From the Department of Pharmacology, University of
Minnesota Medical School, Minneapolis, Minnesota 55455 and the
¶ Microbial, Musculoskeletal, and Proliferative Diseases Center of
Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals,
Collegeville, Pennsylvania 19426
Received for publication, September 9, 2002, and in revised form, December 20, 2002
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ABSTRACT |
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Type II topoisomerases bind to DNA at the
catalytic domain across the DNA gate. DNA gyrases also bind to DNA at
the non-homologous C-terminal domain of the GyrA subunit, which causes
the wrapping of DNA about itself. This unique mode of DNA binding
allows gyrases to introduce the negative supercoils into DNA molecules.
We have investigated the biochemical characteristics of
Staphylococcus aureus (S. aureus) gyrase.
S. aureus gyrase is known to require high concentrations of potassium glutamate (K-Glu) for its supercoiling activity. However, high concentrations of K-Glu are not required for
its relaxation and decatenation activities. This is due to the
requirement of high concentrations of K-Glu for S. aureus gyrase-mediated wrapping of DNA. These results suggest that
S. aureus gyrase can bind to DNA at the
catalytic domain independent of K-Glu concentration, but high
concentrations of K-Glu are required for the binding of the C-terminal
domain of GyrA to DNA and the wrapping of DNA. Thus, salt modulates the
DNA binding mode and the catalytic activity of S. aureus gyrase. Quinolone drugs can stimulate the
formation of covalent S. aureus gyrase-DNA
complexes, but high concentrations of K-Glu inhibit the formation of
S. aureus gyrase-quinolone-DNA ternary
complexes. In the absence of K-Glu, ternary complexes formed with
S. aureus gyrase cannot arrest replication fork
progression in vitro, demonstrating that the formation of a
wrapped ternary complex is required for replication fork arrest by a
S. aureus gyrase-quinolone-DNA ternary complex.
Because of the helical structure of DNA, DNA unlinking is an
essential issue in many aspects of DNA metabolism. DNA topoisomerases are the enzymes responsible for unlinking the parental strands during
DNA replication (1, 2). Topoisomerase function is essential for removal
of the topological constraint to maintain replication fork progression.
Biochemical studies have revealed two distinct modes of DNA unlinking
during DNA replication (3). DNA gyrase removes the positive supercoils
in front of the advancing replication forks, whereas topoisomerase IV
(Topo IV)1 decatenates the
precatenanes behind the replication forks. Recent studies have
demonstrated that both gyrase and Topo IV can support replication fork
progression during chromosome replication in Escherichia coli
(E. coli) (4). Thus, the combined efforts of gyrase and
Topo IV ensure the completion of DNA unlinking during DNA replication
and chromosome segregation.
Both DNA gyrase and Topo IV are the cellular targets of the quinolone
antibacterial drugs (5-7). It has been shown that quinolone drugs
block DNA replication not by depriving the cell of gyrase but by
converting gyrase into a poison of DNA replication (8). The poisoning
of topoisomerases is mediated by trapping of a covalent topoisomerase-DNA complex as a topoisomerase-drug-DNA ternary complex,
which leads to the inhibition of DNA replication and the generation of
double-strand breaks (9-11). The cytotoxicity of quinolone drugs
correlates with the inhibition of DNA replication (7, 12). Some
anticancer drugs that target human topoisomerases also convert their
targets into poisons in a similar manner (11, 13).
Interestingly, DNA gyrase is shown to be the primary target of
quinolone drugs in E. coli (8, 9, 14, 15),
whereas Topo IV becomes the primary target in other bacteria, such as Staphylococcus aureus (S. aureus) (16-18) and
Streptococcus pneumoniae (S. pneumoniae) (19,
20). These observations have suggested that DNA gyrase and Topo IV are
the primary targets in Gram-negative and Gram-positive bacteria,
respectively. However, more recent studies run counter to this view. It
has been demonstrated that each quinolone drug has a preferred target
and the target selection can be altered by changes in quinolone
structure (21, 22). Thus, it is not clear what determines the primary
target in cells. One hypothesis, proposed based on the action of
quinolone drugs in E. coli, is that the locations
of gyrase and Topo IV, relative to advancing replication forks, may
affect the effectiveness of cell killing (3, 7, 23). In E. coli, gyrase is thought to act in front of replication
forks, whereas Topo IV acts behind forks. Thus, ternary complexes
formed with gyrase more frequently collide with replication forks than
those formed with Topo IV.
Despite of the clinical importance of Gram-positive bacteria, the
majority of biochemical studies on topoisomerases and quinolone antibacterial drugs have been carried out with E. coli topoisomerases (1, 2, 9-11). It is not clear if other
topoisomerases act in the same manner as E. coli
topoisomerases and if the findings made in the E. coli system can be generalized. Some studies have suggested
that quinolone drugs affect S. aureus
gyrase in a distinct manner and S. aureus
gyrase-quinolone-DNA ternary complexes contain no broken DNA strand
(24).
