of the procedure of Hallett
et al. (36). Briefly, cells from log-phase cultures were
pelleted, resuspended in TED buffer (50 mM Tris-HCl, pH
7.6, 1 mM EDTA, and 5 mM dithiothreitol), lysed
by sonication, and repelleted by centrifugation at 18,000 × g for 30 min. Topoisomerase IV subunits were extracted from both the soluble fraction and the cell pellet. The two extracts were
combined and dialyzed against TEDG buffer (TED plus 10% glycerol) and
the subunits were purified by the method of Hallett et al. (36) with the following changes. In the first step, the crude fraction
was applied to a heparin-Sepharose column, washed with TEDG plus 0.25 M NaCl, followed by a linear gradient of 0.25 to 1.0 M NaCl in TEDG. Active fractions were combined,
concentrated, brought to a final concentration of 1 M
(NH4)2SO4 and loaded on a
phenyl-Superose FPLC column. Samples were eluted with a linear gradient
starting at 1 M
(NH4)2SO4. Individual subunits were
assayed for catalytic activity using an excess of the complementing
subunit. The specific activity of S. aureus topoisomerase IV
was 3.1 × 104 decatenation units/mg of protein
(decatenating 200 ng of catenated kinetoplast DNA in 30 min at
37 °C). Human topoisomerase II Etoposide and ciprofloxacin were obtained from Sigma. The quinolone
CP-115,953 was synthesized at Pfizer Central Research. Etoposide was
stored as a 10 mM stock in dimethyl sulfoxide at 4 °C.
Ciprofloxacin and CP-115,953 were stored as 40 and 30 mM stock solutions, respectively, in 0.1 N NaOH at Preparation of DNA Substrates--
Negatively supercoiled pBR322
DNA was isolated from E. coli as described previously (39).
A uniquely end-labeled 564-base pair DNA substrate (residues 376-939
in pBR322) was prepared as described by Burden et al.
(40).
DNA Cleavage--
DNA cleavage assays in the absence or presence
of drugs were performed as described by Corbett et al. (41).
Briefly, 5 nM negatively supercoiled pBR322 DNA was
incubated with 15 nM topoisomerase IV in 20 µl of
cleavage buffer (35 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 5 mM dithiothreitol, and 50 µg/ml
bovine serum albumin) at 37 °C. Unless stated otherwise, reactions
were carried out for 30 min. In drug competition assays, ciprofloxacin
and etoposide both were present in the reaction mixture prior to the
addition of topoisomerase IV, so that the enzyme was exposed
simultaneously to both compounds. DNA cleavage reactions were stopped
by the addition of SDS (0.5% final concentration) followed by EDTA (15 mM final concentration). Samples were digested with
proteinase K (80 µg/ml final concentration) for 30 min at 45 °C.
Following the addition of 60% sucrose, 0.5% bromphenol blue, and
0.5% xylene cyanole FF in 10 mM Tris-HCl, pH 7.9, DNA
products were resolved by electrophoresis in 1% agarose gels in 40 mM Tris acetate, pH 8.3, 2 mM EDTA, and 0.5 µg/ml ethidium bromide. DNA bands were visualized by UV light,
photographed through Kodak 23A and 12 filters with Polaroid type 665 film, and quantitated by scanning negatives with an E-C apparatus model
EC910 densitometer in conjunction with Hoefer GS-370 Data System
software. The intensity of bands in the negative was proportional to
the amount of DNA present. Double-stranded DNA breaks were monitored by
the conversion of negatively supercoiled plasmid to linear molecules.
Sites of DNA cleavage were determined in the absence or presence of
drugs using the protocol of Burden et al. (40). Assay mixtures contained 1.4 nM labeled linear 564-mer DNA (25 ng) and 6 nM topoisomerase IV in cleavage buffer, and were
incubated at 37 °C for 15 min. Alternatively, assays contained 60 nM human topoisomerase II DNA Religation--
Reactions were carried out by the procedure
of Anderson et al. (31). DNA cleavage/religation equilibria
were established in the presence or absence of 5 µM
quinolone using negatively supercoiled pBR322 substrates as described
in the preceding section. Religation was initiated by shifting the
temperature from 37 to 65 °C and stopped at various times up to
120 s by the addition of SDS (0.5% final concentration). Samples
were processed and analyzed by agarose gel electrophoresis as described
in the preceding section. The apparent first-order rate of religation
was determined by quantifying the loss of linear DNA.
