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J Biol Chem, Vol. 275, Issue 19, 14649-14658, May 12, 2000
From the Department of Pharmacology, University of Minnesota
Medical School, Minneapolis, Minnesota 55455
Quinolone antibacterial drugs target both DNA
gyrase (Gyr) and topoisomerase IV (Topo IV) and form
topoisomerase-quinolone-DNA ternary complexes. The formation of ternary
complexes results in the inhibition of DNA replication and leads to the
generation of double-strand breaks and subsequent cell death. Here, we
have studied the consequences of collisions between the UvrD helicase and the ternary complexes formed with either Gyr, Topo IV, or a mutant
Gyr, Gyr (A59), which does not wrap the DNA strand around itself. We
show (i) that Gyr-norfloxacin (Norf)-DNA and Topo IV-Norf-DNA, but not
Gyr (A59)-Norf-DNA, ternary complexes inhibit the UvrD-catalyzed strand-displacement activity, (ii) that a single-strand break is
generated at small portions of the ternary complexes upon their collisions with UvrD, and (iii) that the majority of Topo IV-Norf-DNA ternary complexes become nonreversible when UvrD collides with the Topo
IV-Norf-DNA ternary complexes, whereas the majority of Gyr-Norf-DNA
ternary complexes remain reversible after their collision with the UvrD
helicase. These results indicated that different DNA repair mechanisms
might be involved in the repair of Gyr-Norf-DNA and Topo IV-Norf-DNA
ternary complexes.
Topoisomerases are responsible for altering the linking number of
DNA. As such, they play essential roles in DNA replication, transcription, and DNA recombination (1). There are two classes of
topoisomerases, type I and type II enzymes. Type I topoisomerases alter
the linking number in steps of one by breaking one strand of the duplex
DNA, passing the other strand through the break, and then resealing the
broken strand. On the other hand, type II topoisomerases alter the
linking number in steps of two by breaking both strands, passing
another segment of the helix through the break, and then resealing the
broken strands (1). Both DNA gyrase
(Gyr)1 and topoisomerase IV
(Topo IV) are type II topoisomerases.
Quinolones are synthetic antibacterial drugs based on the
4-oxo-1,4-dihydroquinolone skeleton (2). After the discovery of
nalidixic acid as an antibacterial agent, successive generations of
drugs have brought orders of magnitude increases in antibacterial activity. Quinolone drugs, such as ciprofloxacin, are now one of the
most widely prescribed antibacterial drugs. It has been demonstrated
that, shortly after the identification of Escherichia coli
Gyr (3-5), quinolones target Gyr (3, 6) and convert it to a poison
that inhibits DNA replication (7). These drugs form a
topoisomerase-quinolone-DNA ternary complex. As a result, the covalent
topoisomerase-DNA complex (often referred to as the "cleavable
complex") that contains broken DNA strands can persist on the DNA, as
if the topoisomerase were trapped in the cleavable complex. Ultimate
denaturation or disruption of the topoisomerase in the ternary complex
therefore results in the generation of a double-strand break (DSB) and
subsequent cell death (8-10). Based on this unique mode of action,
this class of topoisomerase inhibitors is often called the
"topoisomerase poison." Some anticancer drugs targeting human type
II topoisomerases also convert these enzymes to poisons in the same
fashion (11).
The formation of topoisomerase-quinolone-DNA ternary complexes is
necessary but not sufficient for the cytotoxicity of topoisomerase poisons. These ternary complexes are normally reversible. In addition to the formation of the topoisomerase-quinolone-DNA ternary complexes, an active DNA transaction is required for the disruption of ternary complexes and the generation of a DSB (8-10). The reconstitution of
the collision between a replication fork and a Topo IV-norfloxacin (Norf)-DNA ternary complex has revealed that the collision of a
replication fork with a Topo IV-Norf-DNA ternary complex converts the
ternary complex to a nonreversible form but does not generate a DSB
(12). An additional step, perhaps an aborted repair attempt, is
required for the generation of a DSB.
Khodursky and Cozzarelli (13) have recently demonstrated that quinolone
drugs poison both Gyr and Topo IV in the same manner and that the
cytotoxicity of these drugs correlates with the inhibition of DNA
replication. These studies (13) have also shown that the
recombinational repair system is the main pathway for the repair of
quinolone-induced covalent topoisomerase-DNA complex. Interestingly,
the loss of the UvrD function affects Topo IV-targeted cell killing but
not Gyr-targeted cell killing. These results suggest that the
postreplicative repair system is effective for the repair of ternary
complexes formed with Topo IV but is not effective for the repair of
ternary complexes formed with Gyr. It is concluded that the
UvrD-directed postreplicative repair system can repair Topo
IV-quinolone-DNA ternary complexes, but not Gyr-quinolone-DNA ternary
complexes, because of the positions of Gyr and Topo IV during DNA
replication (13). Gyr acts ahead of the advancing replication forks,
and Topo IV acts behind the forks (14, 15). Thus, ternary complexes
formed with Topo IV have a greater chance to be repaired by the
postreplicative repair system than those formed with Gyr. In addition,
the ternary complexes formed with Gyr more frequently collide with the
replication forks than those formed with Topo IV to trigger the
quinolone-induced cytotoxic events.
We have previously studied the interactions between Topo IV-Norf-DNA
ternary complexes and various DNA helicases (16). We have found that
the collision of the UvrD helicase with a Topo IV-Norf-DNA ternary
complex results in the conversion of the ternary complex to a
nonreversible form, which seems to be critical for the generation of a
DSB. It is suggested that this conversion of Topo IV-Norf-DNA ternary
complex to a nonreversible form may be a critical step to initiate the
removal of the ternary complex and the repair of the DNA damage by the
postreplicative repair system (16).
Here, we further investigated the effects of the UvrD helicase on the
topoisomerase-quinolone-DNA ternary complexes formed with either Gyr,
Topo IV, or Gyr (A59), a mutant Gyr that does not wrap the DNA strand
around itself (17). The ternary complexes formed with either Gyr or
Topo IV, but not those formed with Gyr (A59), inhibited the UvrD
helicase activity. Collisions of UvrD with these ternary complexes
resulted in the formation of a single-strand break (SSB) at small
portions of ternary complexes. Interestingly, the majority of
Gyr-Norf-DNA ternary complexes remained reversible after their
collisions with UvrD, whereas the majority of Topo IV-Norf-DNA ternary
complexes was converted to a nonreversible form upon their collisions
with UvrD. These results demonstrated that the UvrD helicase could
affect the ternary complexes formed with Gyr and those formed with Topo
IV in a distinct manner. Furthermore, these results indicated that
different DNA repair mechanisms might be involved in the repair of
Gyr-Norf-DNA and Topo IV-Norf-DNA ternary complexes.