Here, we investigated the biochemical characteristics of S. aureus gyrase and compare them with those of E. coli gyrase. We found that catalytic activities of
S. aureus gyrase were, in general, more resistant
to salt than those of E. coli gyrase.
S. aureus gyrase required high concentrations of
potassium glutamate (K-Glu) for its supercoiling activity but not for
its relaxation and decatenation activities. We also found that high
concentrations of K-Glu were required for S. aureus gyrase-mediated wrapping of DNA. Thus, the unique
requirement of high concentrations of K-Glu for S. aureus-catalyzed supercoiling reaction was due to the
requirement of high concentrations of K-Glu for S. aureus gyrase-mediated wrapping of DNA. These results
demonstrated that salt could modulate the DNA binding mode and thus the
catalytic activity of S. aureus gyrase.
Quinolone drugs stimulated the formation of covalent gyrase-DNA formed
with either E. coli or S. aureus gyrase. Both E. coli gyrase-quinolone-DNA
and S. aureus gyrase-quinolone-DNA ternary complexes were
sensitive to high concentrations of K-Glu. Interestingly, in the
absence of K-Glu, ternary complexes formed with S. aureus gyrase failed to arrest replication fork progression in
vitro. In contrast, ternary complexes formed with S. aureus Topo IV could arrest replication fork progression. These
results suggested that collisions between replication forks and
S. aureus gyrase-quinolone-DNA ternary complexes would not
cause the inhibition of DNA replication and trigger the cytotoxic
events. This may explain why Topo IV is the primary target of quinolone
drugs in S. aureus.
DNAs and Proteins--
An oriC plasmid, pBROTB535
type I, was prepared according to Hiasa and Marians (25). pBR322 form I
(negatively supercoiled) DNA and kinetoplast DNA (kDNA) were purchased
from New England BioLabs and Topogen, respectively.
E. coli and S. aureus gyrA and
gyrB genes were generated by PCR using E. coli
C600 and S. aureus WCUH29 genomic DNA as a template, respectively, and cloned into pET vectors (Novagen). Overproduction and
purification of E. coli GyrA and GyrB proteins were done as described previously (25-27). S. aureus GyrA and
GyrB were overexpressed in E. coli Rosetta(DE3)
(Novagen) and purified according to unpublished protocols,2 similar to those
described previously (24, 28). Hydroxyapatite (BioRad) and
phenyl-Sepharose (Amersham Biosciences) columns, and heparin-Sepharose
(Amersham Biosciences) and phenyl-Sepharose columns were used for the
purification of S. aureus GyrA and GyrB, respectively. The final preparations of S. aureus
GyrA and GyrB were greater than 90% homogeneous for a single band on
SDS-PAGE (data not shown). Purified E. coli GyrA and GyrB
and S. aureus GyrA and GyrB were mixed at a molar
ratio of 1:1 (addition of a 5-fold excess of either subunit did not
change the specific activity) to reconstitute E. coli and S. aureus gyrase,
respectively. Mixtures of either E. coli GyrA and S. aureus GyrB or S. aureus GyrA and E. coli
GyrB did not yield an active gyrase (data not shown). This indicated
that the preparations of S. aureus GyrA and GyrB
were not contaminated significantly with E. coli
GyrA and GyrB, respectively.
S. aureus grlA (parC) and
grlB (parE) genes were generated by PCR using
S. aureus RN4220 genomic DNA as a template and cloned into
pET vectors (Novagen). S. aureus GrlA (ParC) and GrlB (ParE) proteins were overexpressed in E. coli BL21(DE3)
(Novagen) and purified according to protocols used for purification of
E. coli ParC and ParE proteins, respectively (6,
29).3 The final preparations
of S. aureus GrlA and GrlB were greater than 90%
homogeneous for a single band on SDS-PAGE (data not shown). Purified
S. aureus GrlA and GrlB were mixed, at a molar ratio of 1:1
to reconstitute S. aureus Topo IV. Mixtures of
either E. coli ParC and S. aureus GrlB or
S. aureus GrlA and E. coli ParE did not
yield an active gyrase (data not shown), indicating that the
preparations of S. aureus GrlA and GrlB were not
contaminated significantly with E. coli ParC and
ParE, respectively.
E. coli replication proteins, generous gifts of Kenneth
Marians (Memorial Sloan-Kettering Cancer Center), were as described previously (30-32). Calf thymus Topo I was obtained from Invitrogen.
