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J Biol Chem, Vol. 274, Issue 32, 22747-22754, August 6, 1999
From the Department of Pharmacology, University of Minnesota
Medical School, Minneapolis, Minnesota 55455
Collisions between replication forks and
topoisomerase-drug-DNA ternary complexes result in the inhibition of
DNA replication and the conversion of the normally reversible ternary
complex to a nonreversible form. Ultimately, this can lead to the
double strand break formation and subsequent cell death. To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes. A strand displacement assay was employed to assess the effect of topoisomerase IV (Topo IV)-norfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-antigen, and UvrD DNA helicases. The ternary complexes
inhibited the strand displacement activities of these DNA helicases.
Unlike replication fork arrest, however, this general inhibition of DNA
helicases by Topo IV-norfloxacin-DNA ternary complexes did not require
the cleavage and reunion activity of Topo IV. We also examined the
reversibility of the ternary complexes after collisions with these DNA
helicases. UvrD converted the ternary complex to a nonreversible form,
whereas DnaB, T7 gene 4 protein, and SV40 T-antigen did not. These
results suggest that the inhibition of DnaB translocation may be
sufficient to arrest the replication fork progression but it is not
sufficient to generate cytotoxic DNA lesion.
Topoisomerases are the essential enzymes found in all living
cells. They are responsible for altering the topology of DNA by
catalyzing the passage of DNA strands and helices through each other.
These enzymes are required for DNA replication, transcription, and
recombination. Topoisomerases also play critical roles in chromosome
structure, condensation/decondensation, and segregation (1).
The importance of these enzymes is underscored by the fact that they
are the primary cellular targets for many clinically important
antibacterial and anticancer drugs. In eukaryotes, these enzymes are
the cellular targets of potent anticancer drugs, whereas in prokaryotes
both DNA gyrase and topoisomerase IV (Topo
IV)1 are targets of the
quinolone antibacterial agents (2-4).
Topoisomerases break and rejoin DNA strands by forming a covalent
linkage between the enzyme and the DNA at the site of the strand
breakage (1). This covalent topoisomerase-DNA complex (often referred
to as a "cleavable complex") is normally a fleeting catalytic
intermediate. The DNA helix is thought to be broken in the cleavable
complex with the linear integrity of the DNA maintained by the
topoisomerase bridge. Some topoisomerase inhibitors convert these
essential enzymes into cellular poisons by trapping a cleavable complex
as a topoisomerase-drug-DNA ternary complex. Because of this unique
mode of action, these topoisomerase inhibitors are often called
"topoisomerase poisons" (2-4). The poisoning of topoisomerases
results in the inhibition of chromosomal DNA replication, formation of
double strand breaks (DSBs), and subsequent cell death. Although the
formation of the ternary complex is critical for cytotoxicity, these
complexes are completely reversible, and the DNA strands can be
religated. It has been proposed that an active DNA transaction, such as
passage of the DNA replication fork, is required for disruption of the
ternary complex to generate a nonreversible cytotoxic DNA lesion, a DSB
(2-4). However, the exact mechanisms of the replication fork arrest
and the DSB formation are not yet clear.
We have modeled the collision between a Topo IV-norfloxacin (Norf)-DNA
ternary complex and a replication fork in vitro, using the
oriC replication system and either wild type or mutant Topo IV proteins (5). An active strand cleavage and reunion activity of Topo
IV is required for the formation of a ternary complex that can arrest
replication fork progression. Interestingly, the collision between a
topoisomerase-quinolone-DNA ternary complex and a replication fork
converted the ternary complex to a nonreversible form but did not
generate a DSB. An additional step was required for the generation of a
DSB. Thus, the cytotoxicity associated with these topoisomerase poisons
is likely to require two steps: (i) conversion of a
topoisomerase-quinolone-DNA ternary complex to a nonreversible form as
a result of the collision between a ternary complex and a replication
fork and (ii) generation of a DSB by subsequent denaturation of the
topoisomerase in the dead end complex, perhaps by an aborted repair
attempt (5).
To further determine molecular mechanisms of replication fork arrest by
topoisomerase-quinolone-DNA ternary complexes and generation of DSBs,
we investigated the consequences of the collisions between
topoisomerase complexes and DNA helicases. We found that a Topo
IV-Norf-DNA ternary complex inhibited the activities of the DnaB, T7
gene 4 protein, SV40 large T-antigen (T-ag), and UvrD DNA helicases.