DNAs and Proteins--
The construction of a recombinant M13
containing a defined Topo IV-binding site (M13-T440) (16) and the
preparation of the single-stranded circular DNA of M13-T440 (18, 19)
were as described previously.
Purified UvrD and the subunits of E. coli Gyr and Topo IV
were generous gifts of Kenneth Marians (Memorial Sloan-Kettering Cancer
Center). GyrA and GyrB and ParC and ParE proteins were mixed on ice to
form active tetramers of Gyr and Topo IV, respectively. Purified UvrD,
a gift of Steve Matson (University of North Carolina), was also used.
A deletion mutant of gyrA encoding the amino acids from 1 to
523 (17) was generated by PCR and cloned into pET-11c. The DNA sequence
analysis confirmed that this deletion mutant of gyrA gene
encoded a 59-kDa fragment of the GyrA subunit, GyrA (59). The GyrA (59)
protein was overproduced in E. coli BL21(DE3) and purified
by an unpublished procedure.2
Briefly, this purification procedure consisted of preparation of a
soluble lysate, removal of nucleic acids by precipitation with 0.075%
Polymin P, precipitation of proteins with ammonium sulfate (60%
saturation), and then chromatography on Q Sepharose FF (Amersham
Pharmacia Biotech) followed by chromatography on diethylaminoethyl
cellulose DE52 (Whatman). This preparation of GyrA (59) was judged to
have a purity of greater than 90% by denaturing gel electrophoresis
(data not shown). GyrA (59) was mixed with the wild type GyrB to
reconstitute a mutant Gyr, and this mutant Gyr was referred to as Gyr (A59).
Restriction enzymes and DNA modifying enzymes were from New England
Biolabs, Stratagene, Roche Molecular Biochemicals, and Amersham
Pharmacia Biotech.
Preparation of the Helicase Substrate--
The partial duplex
DNA was prepared as described previously (16). Briefly, a 62-nucleotide
(nt) oligonucleotide (oligo), T440C,
5'-GCTCGTATCTAGACTCCTAAAAATCCGGGGTATACCCCGGATTTTTAGGAGTGTGTCGCGAT-3' was synthesized and hybridized to the M13-T440 single-stranded DNA to
prepare a primer-template. The oligo in the primer-template was then
3'-end-labeled by incorporation of 2 residues of
[32P]dAMP (Amersham Pharmacia Biotech) with the Klenow
enzyme. This DNA was referred to as "T440" in this study
(originally referred to as "3'-T440" in Ref. 16). The
3'-end-labeled oligo was elongated by the Klenow enzyme in the presence
of four deoxyribonucleotides to an average length of 175 nt with a
range of 150-200 nt (see Fig. 1). We referred to this elongated
partial duplex DNA as "T440E."
Topoisomerase-catalyzed DNA Cleavage Assay--
Reaction
mixtures (12.5 µl) containing 50 mM Tris-HCl (pH 7.5 at
23 °C), 10 mM magnesium chloride, 10 mM
dithiothreitol (DTT), 50 µg/ml bovine serum albumin (BSA), 2 mM ATP, 10 fmol (as molecule) of DNA substrate (either T440
or T440E), indicated amounts (as tetramer) of Gyr, Topo IV, or Gyr
(A59), and the indicated concentrations of Norf were incubated at
37 °C for 5 min. SDS was added to 1% to terminate the reactions,
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), heat-denatured by
heating at 100 °C for 5 min, and then analyzed by electrophoresis
through 8% (for T440E) and 10% (for T440) polyacrylamide (19:1,
acrylamide to bisacrylamide) gels (140 × 160 × 1.2 mm) at
12 V/cm for 2 h using 50 mM Tris borate (pH 8.3) and 1 mM EDTA as the electrophoresis buffer (TBE buffer). Gels
were dried under vacuum onto DE81 papers (Whatman) and autoradiographed
with Hyperfilm MP films (Amersham Pharmacia Biotech). Amounts of
cleaved DNA strands were quantitated by scanning images by a STORM 840 PhosphorImager (Molecular Dynamics).
The UvrD Strand-displacement Assay--
The standard reaction
mixtures (12.5 µl) contained 50 mM Tris-HCl (pH 7.5 at
23 °C), 10 mM magnesium chloride, 10 mM DTT, 50 µg/ml BSA, 2 mM ATP, 10 fmol (as molecule) of either
T440 or T440E, and the indicated concentrations of Norf. Three hundred fmol (as tetramer) of Gyr, Topo IV, or Gyr (A59) was added to the
reaction mixtures, and the mixtures were incubated at 37 °C for 5 min to form topoisomerase-DNA complexes. After the first incubation
stage, 250 fmol (as monomer) of UvrD was added and then the reaction
mixtures were incubated at 37 °C for an additional 10 min. Reactions
were terminated by adding EDTA to 25 mM, followed by the
addition of a 0.25 volume of a dye mixture containing 15% glycerol,
2% Sarkosyl, 0.05% xylene cyanol, 0.05% bromphenol blue, and 50 mM EDTA.
Aliquots were analyzed by electrophoresis through vertical 1% agarose
(Seakem ME, FMC) gels (140 × 120 × 3 mm) at 5 V/cm for 2 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). Portions of reaction mixtures were also analyzed by
electrophoresis through 8 (for T440E) and 10% (for T440)
polyacrylamide (19:1, acrylamide to bisacrylamide) gels (140 × 160 × 1.2 mm) at 12 V/cm for 2 h using TBE buffer. Gels were
dried under vacuum onto DE81 papers (Whatman) and autoradiographed with
Hyperfilm MP films (Amersham Pharmacia Biotech). Strand displacement
was quantitated by scanning images by a STORM 840 PhosphorImager
(Molecular Dynamics).