Supercoiling Reaction--
pBR322 form I' (relaxed) DNA was
prepare by incubating pBR322 form I DNA with E. coli Topo I and used as a substrate in the supercoiling
reaction. Standard reaction mixtures (20 µl) contained 40 mM HEPES-KOH (pH 7.6), 10 mM magnesium acetate
(MgOAc2), 10 mM dithiothreitol (DTT), 50 µg/ml bovine serum albumin (BSA), 2 mM ATP, 0.29 µg
(100 fmol as molecule) pBR322 form I' DNA, the indicated concentrations
of K-Glu, and the indicated amounts (as tetramer) of either
E. coli or S. aureus
gyrase. Reaction mixtures were incubated at 37 °C for 15 min and
terminated by adding EDTA to 25 mM and incubating at
37 °C for 5 min. The DNA products were analyzed by electrophoresis
through vertical 1.2% Seakem-agarose (BMA) gels (14 × 10 × 0.3 cm) at 2 V/cm for 12 h in a running buffer of 50 mM Tris-HCl (pH 7.9 at 23 °C), 40 mM sodium
acetate, and 1 mM EDTA (TAE buffer). Gels were stained with
ethidium bromide and photographed using an Eagle Eye II system (Stratagene).
ATP-independent Relaxation of Negatively Supercoiled Plasmid
DNA--
Reaction mixtures (20 µl) containing 40 mM
HEPES-KOH (pH7.6), 10 mM MgOAc2, 10 mM DTT, 50 µg/ml BSA, 0.29 µg (100 fmol as molecule)
pBR322 form I DNA, the indicated concentrations of K-Glu, and the
indicated amounts (as tetramer) of either E. coli
or S. aureus gyrase were incubated at
37 °C for 30 min. Reactions were terminated by adding EDTA to 25 mM and incubating at 37 °C for 5 min. The DNA products
were analyzed and photographed as described in the previous section.
Decatenation of kDNA--
Reaction mixtures (20 µl) contained
40 mM HEPES-KOH (pH7.6), 10 mM
MgOAc2, 10 mM DTT, 50 µg/ml BSA, 2 mM ATP, 0.5 µg of kDNA, the indicated concentrations of
K-Glu, and the indicated amounts of either E. coli or S. aureus gyrase (as
tetramer). Reaction mixtures were incubated at 37 °C for 30 min and
terminated by adding EDTA to 25 mM and incubating at
37 °C for 5 min. The DNA products were analyzed and photographed as
described in the previous section.
DNA Cleavage Reaction--
pBR322 form I DNA was linearized by
digestion with EcoRI endonuclease and then 3'-end-labeled by
incorporation of two residues of [32P]dAMP with Klenow
enzyme. This DNA fragment was used as the substrate in the DNA cleavage reaction.
Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH
7.5), 10 mM MgCl2, 10 mM DTT, 50 µg/ml BSA, 1 mM ATP, 20 fmol (as molecule) DNA substrate,
200 fmol (as tetramer) of either E. coli or S. aureus gyrase, and 50 µM norfloxacin were incubated
at 37 °C for 10 min. SDS was added to 1%, and the reaction mixtures were further incubated at 37 °C for 5 min. EDTA and proteinase K
were then added to 25 mM and 100 µg/ml, respectively, and
the incubation was continued for an additional 15 min. The DNA products were purified by extraction of the reaction mixtures with
phenol-chloroform (1:1, v/v) and then analyzed by electrophoresis
through 1.2% Seakem-agarose (BMA) gels (14 × 10 × 0.3 cm)
at 5 V/cm for 2.5 h in TAE buffer. Gels were dried under vacuum
onto #3 filter papers (Whatman) and autoradiographed with Hyperfilm MP
films (Amersham Biosciences).
Gyrase-induced Constraint of Supercoils in DNA--
Reaction
mixtures (20 µl) containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 100 µg/ml BSA, 10% glycerol, 100 fmol (as molecule) of pBR322 form I' DNA, either 100 or 400 mM K-Glu, and the indicated amounts (as tetramer) of either
E. coli or S. aureus gyrase
were incubated at 37 °C for 5 min. Calf thymus Topo I, either 2 units (for reaction mixtures containing 100 mM K-Glu) or 20 units (for reaction mixtures containing 400 mM K-Glu), was
added to the reaction mixtures, and the incubation was continued at
37 °C for 30 min. SDS was added to 1% to terminate the reaction,
and the reaction mixtures were further incubated at 37 °C for 5 min.
EDTA and proteinase K were then added to 25 mM and 100 µg/ml, respectively, and the incubation was continued for an
additional 15 min. The DNA products were purified by extraction of the
reaction mixtures with phenol-chloroform (1:1, v/v) and then analyzed
by electrophoresis through 1.2% Seakem-agarose (BMA) gels (14 × 10 × 0.3 cm) at 2 V/cm for 16 h in TAE buffer. Gels were
stained with ethidium bromide and photographed using an Eagle Eye II
system (Stratagene).
Staged Nascent Chain Elongation during oriC DNA
Replication--
The modified pulse-chase protocol was performed,
using pBROTB535 type I DNA as the DNA template, as described previously
(33).