Ciprofloxacin is the most widely prescribed quinolone in clinical
use and represents one of the most active oral antibacterial agents
currently available (2-6). It is cytotoxic to a broad range of
bacteria and has well documented activity against topoisomerase IV from
Gram-negative species (2, 4-6, 9, 12, 31). Therefore, it was chosen as
the model quinolone for the present study.
Ciprofloxacin Stimulates DNA Cleavage Mediated by S. aureus
Topoisomerase IV--
As previously observed for its Gram-negative
counterpart (31), topoisomerase IV from S. aureus displayed
a robust DNA scission activity with negatively supercoiled substrates.
A time course for DNA cleavage in the absence of drugs is shown in Fig.
1. The DNA cleavage/religation
equilibrium of the enzyme was established within 30 s of the start
of the reaction, and at a 3:1 ratio of topoisomerase IV:plasmid
molecule, ~5% of the initial DNA substrate was cleaved. This is in
contrast to human topoisomerase II
The DNA cleavage/religation equilibrium of S. aureus
topoisomerase IV was profoundly affected by addition of ciprofloxacin (Fig. 1, inset, and Fig. 2).
DNA scission increased as much as 9-fold in the presence of 5 µM drug. The concentration of quinolone required to
double levels of cleavage (i.e. the CC2 value)
was ~150 nM. The effect of ciprofloxacin on the
Gram-positive enzyme was even greater than that previously observed for
E. coli topoisomerase IV (31). With this latter enzyme,
cleavage levels increased only 4.5-fold, and ~500 nM
quinolone was required to double scission (Fig. 2).
Maximal levels of quinolone-induced DNA cleavage are attained within
1-2 min in the presence of eukaryotic type II topoisomerases from
Drosophila and humans (not shown). By comparison, it takes DNA gyrase ~1 h to re-establish its cleavage/religation equilibrium when ciprofloxacin is included in reaction mixtures (43). S. aureus topoisomerase IV is intermediate to these two enzymes; maximal levels of DNA scission were observed within 10 min following incubation with ciprofloxacin (Fig.
3).
Quinolones Act by Inhibiting DNA Religation Mediated by S. aureus
Topoisomerase IV--
As determined by their effects on DNA
religation, drugs enhance DNA cleavage mediated by type II
topoisomerases by two alternative (but not mutually exclusive)
mechanisms (44, 45). While some drug classes strongly inhibit
enzyme-mediated DNA religation, others have little effect on this
reaction. Agents in this latter category presumably act by stimulating
the forward rate of DNA cleavage.
Specific members of the quinolone family (typified by CP-115,953, which
is shown with ciprofloxacin in Fig. 4)
display antineoplastic activity and are potent enhancers of DNA
scission mediated by eukaryotic type II topoisomerases (31, 46-55).
These drugs show little or no ability to inhibit DNA religation
mediated by these enzymes (47, 48). Thus, it has been suggested that
antineoplastic quinolones act primarily by stimulating the forward DNA
scission reaction.
In contrast, antibacterial quinolones have a greater effect on the DNA
religation reaction of Gram-negative topoisomerase IV (31). When
included in reaction mixtures, levels of ciprofloxacin that stimulated
DNA cleavage ~4.5-fold decreased rates of religation mediated by
E. coli topoisomerase IV ~2-fold (31). This finding suggests that inhibition of DNA religation by quinolones plays a more
prominent role in prokaryotic species.
Since ciprofloxacin has a greater effect on the DNA cleavage activity
of S. aureus topoisomerase IV than it does with the Gram-negative enzyme, the ability of this quinolone to inhibit DNA
religation mediated by the Gram-positive enzyme was examined. A
temperature shift assay was used for comparative studies (31). This
assay is based on the finding that type II topoisomerases can religate,
but not cleave DNA at suboptimal temperatures (56, 57).
Ciprofloxacin dramatically reduced levels of DNA religation in this
assay (Fig. 5). At a drug concentration
(5 µM) that stimulated DNA cleavage ~9-fold, rates of
religation decreased ~7-fold. This is in marked contrast to the
effects of antineoplastic quinolones on eukaryotic topoisomerase II,
which display little to no inhibition of religation in this assay (47,
48). Therefore, it appears that ciprofloxacin enhances DNA scission
mediated by S. aureus topoisomerase IV primarily by
inhibiting the ability of the enzyme to religate cleaved DNA
molecules.