Reversal Assay for the Topoisomerase-Quinolone-DNA Ternary
Complex--
The reversibility of the topoisomerase complexes after
their collision with the UvrD helicase was assessed by combining a strand-displacement assay and a reversal assay (7). Reaction mixtures
were assembled as described above, and Gyr, Topo IV, or Gyr (A59) was
bound to the partial duplex DNA in the presence of Norf during the
first stage of incubation at 37 °C for 5 min. Reaction mixtures were
then incubated at 37 °C for 10 min in the presence and absence of
UvrD. This stage allowed UvrD, when present, to collide with a
topoisomerase-Norf-DNA ternary complex. Reactions were terminated by
adding either EDTA or SDS to 25 mM and 1%, respectively,
and incubating at 37 °C for 5 min. Then either SDS or EDTA, together
with proteinase K, was added to each reaction mixture to a final
concentration of 1%, 25 mM, and 100 µg/ml, respectively.
The incubation was further continued at 37 °C for an additional 15 min. The DNA products were purified by extraction of the reaction
mixtures with phenol/chloroform (1:1, v/v), heat-denatured by heating
at 100 °C for 5 min, and then analyzed by electrophoresis through
8% (for T440E) and 10% (for T440) polyacrylamide (19:1, acrylamide to
bisacrylamide) gels (140 × 160 × 1.2 mm) at 12 (for 10%)
and 18 (for 8%) V/cm, respectively, for 2 h using TBE buffer. Gels were dried and analyzed as described above.
Partial Duplex DNAs--
One of the basic differences between Gyr
and Topo IV is their mode of DNA binding. Gyr is unique among the type
II topoisomerases. This enzyme wraps the DNA strand around itself
(20-23) and protects about 140 base pairs (bp) of the duplex DNA
(21-23). This unique mode of DNA binding enables Gyr to catalyze the
supercoiling reaction. In contrast, Topo IV and other type II
topoisomerases do not wrap the DNA strand and require shorter DNA
fragments for their binding. The footprinting analysis of Topo IV has
revealed that it protects only about 35-40 bp (24). Kampranis and
Maxwell (17) have demonstrated that a deletion of the C-terminal
DNA-binding domain of the GyrA subunit, GyrA (59), gives rise to an
enzyme that does not wrap the DNA strand around itself and that cannot
catalyze the supercoiling reaction. Thus, the DNA binding mode of Gyr
(A59) is likely to be similar, if not identical, to that of Topo IV (17).
To perform the strand-displacement assay, we prepared two partial
duplex DNAs, which differed only by the length of the duplex region
(Fig. 1A). T440 DNA was
prepared by annealing a 62-nt oligo and labeling the annealed oligo at
the 3'-end. As a result, this partial duplex DNA contained a 64-bp
duplex region, and a defined Topo IV-binding site (25) was located in
the middle of the duplex region (Fig. 1A). We have
previously shown that the T440 DNA serves as a good substrate for Topo
IV binding (16). It has been shown that many of the binding sites, as
Norf-stimulated cleavage sites, of Gyr and Topo IV overlap, although
site preferences of these enzymes are distinct (26). Thus, it seemed
reasonable to assume that the defined Topo IV-binding site (25) serves
as a good binding site for Gyr. However, we expected the 64-bp duplex
region in the T440 DNA to be too short for Gyr to bind. Thus, the
annealed and labeled oligo in the T440 DNA was further elongated using the Klenow enzyme to an average length of 175 nt with a range of
150-200 nt (Fig. 1B). This partial duplex with an elongated duplex region was referred to as T440E.
The occupancy of the topoisomerase on the DNA substrate is one of the
determining factors of the probability of the collision between the
UvrD helicase and the topoisomerase-DNA complex. In order to determine
the amounts of topoisomerase-DNA complexes formed on the partial duplex
DNA, we measured the topoisomerase-catalyzed cleavages in the presence
of various concentrations of Norf. The relative amounts of
topoisomerase-catalyzed cleavages represent the occupancy of the DNA
substrate by the topoisomerase-DNA complex.
The Wrapping of the DNA Strand Prevents Gyr from Binding to a Short
Duplex DNA--
First, we measured Gyr-, Topo IV-, and Gyr
(A59)-catalyzed cleavages using T440 as a substrate. The T440 DNA
contains a 64-bp duplex region including a 40-bp defined Topo
IV-binding site (Fig. 1A). Topo IV cleaved the T440 DNA at a
unique site, and Norf stimulated Topo IV-catalyzed cleavages (Fig.
2A). It seemed to be an
interesting question to ask if Gyr (A59), which does not wrap a DNA
strand around itself (17), could bind to a short duplex. To address this question directly, Gyr- and Gyr (A59)-catalyzed cleavages in the
presence of various concentrations of Norf were measured (Fig.
2A). The wild type Gyr could cleave the T440 DNA at a unique site in the presence of Norf. The relative amounts of Gyr-catalyzed cleavages were about one-fourth of those of Topo IV-catalyzed cleavages. On the other hand, the relative amounts of Gyr
(A59)-catalyzed cleavages were similar to those of Topo IV-catalyzed
cleavages. Thus, when Gyr no longer wraps a DNA strand, it could bind
to a short duplex DNA as well as Topo IV could bind.
Next, we measured the Gyr- and Topo IV-catalyzed cleavages using T440E
as a substrate (Fig. 3). All three
topoisomerases cleaved the elongated duplex region and generated DNA
fragments with various sizes. These cleavages were greatly stimulated
by Norf. The pattern of Gyr (A59)-catalyzed cleavages was somewhat
similar to that of Topo IV-catalyzed cleavages but not that of
Gyr-catalyzed cleavages (Fig. 3). To maximize the formation of covalent
topoisomerase-DNA ternary complexes formed, the highest concentrations
of Norf and the topoisomerases were used in the following experiments.
Under these conditions, more than 80, 80, or 90% of the DNA substrate was bound by at least one molecule of Gyr (Fig. 3, lane 6),
Topo IV (Fig. 3, lane 11), or Gyr (A59) (Fig. 3, lane
16), respectively. The elongated duplex region of T440E could
provide more than one binding site for each topoisomerase. Thus, the
generation of cleaved fragments with various sizes was likely due to
the binding of multiple topoisomerases to one DNA substrate and/or the
binding of one topoisomerase per DNA molecule at different sites.