High Concentrations of K-Glu Are Required for S. aureus
Gyrase-catalyzed Supercoiling Activity--
Previous studies have
shown that high concentrations of K-Glu stimulate S. aureus
gyrase-catalyzed supercoiling activity (24, 28). During the initial
characterization of S. aureus gyrase, we noticed that the
apparent specific activity of this enzyme changed drastically,
depending on the concentrations of K-Glu. To determine the effect of
K-Glu on the catalytic activity of S. aureus gyrase, the
supercoiling assay was performed in the presence of various
concentrations of K-Glu. Because the apparent specific activities of
E. coli and S. aureus
gyrases were different, we performed the assay in the presence of both
a subsaturated amount and an excess amount of gyrases (Fig.
1). E. coli gyrase could catalyze the supercoiling reaction in a wide range of K-Glu concentration, although its activity was optimal in the presence of
100-200 mM K-Glu (Fig. 1A, lanes 3 and 4). An inhibitory
effect of salt on E. coli gyrase-catalyzed
supercoiling reaction was observed when 800 mM K-Glu was
present (Fig. 1A, lanes 6 and 11). In
contrast, S. aureus gyrase was able to catalyze
supercoiling reaction only in the presence of high concentrations
(400-800 mM) of K-Glu (Fig. 1B, lanes
5, 6, 10, and 11). Thus,
E. coli and S. aureus
gyrases exhibited distinct salt sensitivities for their
supercoiling activities.
Quinolone Drugs Can Stimulate the Covalent S. aureus Gyrase-DNA
Complex Formation--
It was possible that high concentrations of
K-Glu were required for S. aureus gyrase to bind
to DNA and catalyze the supercoiling reaction. To examine the effect of
K-Glu on the binding of S. aureus gyrase to DNA,
quinolone-stimulated, gyrase-catalyzed cleavage of DNA was measured in
the presence of various concentrations of K-Glu (Fig.
2). Either E. coli
or S. aureus gyrase-catalyzed cleavage was
observed in the absence of K-Glu (Fig. 2, lanes 2 and
7). Both E. coli
gyrase-norfloxacin-DNA and S. aureus
gyrase-norfloxacin-DNA ternary complexes were sensitive to K-Glu,
although ternary complexes formed with S. aureus
gyrase were more resistant to salt than those formed with E. coli gyrase. These results demonstrated that S. aureus gyrase could bind to DNA in the presence of low
concentrations (0-200 mM) of K-Glu.
S. aureus Gyrase Can Catalyze Relaxation and Decatenation Reactions
in the Presence of Low Concentrations of K-Glu--
S. aureusgyrase was able to bind to DNA when K-Glu concentrations were 0-200
mM (Fig. 2) but could not catalyze the supercoiling reaction under these conditions (Fig. 1B). It was possible
that the DNA binding and the catalytic activity of S. aureus gyrase required distinct K-Glu concentrations. In
this case, S. aureus gyrase could bind to DNA at
low K-Glu concentrations but required high concentrations of K-Glu for
its supercoiling, relaxation, and decatenation activities.
Alternatively, high K-Glu concentrations might be a unique requirement
for S. aureus gyrase-catalyzed supercoiling reaction but not for relaxation and decatenation activities. In this
case, S. aureus gyrase could bind to DNA and
catalyze relaxation and decatenation reactions in the presence of low
concentrations of K-Glu. This might imply that high concentrations of
K-Glu were required only for S. aureus
gyrase-mediated wrapping of DNA and S. aureus
gyrase-catalyzed supercoiling activity. To distinguish between these
two possibilities, we assessed the effects of K-Glu on S. aureus gyrase-catalyzed relaxation and decatenation reactions.
Relaxation (Fig. 3) and decatenation
(Fig. 4) activities of S. aureus gyrase were measured in
the presence of various concentrations of K-Glu. E. coli gyrase catalyzed relaxation and decatenation reactions
the most efficiently when K-Glu was absent and the addition of K-Glu in
the reaction mixtures resulted in inhibitions of these catalytic
activities (Figs. 3 and 4). On the other hand, the optimal K-Glu
concentrations for S. aureus gyrase-catalyzed
relaxation and decatenation reactions were 200-400 mM and
100-200 mM, respectively (Figs. 3 and 4). Higher
concentrations of K-Glu were inhibitory to these activities. These
results showed that, in the presence of low concentrations (0-200
mM) of K-Glu, S. aureus gyrase could bind to DNA and catalyze relaxation and decatenation reactions. Thus,
requirement of high concentrations of K-Glu was unique to S. aureus gyrase-catalyzed supercoiling activity, indicating
that high concentrations of K-Glu might be required for S. aureus gyrase-mediated wrapping of DNA.