A caveat to the above conclusion is the fact that reactions with
prokaryotic and eukaryotic type II topoisomerases utilized different
members of the quinolone family. While ciprofloxacin contains an
aliphatic piperazine ring at the C-7 position, CP-115,953 has a planar
aromatic hydroxyphenyl substituent at this position (Fig. 4).
Therefore, it is possible that findings with the bacterial type II
topoisomerases reflect differences in the drug congeners employed,
rather than evolutionary changes in quinolone mechanism.
To address this critical issue, the effects of CP-115,953 on the DNA
cleavage/religation reaction of S. aureus topoisomerase IV
were examined (Fig. 6). Results were
strikingly similar to those observed for ciprofloxacin. CP-115,953
enhanced DNA scission >7-fold with a CC2 of ~160
nM (left). Moreover, when included in religation
assays, CP-115,953 decreased reaction rates ~7-fold (right). These findings demonstrate that the pronounced
quinolone inhibition of DNA religation mediated by Gram-positive
topoisomerase IV reflects an inherent property of the drug-enzyme
interaction rather than a difference in drug congeners.
Conservation of the Drug Interaction Domain on Type II
Topoisomerases--
Two lines of evidence indicate that the drug
interaction domain on type II topoisomerases has been conserved from
bacterial DNA gyrase to eukaryotic topoisomerase II. First, one of the
most common point mutations in DNA gyrase that confers resistance to quinolones in clinical isolates of Gram-negative bacteria
(i.e. Ser
Genetic evidence suggests that the site of quinolone binding on
topoisomerase IV is comparable to that of DNA gyrase. Indeed, a similar
spectrum of point mutations confers drug resistance to both enzymes (4,
6, 18-20). However, given the striking difference in the mechanistic
basis for quinolone action against topoisomerase IV versus
eukaryotic type II topoisomerases, a series of experiments was carried
out to further compare the drug interaction domain on S. aureus topoisomerase IV to that of its eukaryotic counterpart.
First, the effects of etoposide on the DNA cleavage reaction of
topoisomerase IV were assessed. This drug, which is a widely prescribed
anticancer agent, is a potent enhancer of DNA scission mediated by
eukaryotic type II topoisomerases (45, 51, 60-62). As seen in Figs.
7 (left) and 8A,
etoposide also enhanced DNA cleavage mediated by S. aureus
topoisomerase IV. The drug increased levels of DNA scission ~2.5-fold
(Fig. 8A), and shared many
sites of cleavage enhancement with the antibacterial quinolone
ciprofloxacin or the antineoplastic quinolone CP-115,953 (Fig. 7).
Similar DNA cleavage patterns were also observed for CP-115,953 and
etoposide in reactions that contained human topoisomerase II
Second, drug competition experiments were carried out to further define
relationships between the quinolone and etoposide interaction domains
on S. aureus topoisomerase IV. As seen in Fig.
8B, saturating concentrations of etoposide diminished the ability of ciprofloxacin to stimulate DNA cleavage ~50%. To confirm this finding, drug competition was assessed at the site-specific level.
Experiments took advantage of the small subset of cleavage sites that
were specific for quinolones and were not induced by etoposide.
Representative sites are labeled 1, 2, and 3 in
Fig. 7. In the presence of etoposide, ciprofloxacin-induced scission at
these sites dropped in a concentration-dependent manner (Fig. 8C). These results suggest that etoposide and ciprofloxacin
share a common binding site on topoisomerase IV.
Third, the sensitivity of two common quinolone resistance mutants of
S. aureus topoisomerase IV (Ser80
Taken together, the above findings strongly suggest that the drug
interaction domain on type II topoisomerases has been conserved throughout the evolution from prokaryotic to eukaryotic species. Therefore, it appears likely that the inhibition of topoisomerase IV-mediated DNA religation by quinolones reflects a subtle change in
the enzyme·drug·DNA ternary complex rather than a gross difference in the site of quinolone binding.
Quinolones represent an important class of antibacterial agents
that act by increasing levels of DNA cleavage mediated by prokaryotic
type II topoisomerases (4, 5, 10, 19, 24, 26, 30, 31). In Gram-negative
bacteria, quinolones are targeted primarily to DNA gyrase;
topoisomerase IV serves only as a secondary target (4-9, 12, 20).