Ternary Complexes Formed with Either Gyr or Topo IV Inhibit the
UvrD Helicase Activity--
We have demonstrated that the Topo
IV-Norf-DNA ternary complex on T440 inhibits the strand-displacement
activities of various DNA helicases including UvrD (16). We have
concluded that the Topo IV-Norf-DNA ternary complex blocks the passage
of DNA helicases. Because Gyr could form the ternary complex with the
T440 DNA (Fig. 2), we examined if the Gyr complex formed at the short
duplex region of T440 could inhibit the UvrD-catalyzed
strand-displacement activity.
The UvrD helicase activity was assessed in the absence and presence of
Gyr, Topo IV, and Norf, using T440 as a substrate (Fig. 4). Under the conditions used, UvrD
displaced 78% of the annealed oligo (Fig. 4A, lane 3). Gyr
(Fig. 4A, lane 6), Topo IV (Fig. 4A, lane 9), or
Norf alone (Fig. 4A, lane 4) did not affect the UvrD-catalyzed strand displacement. The Gyr-Norf-DNA ternary complex reduced the UvrD-catalyzed strand-displacement activity by 25% (Fig.
4A, lane 7), whereas the Topo IV-Norf-DNA ternary complex inhibited the UvrD helicase activity by 67% (Fig. 4A, lane
10). The extent of inhibition by Gyr and Topo IV complexes
correlated well with the relative amounts of Gyr- (28%) and Topo
IV-catalyzed (61%) DNA cleavages (Fig. 2). Thus, the passage of UvrD
was blocked when UvrD collided with a topoisomerase-quinolone-DNA
ternary complex formed with either Gyr or Topo IV.
Howard et al. (27) have demonstrated that the collision
between UvrD and a T4 topoisomerase-m-AMSA-DNA ternary
complex results in the generation of an SSB and the release of a broken
DNA strand from the ternary complex. To examine if there were any
strand breaks at the ternary complexes when UvrD collided with the
ternary complexes formed with either Gyr or Topo IV, DNA products were analyzed on polyacrylamide gels (Fig. 4B). Only the DNA
strand detected was the intact annealed oligo displaced by UvrD,
suggesting that UvrD did not disrupt either Gyr-Norf-DNA or Topo
IV-Norf-DNA ternary complex to generate an SSB. One possible
explanation of these apparent differences was that, as proposed by
Howard et al. (27), the disruption of the ternary complex
and the generation of an SSB by UvrD might require the binding of UvrD
to the partially unwound DNA strand. The annealed oligo may be too
short for UvrD to bind when it is partially unwound. Therefore, we
further investigated the molecular events during the collision between
the UvrD helicase and the ternary complexes formed with either Gyr or
Topo IV using T440E, which contains an elongated duplex region, as a substrate.
Collisions between UvrD and the Ternary Complexes Formed with
Either Gyr or Topo IV Result in the Generation of an SSB--
The
strand-displacement assay was performed using T440E as a substrate
(Fig. 5A). Under the
conditions used, UvrD-catalyzed strand-displacement was 67% (Fig.
5A, lane 3). Gyr (Fig. 5A, lane 6), Topo IV (Fig.
5A, lane 9), or Norf alone (Fig. 5A, lane 4) did
not affect the activity of UvrD.
We expected both Gyr-Norf-DNA and Topo IV-Norf-DNA ternary complexes to
block the UvrD-catalyzed strand-displacement activity. However, when we
analyzed the DNA products on 1% agarose gels, we detected the partial
displacement of the DNA strand in the presence of either the
Gyr-Norf-DNA (Fig. 5A, lane 7) or the Topo IV-Norf-DNA
ternary complex (Fig. 5A, lane 10). These apparent displaced
fragments (Fig. 5A, lanes 7 and 10)
migrated faster than the intact elongated DNA strands (Fig. 5A,
lane 1). We suspected that the apparent displaced fragments might
be broken DNA strands released from ternary complexes as a result of
strand breaks. If there were a DSB at the ternary complex, M13 DNA
would become linear, which would change the migration of the substrate
DNA. On the other hand, if a strand break were an SSB, the M13 DNA would remain circular and the migration of the substrate DNA would not
change. Because the migration of the DNA substrate on the agarose gel
did not change (Fig. 5A, lanes 7 and
10), the strand break was likely to be an SSB.
To confirm the formation of an SSB and the release of a DNA strand from
the ternary complex, the DNA products were analyzed on 8%
polyacrylamide gels (Fig. 5B). Short DNA fragments were generated when UvrD collided with the ternary complexes formed with
either Gyr (Fig. 5B, lane 7) or Topo IV (Fig. 5B, lane
10). These results demonstrated that the apparent UvrD-catalyzed
strand displacement in the presence of either Gyr-Norf-DNA or Topo
IV-Norf-DNA ternary complexes was indeed due to the release of broken
DNA strands from the ternary complex as a result of an SSB. Based on
the amounts of broken DNA strands released from the T440E DNA (Fig.
5A, lanes 7 and 10), the frequency of the
formation of an SSB was estimated as 28 and 15% at the ternary complex
formed with Gyr and Topo IV, respectively. It was not clear why strand breaks occurred only at small portions of the ternary complexes. The
majority of the ternary complexes formed with either Gyr or Topo IV
seemed to retain all DNA strands after their collisions with UvrD.
Interestingly, portions of the displaced, intact DNA fragments were
shifted (Fig. 5B, lanes 3, 4, 6, and 9). This
shift was due to the binding of UvrD, which was abolished by either the
heat treatment or the deproteinization by the extraction with phenol/chloroform (data not shown).
UvrD Converts the Topo IV-Norf-DNA Ternary Complex, but Not the Gyr
Norf-DNA Ternary Complex, to a Nonreversible Form--
We have
previously shown that the Topo IV-Norf-DNA ternary complex formed with
the T440 DNA is converted to a nonreversible form when it collides with
UvrD (16). This conversion seems to be critical for the DSB formation,
either to trigger the cytotoxic process or to remove the covalent
topoisomerase-DNA complex and repair DNA damage. It has been
demonstrated that the UvrD-directed postreplicative repair system can
effectively repair Topo IV-Norf-DNA ternary complexes but not
Gyr-Norf-DNA ternary complexes (13). To examine the consequences of the
collisions between UvrD and ternary complexes formed with either Gyr or
Topo IV, we assessed the effect of the UvrD helicase on the
reversibility of the ternary complexes.
The reversal assay was first performed using T440 as a substrate (Fig.