Interestingly, unlike replication fork arrest, the strand cleavage and
reunion activity of Topo IV was not required for this general
inhibition of DNA helicases by the ternary complex. The encounter of
UvrD with a Topo IV-Norf-DNA ternary complex converted the ternary
complex to a nonreversible form, whereas that of the DnaB, T7 gene 4 protein, and SV40 T-ag helicases did not. These results suggest that
inhibition of DnaB translocation by the topoisomerase-quinolone-DNA
ternary complex may arrest the replication fork progression but the
collision of DnaB alone is not sufficient to disrupt the
topoisomerase complex and generate DSBs.
DNAs and Proteins--
Two oligonucleotides containing a
40-nucleotide (nt) defined Topo IV binding site (underlined; see
Ref. 6), T440top
(5'-AATTCTTGTTATCGCGACACACTCCTAAAAATCCGGGGTATACCCCGGATTTTTAGGAGTCTAGATACGAGCCGGA-3') and T440-bottom
(5'-AGCTTCCGGCTCGTATCTAGACTCCTAAAAATCCGGGGTATACCCCGGATTTTTAGGAGTGTGTCGCGATAACAAG-3'), were synthesized and annealed to each other to form a short
duplex (referred to as T440). A recombinant M13 containing a 40-base pair (bp) defined Topo IV binding site was constructed by cloning the
T440 duplex DNA into EcoRI/HindIII-digested
M13mp18 (M13-T440). The single-stranded circular DNA of M13-T440 was
prepared as described previously (7, 8).
The wild type, a quinolone-resistant Topo IV, ParC S80L Topo IV, and a
catalytically inactive Topo IV, ParC Y120F Topo IV, were prepared
according to Hiasa et al. (5). Purified Topo IV, DnaB, and
UvrD were the gift of Kenneth Marians (Memorial Sloan-Kettering Cancer
Center). Purified SV40 T-ag, T7 gene 4 protein, and UvrD helicases were
generous gifts of Jerard Hurwitz (Memorial Sloan-Kettering Cancer
Center), Smita Patel (Ohio State University), and Steve Matson
(University of North Carolina), respectively.
Restriction enzymes and DNA modifying enzymes were from New England
Biolabs, Roche Boehringer Mannheim, Stratagene, and Amersham Pharmacia Biotech.
Preparation of Helicase Substrates--
Partial duplex DNAs were
prepared as described previously (9). Briefly, a 62-nt
oligonucleotide, T440C
(5'-GCTCGTATCTAGACTCCTAAAAATCCGGGGTATACCCCGGATTTTTAGGAGTGTGTCGCGAT-3') was synthesized and hybridized to the M13-T440 single-stranded DNA to prepare a primer-template. The oligonucleotide in the
primer-template was then 3'-end-labeled by incorporation of 2 residues
of [32P]dAMP (ICN) with Klenow enzyme. We referred to
this partial duplex DNA as 3'-T440 (Fig. 1A). When required,
a 3'-heteropolymeric tail having an average length of 30 nt was added
to the annealed oligonucleotide using terminal
deoxynucleotidyltransferase (referred to as 3'-T440T) (Fig.
1A). Oligonucleotides T440C and T440CT
(5'-TCGTATCTAGACTCCTAAAAATCCGGGGTATACCCCGGATTTTTAGGAGTGTGTCGCGATATGGTTCAGCTAGCG-3') were 5'-end-labeled using T4 polynucleotide kinase and
[ Topo IV-catalyzed 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 substrate 3'-T440, 300 fmol of Topo IV, and the
indicated concentrations of Norf were incubated at 37 °C for 10 min.
SDS was then added to 1%, and the reaction mixtures were 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 95 °C for 5 min, and then analyzed by
electrophoresis through 8 and 10% polyacrylamide (19:1,
acrylamide/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 paper (Whatman) and autoradiographed
with Hyperfilm MP (Amersham Pharmacia Biotech). Strand displacement was
quantitated by scanning images by a STORM 840 PhosphorImager (Molecular
Dynamics, Inc., Sunnyvale, CA) and scanning films by the Eagle Eye II
Jr. system (Stratagene).