However, in Gram-positive species, topoisomerase IV appears to be the
primary cytotoxic target for most members of this drug family (4-6,
11, 14-16, 20, 33). Despite the importance of Gram-positive
topoisomerase IV as a significant new drug target, its interactions
with quinolones are not well understood. Therefore, the present study
characterized the effects of quinolones on the DNA cleavage and
religation reactions mediated by S. aureus topoisomerase IV.
Ciprofloxacin displayed high activity against the Gram-positive enzyme.
As determined by DNA cleavage assays, the drug was more potent
(>3-fold) and more efficacious (~2-fold) than it was against
E. coli topoisomerase IV (31). Furthermore, quinolones inhibited DNA religation mediated by S. aureus topoisomerase
IV to a greater extent than observed for any other type II enzyme (31,
45, 48, 51, 57). In fact, the level of inhibition was large enough to
account for essentially all of the DNA cleavage enhancement induced by
these drugs. This finding stands apart from those reported for the
actions of antineoplastic quinolones against eukaryotic topoisomerase
II (in which little inhibition was observed) (45, 48, 51, 57) or
E. coli topoisomerase IV (in which ~50% of the DNA
cleavage enhancement could be attributed to decreases in religation
rates) (31). These results suggest that the mechanistic basis for
quinolone action against type II topoisomerases is not conserved across
evolutionary boundaries. Rather, as drug potency increases across
species, it appears that inhibition of DNA religation plays a
considerably more prominent role.
The apparent change in quinolone mechanism is not caused by an overt
difference in the drug interaction domain on S. aureus topoisomerase IV. All available data suggests that the physical location of drug binding has been maintained throughout the evolution of the type II enzyme. Therefore, the following model is proposed to
explain the inhibition of topoisomerase IV-mediated DNA religation by quinolones.
The model for quinolone action is based on the premise that stimulation
of the forward DNA cleavage reaction and inhibition of the reverse DNA
religation reaction by topoisomerase-targeted drugs do not actually
represent two separate mechanisms; rather they reflect the two extremes
of a mechanistic continuum. This model is based on the "positional
poison model" (64, 65) and centers on two major points. First, it is
postulated that quinolones increase enzyme-mediated DNA scission by
distorting the double helix proximal to the points of cleavage (64,
65). This aspect of the hypothesis is supported by studies that
localized drug-DNA cross-linking within the topoisomerase·drug·DNA
ternary complex (66) and the positional specificity of
cleavage-enhancing drugs and DNA lesions (60, 61, 64). In addition,
chemical oxidation studies by Marians and Hiasa (67) have shown that
the quinolone norfloxacin deforms DNA specifically within a cleavage
site utilized by E. coli topoisomerase IV (67). Moreover,
this distortion abuts the nucleotide bond cut by the enzyme.
Second, the location of quinolone-induced DNA distortion within the
ternary complex determines whether the drug acts primarily by enhancing
rates of DNA cleavage or by inhibiting DNA religation. When type II
topoisomerases cleave DNA, they become covalently joined to the newly
generated 5'-phosphate (22, 45, 56, 68). Because of this enzyme-DNA
linkage, base pairing within the 4-base cleavage overhang is not
required to promote efficient DNA religation (69). Therefore, when
drug-induced distortion is encompassed completely within the cleavage
overhang, rates of religation are unaffected (69). In contrast to the
5' terminus, the 3'-hydroxyl generated by cleavage is not covalently
attached to the enzyme (22, 70, 71). Therefore, when DNA distortion induced by quinolones alters the orientation of the 3'-hydroxyl such
that it can no longer be correctly positioned by the enzyme, the DNA
religation reaction is inhibited. A similar inhibition could also be
envisioned if the DNA-bound quinolone physically occluded the
3'-hydroxyl or blocked appropriate enzyme-nucleotide interactions by
steric hindrance.
Using the above model as a guide, we suggest that the dramatically
different effects of quinolones on DNA religation mediated by
eukaryotic and prokaryotic type II topoisomerases actually result from
a subtle change in the alignment of quinolones within the ternary
complex. In eukaryotic systems, quinolone-induced distortion is located
entirely within the cleavage overhang; thus these drugs do not affect
DNA religation mediated by topoisomerase II. However, in bacterial
systems, the position of DNA-bound quinolone is shifted so that it also
affects the orientation of the 3'-hydroxyl (or interactions of the
enzyme with the 3'-hydroxyl) and inhibits DNA religation. Since the
most severe inhibition was observed with S. aureus
topoisomerase IV, it is further suggested that the effects of
quinolones on the position of the 3'-hydroxyl are more significant for
the Gram-positive enzyme than they are for E. coli
topoisomerase IV.