6A). The Topo IV-Norf-DNA
ternary complex formed with T440 was converted to a nonreversible form
when it collided with UvrD (Fig. 6A, lane 4). In contrast,
the Gyr-Norf-DNA ternary complex remained reversible after its
collision with UvrD (Fig. 6A, lane 8). Two independent
preparations of Gyr, Topo IV, and UvrD were used, and identical results
were obtained (data not shown). These results demonstrated that the
UvrD helicase affected the ternary complexes formed with Gyr and Topo
IV in a distinct manner.
We obtained similar results when T440E was used as a substrate in the
reversal assay (Fig. 6B). The majority of Gyr-Norf-DNA ternary complexes remained reversible after their collisions with the
UvrD helicase. The amount of the reversed DNA strands in the presence
of UvrD (Fig. 6B, lane 4) was less than that in the absence of UvrD (Fig. 6B, lane 2) by 22%. This
correlated well with the observation that about 28% of the
Gyr-Norf-DNA ternary complexes lost a DNA strand as a result of an SSB
(Fig. 5A). In contrast, the Topo IV-Norf-DNA complexes were
completely nonreversible after their collision with UvrD (Fig.
6B, lane 8). Only a small portion (15%) of these
nonreversible ternary complexes became nonreversible because of the
formation of an SSB (Fig. 5A). The majority of nonreversible
complexes seemed to retain all DNA strands. Thus, UvrD could convert
Topo IV-Norf-DNA ternary complexes, but not Gyr-Norf-DNA ternary
complexes, to a nonreversible form. These results suggested that UvrD,
and most likely the UvrD-directed repair system, could distinguish
ternary complexes formed with Topo IV from those formed with Gyr.
The Gyr (A59)-Norf-DNA Ternary Complex Remains Reversible After Its
Collision with UvrD--
It is not clear what makes the difference
between ternary complexes formed with Gyr and those formed with Topo
IV. One obvious possibility is that the Gyr-mediated wrapping of the
DNA strand makes Gyr different from Topo IV and other type II
topoisomerases. We used Gyr (A59) protein to ask this question
directly. If the Gyr-Norf-DNA ternary complex remained reversible after
its collision with UvrD because of the Gyr-mediated wrapping of the DNA
strand, the Gyr (A59)-Norf-DNA ternary complex should be converted to a
nonreversible form. In contrast, if the difference between Gyr and Topo
IV is not due to the Gyr-mediated wrapping of the DNA strand, the
ternary complex formed with Gyr (A59) should remain reversible.
We performed the reversal assay using Gyr (A59) (Fig.
7). The Gyr (A59)-Norf-DNA ternary
complexes remained completely reversible when T440 was used as a
substrate (Fig. 7A, lane 4). These results showed that,
whether Gyr could wrap the DNA strand around itself or not, the
Gyr-Norf-DNA ternary complexes remained reversible after their
collisions with UvrD. The Gyr (A59)-catalyzed cleavages were inhibited
when UvrD was present in the reaction mixtures (Fig. 7, lane
5).
We further examined the consequences of the collisions between UvrD and
the Gyr (A59)-Norf-DNA ternary complex using T440E as a substrate (Fig.
7B). Two distinct DNA products, a short DNA strand (38%)
and the intact fragments (62%), were generated after the encounter of
UvrD with the Gyr (A59)-Norf-DNA ternary complex (Fig. 7B, lane
4). These results were very similar to those using the wild type
Gyr (Fig. 6B). These results demonstrated that the collision
between UvrD and the Gyr-Norf-DNA ternary complex did not affect the
reversibility of the ternary complex formed with Gyr, even when Gyr
could no longer wrap the DNA strand around itself.
The Gyr (A59)-Norf-DNA Ternary Complex Does Not Inhibit
UvrD-catalyzed Strand Displacement--
To confirm the formation of an
SSB at the Gyr (A59)-Norf-DNA ternary complex, we performed the
strand-displacement assay using Gyr (A59). First, T440 was used as a
DNA substrate (Fig. 8, A and
B). Under the condition used, about half of the T440 DNA was occupied by the Gyr (A59)-Norf-DNA ternary complex (Fig. 2). Thus, we
expected to observe an inhibitory effect of the ternary complex formed
with Gyr (A59) on the UvrD helicase. Interestingly, UvrD-catalyzed strand displacement was not inhibited at all by the Gyr (A59)-Norf-DNA ternary complex (Fig. 8, A and B). UvrD was
capable of displacing 76% of the annealed oligo (Fig. 8A, lane
3). In the presence of Norf alone (Fig. 8A, lane 4),
Gyr (A59) alone (Fig. 8A, lane 5), or both Gyr (A59) and
Norf (Fig. 8A, lane 6), UvrD-catalyzed strand displacement
was 73, 72, or 70%, respectively. These results demonstrated that,
unlike the ternary complex formed with either the wild type Gyr or Topo
IV, the Gyr (A59)-Norf-DNA ternary complex did not inhibit the UvrD
helicase activity. No short fragment was generated when UvrD collided
with Gyr (A59)-Norf-DNA ternary complexes (Fig. 8B),
indicating that no SSB was generated at the ternary complex formed with
T440.
The strand-displacement assay was also performed using T440E as a
substrate (Fig. 8, C and D). Under the conditions
used, 60% of the elongated DNA strand was displaced by UvrD (Fig.
8C, lane 3). Either Norf alone (Fig. 8C, lane,
4), Gyr (A59) alone (Fig. 8C, lane 5), or Gyr
(A59)-Norf-DNA ternary complex (Fig. 8C, lane 6) did not
affect UvrD-catalyzed strand-displacement activity. When the Gyr
(A59)-Norf-DNA ternary complex was present, however, in addition to the
intact DNA strands displaced by UvrD, a short DNA strand was generated
(Fig. 8D, lane 5). We used two preparations of Gyr (A59) and
obtained essentially identical results (data not shown). These results
showed that an SSB was generated at a portion (58%) of the Gyr
(A59)-Norf-DNA ternary complexes formed with T440E and that UvrD could
displace some (42%) of the intact DNA fragment even in the presence of
the Gyr (A59)-Norf-DNA ternary complex. Thus, the UvrD helicase, upon
its collision with the Gyr (A59)-Norf-DNA ternary complex, seemed to be
able to force Gyr (A59) to reverse the ternary complex formation and
religate the DNA strands.