Strand Displacement Assays--
For the DnaB (9, 10) and T7 gene
4 protein helicase assays, standard reaction mixtures (12.5 µl)
contained 40 mM Hepes-KOH (pH 7.6 at 23 °C), 10 mM magnesium acetate, 3.5 mM ATP, 10 mM DTT, 50 µg/ml BSA, 10 fmol (as molecule) of DNA
substrate (either 3'-T440T or 5'-T440T), and the indicated amounts of
Norf. Three hundred fmol (as tetramer) of Topo IV was first bound to
the DNA in a first stage incubation of 5 min at 37 °C. DnaB (250 fmol as hexamer) or T7 gene 4 protein (250 fmol as hexamer) was then added, and the reaction mixtures were incubated during the second stage
for 10 min at 37 °C.
SV40 T-ag helicase activity was assayed (11) in reaction mixtures (12.5 µl) containing 50 mM Tris-HCl (pH 7.5 at 23 °C), 50 mM potassium glutamate, 10 mM magnesium
chloride, 100 mM DTT, 10 µg/ml BSA, 2 mM ATP,
and 10 fmol of DNA substrate (either 3'-T440 or 5'-T440). During the
first stage of incubation, 300 fmol (as tetramer) of Topo IV was bound
to the DNA. Then, 500 fmol (as hexamer) of SV40 T-ag helicase was
added, and the second stage of the incubation was continued at 37 °C
for 10 min.
The helicase activity of UvrD was assessed (9, 12) in standard reaction
mixtures (12.5 µl) containing 50 mM Tris-HCl (pH 7.5 at
23 °C), 10 mM magnesium chloride, 10 mM DTT,
50 µg/ml BSA, and 2 mM ATP. Ten fmol of DNA substrate
(either 3'-T440 or 5'-T440) was added, to which 300 fmol (as tetramer)
of Topo IV protein was bound by incubating at 37 °C for 5 min. After
the first incubation stage, 125 fmol (as dimer) of UvrD helicase was added, and then the reaction mixtures were incubated at 37 °C for an
additional 10 min.
As described above, substrates 5'-T440T and 3'-T440T were used in the
helicase assays for DnaB and T7 gene 4 protein, and 5'-T440 and 3'-T440
were used for SV40 T-ag and UvrD. We obtained virtually identical
results with either substrate (data not shown).
Reactions were terminated by adding EDTA to 25 mM, followed
by the addition of a quarter 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). Gels were dried under
vacuum onto DE81 paper (Whatman) and autoradiographed with Hyperfilm MP
(Amersham Pharmacia Biotech). Strand displacement was quantitated by
scanning images by a STORM 840 PhosphorImager (Molecular Dynamics) and
scanning films by the Eagle Eye II Jr. system (Stratagene).
Reversal Assay for the Topo IV-Quinolone-DNA Ternary
Complex--
The reversibility of the topoisomerase complexes after
their collision with DNA helicases was assessed by combining the strand displacement assay and the reversal assay (5). Reaction mixtures were
assembled as described above, and Topo IV 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 a DNA helicase. This
assay allowed a DNA helicase to collide with a Topo IV-Norf-DNA ternary
complex formed at the defined Topo IV binding site. Reactions were
terminated by adding either EDTA or SDS to 25 mM and 1%,
respectively, and incubating at 37 °C for 5 min. Either SDS or EDTA,
together with proteinase K, was then 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 95 °C for 5 min, and then analyzed by electrophoresis
through 10% polyacrylamide (19:1, acrylamide/bisacrylamide) gels
(140 × 160 × 1.2 mm) at 12 V/cm for 2 h using TBE
buffer. Gels were dried and analyzed as described above.
Partial Duplex DNAs--
In our previous studies (6), we defined a
preferred Topo IV binding site based on the DNA sequences of
Norf-induced, Topo IV-catalyzed DNA cleavage sites on a plasmid DNA.
Two oligonucleotides of 24 and 50 nt in length containing this defined
Topo IV binding site have been designed as perfect palindromes with the
cleavage sites offset 2 nt from the center. The wild type and various
mutant Topo IV proteins bind to these short duplex DNAs, and their
bindings are stimulated by Norf (6).