Among all the drug classes that target type II topoisomerases,
quinolones are the only family that includes members that display high
activity against enzymes from prokaryotic and eukaryotic sources (4-6, 45, 51, 52). For example, the quinolone CP-115,953 is a
potent enhancer of DNA cleavage mediated by such varied enzymes as
E. coli DNA gyrase (47), Gram-positive and -negative
topoisomerase IV (31), and topoisomerase II from thermophilic
archaebacteria (72), slime mold (73), yeast (74), Drosophila
(47), and mammals (53, 75). This broad range of affected species
affords a unique opportunity to examine drug mechanism and enzyme
interactions over widely divergent evolutionary boundaries. Results of
the present study strongly suggest that the mechanistic basis for quinolone action has not been conserved over the course of evolution. This finding undercuts the universal application of drug mechanism from
species to species and argues for further mechanistic studies with
divergent type II topoisomerases.
We are grateful to Dr. Paul R. McGuirk
(Pfizer Central Research) for the synthesis of CP-115,953 and Dr. D. Andrew Burden, Susan D. Cline, and John M. Fortune for critical reading
of the manuscript.
*
This work was supported by National Institutes of Health
Grant GM33944.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Trainee under National Institutes of Health Grant 5 T32 CA09385.
**
To whom reprint requests should be addressed at: Dept. of
Biochemistry, 654 Medical Research Bldg. I, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-322-4338; Fax:
615-343-1166; E-mail: osheron@ctrvax.vanderbilt.edu.
Quinolones Inhibit DNA Religation Mediated by
Staphylococcus aureus Topoisomerase IV
CHANGES IN DRUG MECHANISM ACROSS EVOLUTIONARY BOUNDARIES*
§,
**
Biochemistry and
Medicine (Hematology/Oncology), Vanderbilt University School of
Medicine, Nashville, Tennessee 37232-0146 and the ¶ Department of
Cancer, Immunology, and Infectious Diseases, Pfizer Central Research,
Pfizer, Inc., Groton, Connecticut 06340
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was expressed in
Saccharomyces cerevisiae (37) and purified by the protocol
of Kingma et al. (38).
20 °C,
then diluted one-fifth with 10 mM Tris-HCl, pH 7.9, immediately prior to use. Tris and ethidium bromide were obtained from
Sigma; SDS and proteinase K were from Merck; restriction endonucleases,
calf intestine alkaline phosphatase, and T4 polynucleotide kinase were
from New England BioLabs; ATP and [
-32P]ATP (6000 Ci/mmol) were from Amersham Pharmacia Biotech. All other chemicals were
analytical reagent grade.
in 10 mM Tris-HCl,
pH 7.9, 100 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 2.5% glycerol, and were incubated at 37 °C
for 15 min. In both cases, DNA cleavage complexes were trapped by the
addition of SDS and samples were digested with proteinase K in the
presence of EDTA, as above. DNA cleavage products were precipitated
twice with ethanol, dried, and resuspended in 40% formamide, 8.4 mM EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol
FF. Samples were subjected to electrophoresis in 8% sequencing gels
(42), fixed in 10% methanol, 10% acetic acid, and dried. Reaction
products were visualized using a Molecular Dynamics PhosphorImager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which cleaves <1% of the DNA
molecules at a 30:1 ratio of enzyme:plasmid (31).
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Fig. 1.
S. aureus topoisomerase IV
establishes a rapid DNA cleavage/religation equilibrium. A time
course for topoisomerase IV-mediated DNA cleavage in the absence of
quinolones is shown. Data represent the average of two independent
experiments. Standard deviations are shown as error bars.
The inset depicts an ethidium bromide-stained agarose gel
showing products of enzyme-mediated DNA cleavage. Reactions were
carried out in the absence (Topo IV) or presence of 5 µM ciprofloxacin (Cipro). Double-stranded DNA
cleavage converts negatively supercoiled plasmid (form I,
FI) to linear molecules (form III, FIII). The
position of nicked circular DNA (form II, FII) is shown for
reference.