Topoisomerases are responsible for unlinking the DNA molecules
during DNA replication (1). It has been thought that the topological
constraint accumulates as positive supercoils in the unreplicated
region. However, recent studies have demonstrated that, as originally
proposed by Champoux and Been (28), the topological constraint can take
two forms, positive supercoils in the unreplicated region and
precatenanes in the replicated region (15, 29, 30). Gyr removes
positive supercoils in front of the replication forks to support the
nascent chain elongation, whereas Topo IV decatenates precatenanes
behind the forks to support the nascent chain elongation and the
decatenation of daughter DNA molecules (15).
Gyr binds to the DNA by wrapping a DNA strand around itself, which
requires about 140-bp duplex DNA (20-23). Thus, we did not expect Gyr
to bind to the T440 DNA, because the duplex region of T440 is only 64 bp long (Fig. 2). A mutant Gyr, Gyr (A59), which does not wrap a DNA
strand around itself (17), was used to examine the effect of
Gyr-mediated wrapping on the ability of Gyr to bind to a short duplex
DNA. Gyr (A59) could bind to T440 as well as Topo IV (Fig. 2). Thus,
Gyr-mediated wrapping of the DNA strand prevents Gyr from binding to a
short duplex DNA. Furthermore, these results may support the finding
that the binding sites of Gyr and Topo IV overlap, although site
preference for cleavages is distinct for these enzymes (26). In
contrast, yeast topoisomerase II could not bind to T440 in the presence of m-AMSA or
etoposide.3
Topoisomerases are the cellular targets of some antibacterial agents
and potent anticancer drugs (8-10). Both Gyr and Topo IV are the
targets of quinolone antibacterial drugs. These drugs form a
topoisomerase-quinolone-DNA ternary complex, in which the topoisomerase
is trapped as a cleavable complex. These ternary complexes are normally
reversible, and the formation of ternary complexes is not sufficient,
although necessary, for the cytotoxicity of these drugs. An active DNA
transaction, such as the passage of replication forks, is required to
convert a ternary complex to a permanent cytotoxic lesion (8-10). It
has been proposed that the collision between a replication fork and a
topoisomerase-drug-DNA ternary complex disrupts the ternary complex and
generates a DSB. We have previously demonstrated that, in fact, the
Topo IV-Norf-DNA ternary complex blocks the replication fork
progression in vitro and this collision converts the ternary
complex to a nonreversible form (12). However, an additional step is
required to remove the topoisomerase in the dead-end complex and to
generate a DSB (12).
In E. coli, although both Gyr and Topo IV are the targets of
the quinolone drugs, Gyr becomes the primary target and Topo IV is the
secondary target (14). The order of the targets seems to be reversed in
Staphylococcus aureus, where Topo IV becomes the primary
target (31). It is not clear what determines which topoisomerase is the
primary target in the cell. One possibility is that E. coli
Gyr is more sensitive to the quinolone drugs than E. coli
Topo IV. However, this is not likely to be the case. Only a slight
difference is found between Gyr and Topo IV when the drug sensitivities
of these enzymes are measured in vitro (14, 26).
Alternatively, although both topoisomerases are poisoned in the same
manner, the ternary complex formed with Gyr is more cytotoxic than that
formed with Topo IV. It has been proposed that Gyr becomes the primary
target in E. coli because of the position of Gyr-Norf-DNA ternary complexes relative to the advancing replication forks (13). Gyr
functions in front of the replication forks, whereas Topo IV binds
behind the forks during the chromosomal DNA replication (14, 15). Thus,
the ternary complexes formed with Gyr collide with the replication
forks more frequently than those formed with Topo IV. Another
possibility is that the differences in repairing the ternary complexes
formed with Gyr and those formed with Topo IV make the
Gyr-quinolone-DNA ternary complex more cytotoxic than the Topo
IV-quinolone-DNA ternary complex. Let us assume that the same number of
ternary complexes is formed with Gyr and Topo IV on the chromosome. If
the ternary complexes formed with Topo IV, but not those formed with
Gyr, can be repaired efficiently, Gyr-quinolone-DNA ternary complexes
become more cytotoxic than Topo IV-quinolone-DNA ternary complexes do.
Note that, once the collision takes place between a replication fork
and a ternary complex formed with either Gyr or Topo IV, both
Gyr-Norf-DNA and Topo IV-Norf-DNA ternary complexes block the
replication fork progression (12).
We detected an SSB as a result of the collision between the UvrD
helicase and the ternary complex formed with either Gyr or Topo IV
(Figs. 6 and 7). How is an SSB formed at the ternary complex? One
possible mechanism is that the ternary complex formed with either Gyr
or Topo IV could block the UvrD-catalyzed unwinding of the duplex DNA.
Binding of the UvrD to the displaced DNA strand seems to be essential
for the destabilization of the interaction between the displaced DNA
strand and the topoisomerase in the ternary complex. The displaced DNA
strand is not covalently attached to the topoisomerase. Thus, release
of this DNA strand from the ternary complex results in the formation of
an SSB. A similar model has been proposed by Howard et al.
(27).
What is the role of the UvrD-mediated SSB formation in the repair of
the topoisomerase-quinolone-DNA ternary complexes? Ternary complexes
formed with either Gyr or Topo IV are mainly repaired by the
recombinational repair system (13). The formation of an SSB at the
ternary complex may provide a single-stranded DNA with a free 3'-OH. It
is interesting to speculate that the SSB formation provides an invading
strand to initiate the recombination process, which leads to the repair
of the quinolone-induced, topoisomerase-mediated DNA damage.
We found that SSBs were generated only at small portions of the ternary
complexes (Figs. 5 and 8). It is not clear what determines if an SSB
generates at the ternary complex upon its encounter with the UvrD
helicase. One possible explanation is that the DNA sequences of
topoisomerase-binding sites, in which the topoisomerase-quinolone-DNA ternary complexes are formed, affect the stability of each ternary complex. Some ternary complexes are more stable than others. The formation of an SSB could occur only at unstable ternary complexes. Strumberg et al. (32) have recently demonstrated that, using yeast topoisomerase II, the DNA sequences of the topoisomerase-binding sites affect the stability of the ternary complex.