We constructed a recombinant M13 that contained the 40-bp defined Topo
IV binding site (M13-T440) and prepared its single-stranded DNA.
Partial duplex DNAs were prepared by annealing short oligonucleotides complementary to the cloned Topo IV binding site to the M13-T440 single-stranded DNA. On these partial duplex DNAs, the 40-bp-long Topo
IV binding site was located in the middle of the duplex region (61-64
bp long) flanked by 10-12-bp spacer sequences at both sides (Fig.
1).
First, we examined the stimulation of Topo IV-catalyzed cleavages by
Norf using a partial duplex DNA 3'-T440 as the substrate (Fig.
2). As expected, we observed a unique
cleavage of the annealed oligonucleotide as a result of a
Norf-stimulated, Topo IV-catalyzed cleavage (Fig. 2A). There
was an additional weak cleavage (about 5-15% of the cleaved DNA) that
was observed only at high concentrations of Norf. The stimulation of
Topo IV-catalyzed cleavage of a partial duplex by Norf (Fig.
2B) was very similar to that of a short duplex DNA
containing the same defined Topo IV binding site (6). These results
demonstrated that the Norf-stimulated Topo IV-catalyzed DNA cleavages
(Fig. 2) and the Norf-stimulated Topo IV binding to the DNA (6)
coincided with each other. Thus, we assumed that the Topo IV-catalyzed
DNA cleavages in the presence of various concentrations of Norf shown
in Fig. 2 correlated with the relative amounts of Topo IV-Norf-DNA
ternary complexes formed on the partial duplex DNA. The occupancy of
the DNA substrate by the Topo IV complex determines the probability of
the collision between a DNA helicase and a Topo IV complex. These
results also showed that the long single-stranded DNA region of the
partial duplex DNA neither sequestered the Topo IV protein nor
inhibited Topo IV binding to the short duplex region. In addition, we
did not observe DNA cleavages of the long single-stranded region of the partial duplex DNA (data not shown). These results were consistent with
the observation that Topo IV does not bind to single-stranded DNA (13).
Thus, we concluded that Topo IV could bind to and form both a binary
and ternary complex at the short duplex region of the partial duplex
DNA. Furthermore, because of the length of the duplex region, it is
likely that only one molecule of Topo IV protein could bind to each
partial duplex DNA. Thus, this system allowed us to monitor the
molecular events during the collision of a DNA helicase with a Topo
IV-Norf-DNA ternary complex at the defined position. We therefore
investigated the effects of topoisomerase complexes on the activities
of DNA helicases by using the strand displacement assay.
Topo IV-Quinolone-DNA Ternary Complexes Inhibit Strand Displacement
Activities of DNA Helicases--
Using oriC replication
reconstituted with purified proteins, we have shown that the collision
between a replication fork and a Topo IV-Norf-DNA ternary complex
results in both replication fork arrest and the conversion of a
normally reversible ternary complex to a nonreversible form (5). To
further investigate the mechanisms of replication fork arrest, we
examined the effects of a Topo IV-Norf-DNA ternary complex on DNA
helicases. Among the components of the replication fork, the DNA
helicase is first to encounter any roadblock on the parental duplex DNA
during chromosomal DNA replication. The DnaB protein is the replicative
helicase in Escherichia coli (10). Recent studies have
demonstrated that DnaB forms a hexameric ring around the lagging strand
template while translocating in the 5'
We first assessed the effects of the Topo IV-Norf-DNA ternary complex
on the DnaB helicase using a strand displacement assay (Fig.
3A). DnaB could displace about
60% of a short oligonucleotide from the partial duplex DNA under the
conditions used (Fig. 3A, lane 4).
Norf alone did not affect the activity of DnaB. In the absence of Norf,
the binding of Topo IV inhibited DnaB activity by 50% in this assay
(Fig. 3A, lane 6). A catalytically
inactive Topo IV alone also partially inhibited DnaB activity (data not shown). This may be because of the sensitivity of the DnaB
translocation activity to the presence of proteins bound to duplex DNA
that we have observed during previous studies on the effect of Tus protein on DNA helicases (9). Formation of a Topo IV-Norf-DNA ternary
complex resulted in an 80% inhibition (Fig. 3A,
lane 7). These results showed that a Topo
IV-Norf-DNA ternary complex blocked the passage of the DnaB helicase.