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Fig. 2.
Ciprofloxacin stimulates DNA scission
mediated by S. aureus topoisomerase IV. Assays
contained 15 nM S. aureus topoisomerase IV ( 
)
and 0 to 10 µM ciprofloxacin. Data for reactions that
contained 7.5 nM E. coli topoisomerase IV (
)
are shown for comparison (31). The relative level of DNA cleavage in
the absence of drug was set to 1.0. Data represent the average of three
independent experiments. Standard deviations are shown as error
bars.
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Fig. 3.
Time course for ciprofloxacin-induced DNA
cleavage mediated by S. aureus topoisomerase IV.
Assays contained 5 µM ciprofloxacin. The relative level
of DNA cleavage in the absence of drug was set to 1.0. Data represent
the average of two independent experiments. Standard deviations are
shown as error bars.
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Fig. 4.
Quinolone structures. The ring numbering
system shown for ciprofloxacin is applicable to CP-115,953.
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Fig. 5.
Ciprofloxacin inhibits DNA religation
mediated by S. aureus topoisomerase IV. Assays
were carried out in the absence ( ) or presence () of 5 µM ciprofloxacin. Religation was initiated by shifting
the temperature from 37 to 65 °C. At time 0, levels of
double-stranded cleavage were set to 100%. Data represent the average
of two independent experiments. Standard deviations are shown as
error bars.
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Fig. 6.
Effects of the antineoplastic quinolone
CP-115,953 on the DNA cleavage and religation reactions of S. aureus topoisomerase IV. The left panel
depicts the effects of CP-115,953 on enzyme-mediated DNA scission. The
right panel shows the effect of CP-115,953 on DNA religation
mediated by the enzyme. Assays were incubated in the absence ( ) or
presence () of 5 µM CP-115,953. Religation was
initiated by shifting the temperature from 37 to 65 °C. At time 0, levels of double-stranded cleavage were set to 100%. Data represent
the average of two to three independent experiments. Standard
deviations are shown as error bars.
Trp at position 83 in GyrA) (5, 18, 19) also
confers resistance to antineoplastic quinolones when introduced at the corresponding residue in yeast topoisomerase II (58). Second, the
antibacterial quinolone ciprofloxacin displays a weak ability to
stimulate DNA scission mediated by eukaryotic topoisomerase II and acts
as a competitive inhibitor of cleavage-enhancing anticancer drugs
targeted to the enzyme (46, 53, 59).
(Fig.
7, right).
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Fig. 7.
Effects of drugs on site-specific DNA
cleavage mediated by S. aureus topoisomerase IV or
human topoisomerase II . Assays contained
either 6 nM topoisomerase IV (S. aureus Topo IV)
or 60 nM human topoisomerase II (Human Topo
II) and 1.4 nM labeled linear 564-mer DNA. Control
reactions contained 8 mM Tris-HCl, pH. 7.9, 0.02 N NaOH (Diluent) or dimethyl sulfoxide
(DMSO). Drug concentrations employed were 10 µM ciprofloxacin (Cipro), 10 µM
CP-115,953 (953), 250 µM etoposide
(Etop, topoisomerase IV), or 20 µM etoposide
(Etop, human topoisomerase II), as indicated.
View larger version (20K):
[in a new window]
Fig. 8.
Etoposide attenuates ciprofloxacin-induced
DNA cleavage enhancement. DNA cleavage assays with supercoiled
pBR322 plasmid (panel A) contained 15 nM
topoisomerase IV and 0 to 1000 µM etoposide. Competition
experiments with supercoiled pBR322 (panel B) utilized
saturating concentrations of ciprofloxacin (10 µM,
Cipro) or etoposide (500 µM, Etop)
or both compounds. The relative level of DNA cleavage in the absence of
drug was set to 1.0. Data represent the average of two to three
independent experiments. Standard deviations are shown as error
bars. Site-specific competition experiments (panel C)
monitored DNA cleavage in the presence of 10 µM
ciprofloxacin and 0-1,000 µM etoposide. Data are typical
of two independent experiments.
Phe and
Glu84 Lys in the GrlA subunit) to etoposide was
examined (5, 63). As seen in Table I,
both mutations displayed cross-resistance to the anticancer drug.
Relative resistance of drugs to S. aureus topoisomerase IV
mutants
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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