Recently, an interesting phenotype of the uvrD deletion has
been reported (13). Khodursky and Cozzarelli (13) have developed an
assay system to assess the efficiencies of the Gyr- and Topo IV-targeted cell killing by the quinolone drug. The loss of the UvrD
function has no effect on the Gyr-targeted cell killing, whereas the
efficiency of the Topo IV-targeted cell killing is drastically
increased when the uvrD gene is deleted. It is concluded that the postreplicative repair system can repair the Topo IV-Norf-DNA ternary complexes but not Gyr-Norf-DNA ternary complexes in E. coli (13). The observed Topo IV-specific repair by the
postreplicative repair system is explained by the positions of Gyr and
Topo IV relative to the advancing replication forks. Topo IV-Norf-DNA ternary complexes are formed behind the replication forks, and thus
these complexes are repaired by the postreplicative repair system.
We showed here that the UvrD helicase affected the Gyr-Norf-DNA and
Topo IV-Norf-DNA ternary complexes in a distinct manner. The
UvrD-mediated conversion of Topo IV-Norf-DNA ternary complex to a
nonreversible form may be a critical step for the removal of the
covalent Topo IV-DNA complex and the repair of the DNA damage. It is
likely that the UvrD helicase can distinguish between Gyr and Topo IV
in the ternary complexes. At the Topo IV-Norf-DNA ternary complexes,
UvrD convert the ternary complex to a nonreversible protein-DNA adduct,
which can be subsequently repaired by the postreplicative repair
system. In contrast, when UvrD collides with the Gyr-Norf-DNA ternary
complexes, the ternary complexes remain reversible and eventually fall
off the DNA. The possibility exists that collisions between UvrD and
the Gyr-Norf-DNA ternary complex reverse the ternary complex formation
and force Gyr to religate the DNA strands. The reversal of Gyr
(A59)-Norf-DNA ternary complexes upon their collisions with UvrD (Figs.
7 and 8) supports this possibility. In any case, in addition to the
positions of these ternary complexes formed with either Gyr or Topo IV
relative to the replication forks, the different responses between Topo IV-Norf-DNA and Gyr-Norf-DNA ternary complexes to the UvrD helicase might contribute to the Topo IV-specific repair of DNA damage by the
postreplicative repair system.
After the collisions between UvrD and the ternary complexes, the
majority of Gyr-Norf-DNA ternary complexes remained reversible, whereas
the majority of Topo IV-Norf-DNA ternary complexes was converted to a
non-reversible form (Fig. 6). It is not clear what makes the difference
between the ternary complexes formed with Gyr and those formed with
Topo IV. The fact that the ternary complexes formed with Gyr (A59)
remained reversible (Fig. 7) suggests that the Gyr-mediated wrapping of
the DNA strand does not make the Gyr-Norf-DNA ternary complex different
from the Topo IV-Norf-DNA ternary complex. It is interesting to
speculate that there is a specific protein-protein interaction between
UvrD and the topoisomerase in the ternary complex. Thus, the UvrD
helicase can interact with some topoisomerases but not others. This
protein-protein interaction determines the fate of the ternary complex
after the collision of UvrD with the ternary complex. The observations
that UvrD disrupts the ternary complexes formed with phage T4
topoisomerase (27), but this helicase had no effect on the
reversibility of the ternary complexes formed with yeast topoisomerase
II,3 support this possibility.
How does the collision between the UvrD helicase and a
topoisomerase-quinolone-DNA ternary complex take place on the
chromosome? Unlike the replication fork, UvrD does not translocate
throughout the chromosome. One likely explanation is that some tracking
and scanning system for DNA damage recognizes the
topoisomerase-quinolone-DNA ternary complex as a protein-DNA adduct and
recruits the UvrD helicase as a part of the repair machinery. MutS
protein, which scans for mismatches in the replicated region, seems to
be a good candidate. A recent demonstration (33) of the stimulation of the UvrD activity by MutS and MutL proteins supports this model.
We thank Dr. Kenneth Marians for the generous
gifts of purified proteins and the comments on these studies. We also
thank Dr. Steve Matson for the gift of purified UvrD helicase.
*
This work was supported by National Institutes of Health
Grant GM59465-01.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.
2
M. E. Shea and H. Hiasa, unpublished results.
3
H. Hiasa, M. E. Shea, and J. L. Nitiss, unpublished results.
The abbreviations used are:
Gyr, DNA gyrase;
bp, base pair(s);
BSA, bovine serum albumin;
DSB, double strand break;
DTT, dithiothreitol;
m-AMSA, 4'-(9-acridinylamino)methanesulfon-m-anisidide;
Norf, norfloxacin;
nt, nucleotide(s);
oligo, oligonucleotide;
SSB, single
strand break;
Topo, topoisomerase.
Distinct Effects of the UvrD Helicase on
Topoisomerase- Quinolone-DNA Ternary Complexes*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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
View larger version (19K):
[in a new window]
Fig. 1.
Schematic presentations of partial duplex
DNAs. A, annealed oligo was 3'-end-labeled by Klenow
enzyme to prepare T440 (top panel). Annealed and labeled
oligo was further elongated by Klenow enzyme to prepare T440E
(bottom panel). Closed circle and open
square represent 32P-nucleotide and the 40-nt defined
Topo IV-binding site (25), respectively. Details were described under
"Materials and Methods." B, T440 and T440E were
heat-denatured, and the DNA products were analyzed by electrophoresis
through 8% polyacrylamide gels. Size markers were 5'-end-labeled
100-bp ladders (New England Biolabs).
View larger version (23K):
[in a new window]
Fig. 2.
Norf-induced, Gyr- and Topo IV-catalyzed
cleavage of the T440 DNA. A, DNA cleavage assays using
either Gyr, Topo IV, or Gyr (A59) (24 nM), and T440 as a
DNA substrate (0.8 nM) were performed in the presence of
indicated concentrations of Norf, and the DNA products were analyzed as
described under "Materials and Methods." B, the extent
of DNA cleavages as a function of the Norf concentration was
quantitated using a phosphorimager. Open circles,
closed circles, and open squares represent Gyr-,
Gyr (A59)-, and Topo IV-catalyzed cleavages, respectively.
View larger version (59K):
[in a new window]
Fig. 3.
Norf-induced, Gyr- and Topo IV-catalyzed
cleavage of the T440E DNA. DNA cleavage assays using either Gyr,
Topo IV, or Gyr (A59) and T440E as a substrate were performed in the
presence of the indicated concentrations of Norf, and the DNA products
were analyzed as described under "Materials and Methods."
View larger version (32K):
[in a new window]
Fig. 4.