S. aureus Gyrase-mediated Wrapping of DNA Requires High
Concentrations of K-Glu--
DNA gyrase binds to DNA at the catalytic
domain across the DNA gate as well as the C-terminal domain of the GyrA
subunit. It is the binding of C-terminal domain of GyrA to DNA that
causes the wrapping of DNA about itself and enables gyrase to catalyze supercoiling reaction (1). Results described in the previous sections
suggested that S. aureus gyrase was able to bind
to DNA at the catalytic domain at a wide range of K-Glu concentration. However, the binding of the C-terminal domain of GyrA to DNA and wrapping of DNA could take place only in the presence of high concentrations of K-Glu. Thus, S. aureus
gyrase-catalyzed supercoiling reaction required high concentrations of
K-Glu.
To directly test this possibility, we measure the constraint of
supercoils in DNA induced by gyrase-mediated wrapping of DNA in the
presence of various concentrations of K-Glu. When positive supercoils
are generated as a result of gyrase-mediated wrapping of DNA, negative
supercoils must be generated in other regions of the DNA molecule to
maintain the overall linking number (1). Addition of Topo I would
result in the relaxation of negative supercoils, and the subsequent
removal of proteins from the DNA would leave positive supercoils in the
DNA. Thus, by measuring the constraint of supercoils introduced in DNA,
we can determine the extent of gyrase-mediated wrapping of DNA. Form I'
DNA was bound by either E. coli or S. aureus gyrase in the absence of ATP and the presence of
either 100 or 400 mM K-Glu (Fig.
5). E. coli gyrase
could wrap DNA about itself in the presence of either 100 or 400 mM K-Glu (Fig. 5, lanes 3 and 4),
which correlated with its ability to catalyze supercoiling reaction at
various K-Glu concentrations (Fig. 1A). In contrast,
S. aureus gyrase-mediated wrapping of DNA was
detected only when 400 mM K-Glu was present (Fig. 5,
lane 6). These results demonstrated that S. aureus gyrase-mediated wrapping required high concentrations
of K-Glu. Thus, it was likely that S. aureus
gyrase required high concentrations of K-Glu for its supercoiling
activity, because its interaction with DNA at the C-terminal domain of
GyrA and wrapping of DNA could occur only in the presence of high
concentrations of K-Glu.
S. aureus Gyrase-Quinolone-DNA Ternary Complexes Can Not Arrest
Replication Fork Progression in Vitro--
Our previous studies have
demonstrated that E. coli gyrase-mediated wrapping of DNA is required
for the formation of gyrase-quinolone-DNA ternary complexes that can
arrest replication fork progression in vitro (27). Thus, the
formation of a wrapped ternary complex is required for replication fork
arrest by a ternary complex formed with E. coli gyrase. We
examined if this was also the case with S. aureus gyrase. If
S. aureus gyrase-mediated wrapping were required for
replication fork arrest by a ternary complex, S. aureus
gyrase could not wrap DNA and thus would fail to halt replication fork progression at low K-Glu concentrations. In contrast, wrapped ternary
complexes would be formed at high K-Glu concentrations and replication
fork progression would be arrested. Unfortunately, high concentrations
of K-Glu inhibited oriC replication reaction (roughly 50 and
90% inhibition by 200 and 400 mM K-Glu, respectively (25))
and reversed formation of gyrase-quinolone-DNA ternary complexes (Fig.
2) (8). Thus, the ability of S. aureus gyrase to arrest
replication fork progression could not be assessed at high K-Glu concentrations.
The modified oriC pulse-chase protocol (33) was employed to
assess the ability of the ternary complex formed with either E. coli or S. aureus gyrase to arrest
replication fork progression in vitro (Fig.
6A). Early replicative
intermediates (ERI) were formed and labeled. The paused replication
forks in the ERI were released by linearizing the DNA template with
the SmaI restriction endonuclease, which digested the DNA
template once at oriC. Linearization of the DNA template was
sufficient to release the paused replication forks and generate the
full-length product as a result of the run-off DNA replication (Fig.
6A, lane 1). Because no topoisomerase was
required to relieve topological constraint, this reaction was
insensitive to the presence of norfloxacin (Fig. 6A,
lane 2).
Either E. coli or S. aureus gyrase was added,
together with norfloxacin, to the reaction mixtures to form ternary
complexes prior to the linearization of the template DNA. The
subsequent addition of the SmaI restriction endonuclease
released paused replication forks, which collided with ternary
complexes. The ternary complexes that arrest replication fork
progression would, therefore, manifest themselves in this assay by
preventing the appearance of the full-length DNA product (33). In the
absence of norfloxacin, neither the presence of E. coli
gyrase nor S. aureus gyrase affected elongation of the
nascent chains in the ERI to full-length product (Fig. 6A,
lanes 3 and 5). When norfloxacin was present,
replication fork progression was blocked in the presence of E. coli gyrase (Fig. 6A, lane 4) but not in the
presence of S. aureus gyrase (Fig. 6A, lane
6). These results demonstrated that, under the conditions where
S. aureus gyrase did not wrap DNA (Fig. 5), S. aureus gyrase-quinolone-DNA ternary complexes could not arrest
replication fork progression. Thus, gyrase-mediated wrapping of DNA was
required for the formation of gyrase-quinolone-DNA ternary complexes
that could block replication fork progression in vitro.
Here, we assessed the effect of S. aureus
gyrase-quinolone-DNA ternary complexes on replication fork progression
in E. coli replication system. It was possible
that the topoisomerase in a ternary complex could interact with the
component(s) of the replication fork, such as the replicative helicase,
and this interaction would influence the fate of a replication fork
upon its collision with a ternary complex. Thus, the inability of
S. aureus gyrase-quinolone-DNA ternary complexes
to arrest E. coli replication fork progression could be due
to the fact that S. aureus gyrase was used in the E. coli replication system. We examined the effect of S. aureus Topo IV-quinolone-DNA ternary complexes on replication fork
progression under the same conditions (Fig. 6B). S. aureus Topo IV exhibited similar drug and salt sensitivities to
E. coli Topo IV (Ref. 24 and data not shown). In the absence
of norfloxacin, S. aureus Topo IV did not affect elongation
of the nascent chains in the ERI to full-length product (Fig.
6B, lane 5). When norfloxacin was present,
replication fork progression was blocked in the presence of either
E. coli or S. aureus Topo IV (Fig. 6B,
lanes 4 and 6). These results showed that a
ternary complex formed with S. aureus Topo IV could arrest
the progression of E. coli replication forks. Thus, it was
likely that the inability of S. aureus gyrase to arrest
replication fork progression in the absence of K-Glu was due to the
lack of the formation of a wrapped ternary complex.
Among the type II topoisomerases, DNA gyrase is the only enzyme
that can introduce negative supercoils into DNA molecules (1, 2). The
binding of conventional type II topoisomerases to DNA takes place at
the catalytic domain across the DNA gate, whereas gyrases bind to DNA
not only at the catalytic domain but also at the non-homologous
C-terminal domain of the GyrA subunit. The topoisomerase-DNA
interaction at the catalytic domain is sufficient for type II
topoisomerases to catalyze relaxation and decatenation reactions. On
the other hand, the binding of the C-terminal domain of GyrA to DNA is
required for wrapping of DNA about itself and catalyzing the
supercoiling reaction (35).
High concentrations of K-Glu were required for S. aureus gyrase-catalyzed supercoiling activity (Fig. 1). In
contrast, S. aureus gyrase was able to
catalyze relaxation and decatenation reactions in the presence of low
concentrations of K-Glu and higher concentrations of K-Glu were
inhibitory to these reactions (Figs. 3 and 4). S. aureus gyrase could wrap DNA only when high concentrations of K-Glu were present (Fig. 5). These results demonstrated that S. aureus gyrase could bind to DNA at the
catalytic domain cross the DNA gate in the presence of either low or
high concentrations of K-Glu. However, high concentrations of K-Glu
were required for the DNA binding of the C-terminal domain of
S. aureus GyrA. Thus, salt could modulate the DNA
binding mode and the catalytic activity of S. aureus gyrase. E. coli gyrase did
not show any requirement of high concentrations of salt for its
wrapping of DNA and supercoiling activity (Figs. 1 and 5). It is not
clear why S. aureus gyrase, but not E. coli gyrase, required high concentrations of K-Glu for
wrapping of DNA. We noticed that the C-terminal domain of S. aureus GyrA was more negatively charged than that of
E. coli GyrA (Table
I). Thus, it is interesting to speculate
that higher ionic environment is required for the DNA binding of the C-terminal domain of S. aureus GyrA than that of
the C-terminal domain of E. coli GyrA.
Alternatively, K-Glu may affect conformations of E. coli and S. aureus gyrases in distinct
manners.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
Materials and Methods
RESULTS
DISCUSSION
REFERENCES
Materials and Methods
TOP
ABSTRACT
INTRODUCTION
Materials and Methods
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
Materials and Methods
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
S. aureus gyrase-catalyzed
supercoiling reaction requires high concentrations of K-Glu. The
strand supercoiling reaction mixtures containing 100 fmol (as molecule)
of pBR322 form I' DNA, the indicated amounts (as tetramer) of either
E. coli (A) or S. aureus (B) gyrase, and the indicated
concentrations of K-Glu were incubated, and the DNA products were
analyzed as described under "Materials and Methods."
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Fig. 2.
Quinolone drugs can stimulate formation of
covalent S. aureus gyrase-DNA
complexes. The strand DNA cleavage reaction mixtures containing 20 fmol (as molecule) of 32P-labeled linear pBR322 DNA, 50 µM norfloxacin, 200 fmol (as tetramer) of either
E. coli (lanes 2-6) or S. aureus (lanes 7-11) gyrase, and the indicated
concentrations of K-Glu were incubated, and the DNA products were
processed analyzed as described under "Materials and
Methods."
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Fig. 3.