Both Gyr-Norf-DNA and Topo IV-Norf-DNA
ternary complexes formed on the T440 DNA inhibit strand-displacement
activity of the UvrD helicase. Helicase assays for UvrD in the
presence and absence of Gyr, Topo IV, and Norf, as indicated, were
performed, and the DNA products were analyzed by electrophoresis
through 1% agarose gels (A) and 10% polyacrylamide gels
(B) as described under "Materials and Methods." T440 was
used as a substrate, and each experiment was triplicated. In the
absence of topoisomerase, UvrD-catalyzed strand displacement, either in
the presence or absence of Norf, was 75% (±4%). Representative
results are shown, but the S.D. was derived from the results of three
experiments. Lane 1 in both panels was
heat-denatured DNA substrate. Gyr (as tetramer), Topo IV (as tetramer),
and UvrD (as monomer) were present at 30-, 30-, and 25-fold molar
excess over DNA substrate, respectively. Norf was at 400 µM when present.
View larger version (37K):
[in a new window]
Fig. 5.
Collisions of UvrD with Gyr-Norf-DNA and Topo
IV-Norf-DNA ternary complexes formed with the T440E DNA result in the
inhibition of UvrD-catalyzed strand-displacement activity and the
generation of strand breaks. The strand-displacement assay for
UvrD in the presence and absence of Gyr, Topo IV, and Norf, as
indicated, were performed, and the DNA products were analyzed by
electrophoresis through 1% agarose gels (A) and 8%
polyacrylamide gels (B) as described under "Materials and
Methods." T440E was used as a substrate, and each experiment was
duplicated. In the absence of topoisomerase, UvrD-catalyzed strand
displacement, either in the presence or absence of Norf, was 64%
(±5%). Representative results are shown, but the S.D. was derived
from the results of two experiments. Lane 1 in both
panels was heat-denatured DNA substrate. Gyr (as tetramer), Topo
IV (as tetramer), and UvrD (as monomer) were present at 30-, 30-, and
25-fold molar excess over DNA substrate, respectively. Norf was at 400 µM when present. Arrows indicated broken DNA
strands released from the ternary complexes.
View larger version (35K):
[in a new window]
Fig. 6.
UvrD converts a Topo IV-Norf-DNA ternary
complex but not a Gyr-Norf-DNA ternary complex to a nonreversible form
upon its collision with the ternary complex. Reversal assays for
the ternary complexes formed with either Gyr or Topo IV in the absence
and presence of the UvrD helicase, as indicated, were performed and the
DNA products were analyzed as described under "Materials and
Methods." Either T440 (A) or T440E (B) was used
as a substrate. Gyr (as tetramer), Topo IV (as tetramer), and UvrD (as
monomer) were present at 30-, 30-, and 25-fold molar excess over the
DNA substrate, respectively. Norf was present at 400 µM.
E and S represent the reactions terminated by the
addition of EDTA or SDS, respectively.
View larger version (21K):
[in a new window]
Fig. 7.
The Gyr (A59)-Norf-DNA ternary complex
remains reversible after its collision with the UvrD helicase.
Reversal assays for the ternary complexes formed with Gyr (A59) in the
absence and presence of UvrD, as indicated, were performed, and the DNA
products were analyzed as described under "Materials and Methods."
Either T440 (A) or T440E (B) was used as a
substrate. Gyr (A59) (as tetramer) and UvrD (as monomer) were present
at 30- and 25-fold molar excess over the DNA substrate, respectively.
Norf was present at 400 µM. E and S
represent the reactions terminated by the addition of EDTA or SDS,
respectively.
View larger version (41K):
[in a new window]
Fig. 8.
The ternary complex formed with Gyr (A59)
does not inhibit the UvrD helicase activity. The
strand-displacement assay for UvrD in the presence and absence of Gyr
(A59) and Norf, as indicated, were performed, and the DNA products were
analyzed by electrophoresis through 1% agarose gels (A and
C) and 8 (D) or 10% (B)
polyacrylamide gels as described under "Materials and Methods."
Either T440 (A and B) or T440E (C and
D) was used as a substrate. Each experiment was either
duplicated or triplicated. In the absence of topoisomerase,
UvrD-catalyzed strand displacement, either in the presence or absence
of Norf, of T440, and T440E were 75 (±4%) and 64% (±5%),
respectively. Representative results are shown, but the standard
deviations were derived from the results of three experiments.
Lane 1 in all panels was heat-denatured DNA substrate. Gyr
(A59) (as tetramer) and UvrD (as monomer) were present at 30- and
25-fold molar excess over the DNA substrate, respectively. Norf was at
400 µM when present. An arrow (D)
indicates a broken DNA strand released from the ternary complex.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Member of the University of Minnesota Comprehensive Cancer Center.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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  K. Drlica, M. Malik, R. J. Kerns, and X. Zhao Quinolone-Mediated Bacterial Death Antimicrob. Agents Chemother., February 1, 2008; 52(2): 385 - 392. [Full Text] [PDF]
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 V. M. Kramlinger and H. Hiasa The "GyrA-box" Is Required for the Ability of DNA Gyrase to Wrap DNA and Catalyze the Supercoiling Reaction J. Biol. Chem., February 10, 2006; 281(6): 3738 - 3742. [Abstract] [Full Text] [PDF]
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M. E. Shea and H. Hiasa The RuvAB Branch Migration Complex Can Displace Topoisomerase IV{middle dot}Quinolone{middle dot}DNA Ternary Complexes J. Biol. Chem., November 28, 2003; 278(48): 48485 - 48490. [Abstract] [Full Text] [PDF]
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X.-S. Pan, G. Yague, and L. M. Fisher Quinolone Resistance Mutations in Streptococcus pneumoniae GyrA and ParC Proteins: Mechanistic Insights into Quinolone Action from Enzymatic Analysis, Intracellular Levels, and Phenotypes of Wild-Type and Mutant Proteins Antimicrob. Agents Chemother., November 1, 2001; 45(11): 3140 - 3147. [Abstract] [Full Text] [PDF]
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H. Hiasa and M. E. Shea DNA Gyrase-mediated Wrapping of the DNA Strand Is Required for the Replication Fork Arrest by the DNA Gyrase-Quinolone-DNA Ternary Complex J. Biol. Chem., October 27, 2000; 275(44): 34780 - 34786. |