High concentrations of K-Glu are not required
for S. aureus gyrase-catalyzed
relaxation reaction. The strand relaxation reaction mixtures
containing 100 fmol (as molecule) of pBR322 form I DNA, either 0.5 pmol
(as tetramer) of E. coli (lanes 2-6)
or 3 pmol (as tetramer) of S. aureus (lanes
7-11) gyrase, and the indicated concentrations of K-Glu were
incubated, and the DNA products were analyzed as described under
"Materials and Methods."
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Fig. 4.
S. aureus gyrase can decatenate
kDNA in the presence of low concentrations of K-Glu. The strand
decatenation reaction mixtures containing 0.5 µg of kDNA, either 0.5 pmol (as tetramer) of E. coli (lanes
2-6) or 3 pmol (as tetramer) of S. aureus
(lanes 7-11) gyrase, and the indicated concentrations of
K-Glu were incubated, and the DNA products were analyzed as described
under "Materials and Methods."
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Fig. 5.
S. aureus
gyrase-mediated wrapping of DNA requires high concentrations of
K-Glu. The gyrase binding reaction mixtures containing 100 fmol
(as molecule) of pBR322 form I' DNA, either 1.5 pmol (as tetramer) of
E. coli (lanes 3 and 4) or
3 pmol (as tetramer) of S. aureus (lanes
5 and 6) gyrase, and either 100 or 400 mM
K-Glu were incubated during the first stage, either 2 units (for
reaction mixtures containing 100 mM K-Glu) or 20 units (for
reaction mixtures containing 400 mM K-Glu) of calf thymus
Topo I was added to the reaction mixtures, and then the second stage of
incubation was continued. Gyrase diluent (lanes 1 and
2) and 100 µg/ml ethidium bromide (EtBr)
(lanes 7 and 8) were used as controls. The DNA
products were processed and analyzed as described under "Materials
and Methods."
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Fig. 6.
S. aureus gyrase-quinolone-DNA
ternary complexes cannot arrest replication fork progression in
vitro. The ability of S. aureus
gyrase-norfloxacin-DNA (A) and S. aureus Topo
IV-norfloxacin-DNA (B) ternary complexes to arrest the
replication fork progression was assessed as described under
"Materials and Methods." The modified pulse-chase analysis was
performed, and the DNA products were analyzed according to Hiasa
et al. (33). Either the E. coli or S. aureus topoisomerase at a molar ratio of 4:1 to the DNA template
and 100 µM norfloxacin were added to the reactions as
indicated. Ec, E. coli; Sa, S. aureus.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
Materials and Methods
RESULTS
DISCUSSION
REFERENCES
Amino acid compositions and overall charges of the C-terminal domains
of GyrA subunits
S. aureus Topo I-catalyzed relaxation activity required high concentrations of K-Glu,4 whereas E. coli Topo I-catalyzed relaxation activity was inhibited by high concentrations of K-Glu (data not shown). Thus, the supercoiling activity of DNA gyrase and the relaxation activity of Topo I, which determine the superhelicity of DNA in bacterial cells (1, 2), exhibit similar salt sensitivities. These observations indicate that gyrase-catalyzed supercoiling activity and Topo I-catalyzed relaxation activity could keep their balance at various salt concentrations to maintain a certain superhelicity of the genome in S. aureus. S. aureus is known to contain high concentrations of dicarboxylic amino acids (36), and thus S. aureus topoisomerases may have evolved to accommodate this particular environment.
Quinolone antibacterial drugs target both DNA gyrase and Topo IV (5-7). These drugs trap covalent topoisomerase-DNA complexes by forming topoisomerase-quinolone-DNA ternary complexes and collisions between replication forks and ternary complexes result in the inhibition of DNA replication (9-11). Although quinolone drugs seem to affect both gyrase and Topo IV in the same manner (23) and these two topoisomerases are highly homologous with each other (1, 2), quinolone drugs select one of these topoisomerases as the primary target in cells. Interestingly, DNA gyrase is the primary target in some bacteria (8, 9, 14, 15) and Topo IV becomes primary target in others (16-20). In addition, each quinolone drug has a preferred target (21, 22). Fisher and his coworkers (21) have conducted extensive studies on S. pneumoniae topoisomerases. Sparfloxacin and ciprofloxacin select Topo IV and gyrase as the primary target in vivo, respectively. In contrast, both of these quinolone drugs form the covalent topoisomerase-DNA complex with S. pneumoniae Topo IV much more efficiently than with S. pneumoniae gyrase in vitro (37). Fournier et al. (22) have demonstrated that norfloxacin and nalidixic acid target Topo IV and gyrase, respectively, in S. aureus. Thus, it is not clear how the primary target is selected between two type II topoisomerases in bacterial cells. Furthermore, it has been shown that only a subset of quinolone-induced covalent topoisomerase-DNA complexes are physiologically relevant for the drug action in S. aureus (22). It is likely that the frequency of the formation of topoisomerase-quinolone-DNA ternary complexes on the genome, the cytotoxicity of each ternary complex, and the efficiency of the repair of ternary complexes affect the target selection in vivo (38).
Blanche et al. (24) have reported that quinolone drugs affect S. aureus gyrase in a distinct manner. It is proposed that quinolone drugs interfere with S. aureus gyrase prior to its strand scission and S. aureus gyrase-quinolone-DNA ternary complexes do not contain any broken DNA strands. This conclusion is based on their observations that quinolone drugs do not stimulate S. aureus gyrase-catalyzed cleavage, although both E. coli gyrase- and S. aureus Topo IV-catalyzed cleavages are stimulated by quinolone drugs. The apparent unique effect of quinolone drugs on S. aureus gyrase is likely to be due to the salt concentrations used in the assays. Blanche et al. (24) have performed the cleavage assay for S. aureus gyrase in the presence of high concentrations of K-Glu and that for either E. coli gyrase or S. aureus Topo IV in the presence of low concentrations of salt. As described here, quinolone drugs stimulated the formation of either S. aureus gyrase-DNA or E. coli gyrase-DNA covalent complexes at low K-Glu concentrations (Fig. 2). However, both S. aureus gyrase-quinolone-DNA and E. coli gyrase-quinolone-DNA ternary complexes were sensitive to salt and no cleavage of DNA was observed when high concentrations of K-Glu were present. Saiki et al. (39) have reported similar results. Thus, quinolone drugs can affect S. aureus gyrase and other bacterial type II topoisomerases in the same manner and trap S. aureus gyrase as a covalent enzyme-DNA complex.
Differences between E. coli and S. aureus gyrases described here do not explain why Topo IV becomes the primary target in S. aureus. It is reasonable to assume that drug sensitivities of gyrase and Topo IV play a critical role in determining the primary target in vivo. Apparent quinolone resistance of S. aureus gyrase observed in the supercoiling assay (24) may contribute to the target selection. However, the cytotoxicity of quinolone drugs is mediated by poisoning of topoisomerases not by depriving the cell of gyrase (8). Thus, the potency of quinolone drugs should correlate better with quinolone-stimulated, topoisomerase-catalyzed cleavage of DNA than with the inhibitory effect of quinolones on the catalytic activity of topoisomerases. The difference between the stimulatory effect of quinolones on E. coli gyrase-catalyzed cleavage and that on S. aureus gyrase-catalyzed cleavage (Fig. 2) was much less than the difference between the inhibition of E. coli gyrase-catalyzed supercoiling reaction by quinolones and that of S. aureus gyrase-catalyzed reaction (24). It is likely that other factors may be involved in the selection of the primary target by quinolone drugs in vivo. One possible factor is the locations of topoisomerase complexes relative to advancing replication forks. In E. coli, the locations of gyrase and Topo IV relative to replication forks are critical for the efficiency of gyrase- and Topo IV-mediated cell killings (23). Further studies are necessary to determine functional activities of S. aureus topoisomerases during DNA replication and mechanisms of cell killing by quinolone drugs.
We found distinct effects of S. aureus gyrase-quinolone-DNA
and S. aureus Topo IV-quinolone-DNA ternary complexes on
replication fork progression. In the absence of K-Glu, S. aureus Topo IV-quinolone-DNA, but not S. aureus
gyrase-quinolone-DNA, ternary complexes could arrest the progression of
replication forks in vitro (Fig. 6). It is likely that, as
it is the case with E. coli gyrase (20), S. aureus gyrase-mediated wrapping of DNA is required for
the formation of a wrapped ternary complex that can arrest replication fork progression. At low K-Glu concentrations, S. aureus gyrase does not wrap DNA to form a wrapped ternary
complex that can arrest replication fork progression. High
concentrations of K-Glu allow S. aureus gyrase to
wrap DNA but also reverse the ternary complex formation. Thus, ternary
complexes formed with Topo IV could be more cytotoxic than those formed
with gyrase in S. aureus.
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ACKNOWLEDGEMENT |
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We thank Dr. Kenneth Marians for his comments on these studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM59465.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.
§ To whom correspondence should be addressed: Dept. of Pharmacology, Medical School, University of Minnesota, 6-120 Jackson Hall, 321 Church St., SE, Minneapolis, MN 55455. Tel.: 612-626-3101; Fax: 612-625-8408; E-mail: hiasa001@tc.umn.edu.
Current address: RiboTargets Ltd., Granta Park, Cambridge CB1
6GB, UK.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M209207200
2 M. Gwynn, unpublished results.
3 M. Shea and H. Hiasa, unpublished results.
4 I. Wildin, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: Topo, topoisomerase; BSA, bovine serum albumin; DTT, dithiothreitol; ERI, early replicative intermediates; form I, covalently closed, negatively supercoiled DNA circular; form I', covalently closed, relaxed DNA circular; kDNA, kinetoplast DNA; K-Glu, potassium glutamate; MgOAc2, magnesium acetate.
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