These results suggest that replication fork arrest by a Topo
IV-Norf-DNA ternary complex could be due to the inhibition of DnaB
translocation.
To examine if the inhibition of helicase activity by a Topo IV-Norf-DNA
ternary complex was specific to DnaB or represented a generalized
inhibition of DNA helicases, we performed the strand displacement assay
with other DNA helicases. The T7 gene 4 protein is another hexameric
replicative helicase that translocates in the 5'
We assessed the activity of T7 gene 4 protein in the absence and
presence of Topo IV and Norf (Fig. 3B). The T7 gene 4 protein-catalyzed strand displacement activity was severely inhibited
(greater than 80%) only when both Topo IV and Norf were present in the
reaction mixtures (Fig. 3B, lane 6).
Neither Topo IV nor Norf alone affected the activity of T7 gene 4 protein, and this helicase displaced about 70% of the annealed
oligonucleotide under these conditions (Fig. 3B,
lane 3). These results demonstrated that
inhibition of helicase activities by a Topo IV-Norf-DNA ternary complex
was not specific to DnaB.
In a cleavable complex, Topo IV forms a covalent enzyme-DNA bond at the
5'-end of the cleaved DNA strand. Although the topoisomerase-DNA complex is symmetrical, DNA helicases that translocate in the 3'
We performed the strand displacement assay with SV40 T-ag and found
that about 30% of the annealed oligonucleotide was displaced (Fig.
3C, lane 3). When Topo IV formed a
ternary complex with Norf and the DNA, the activity of SV40 T-ag was
inhibited greater than 80% (Fig. 3C, lane
6). Unlike DnaB, Topo IV alone had no effect on SV40 T-ag.
However, Norf alone exhibited an inhibitory effect (about 50%) on SV40
T-ag (Fig. 3C, lane 4). We believed that this inhibition was nonspecific inhibition of protein binding to
DNA by Norf. We have noted that Norf specifically inhibits the
activities of DNA gyrase and Topo IV in the oriC replication system, but this drug has an inhibitory effect on the binding of DnaA
proteins to oriC when it is present at high
concentrations.2 Thus, a Topo
IV-Norf-DNA ternary complex could arrest replicative helicases
approaching from either direction.
The three DNA helicases described above are hexameric
replicative helicases that act
processively during the DNA unwinding reaction. In order to determine
if the nature of helicase action modulated interactions between Topo
IV-Norf-DNA ternary complexes and DNA helicases, we examined the effect
of a ternary complex on UvrD, a repair helicase. UvrD is thought to
function as a dimer on the DNA and catalyzes the DNA unwinding reaction
in a distributive manner (12). Unlike DnaB and SV40 T-ag, neither Topo
IV nor Norf alone had an effect on UvrD-catalyzed strand displacement (Fig. 3D). UvrD displaced about 70% of the annealed
oligonucleotide under these conditions (Fig. 3D,
lane 3). However, its activity was severely
inhibited (greater than 80%) by a Topo IV-Norf-DNA ternary complex
formed in the duplex region (Fig. 3D, lane
6). These results showed that a topoisomerase-quinolone-DNA
ternary complex acted as a roadblock that inhibited the translocation of DNA helicases in a generalized manner.
Topo IV-Norf-DNA Ternary Complexes Are Still Reversible after
Collision with Replicative Helicases--
We have demonstrated that
the collision between a replication fork and a Topo IV-Norf-DNA ternary
complex results not only in replication fork arrest but also in the
conversion of the ternary complex to a nonreversible form (5). In light
of this observation, we examined if the encounter of a Topo IV-Norf-DNA
ternary complex with a replicative helicase converts the ternary
complex to a nonreversible form. We modified the strand displacement
assay so that we could also assess the reversibility of the
topoisomerase complex after its collision with a DNA helicase (see
"Materials and Methods"). After incubation with a DNA helicase in
the presence of a ternary complex, reaction mixtures were terminated by
adding either EDTA or SDS. EDTA reverses ternary complex formation, and the topoisomerase religates the DNA strands (21). In contrast, SDS
denatures the enzyme in the ternary complex, resulting in the
generation of a DSB. The DNA products were deproteinized, heat-denatured, and analyzed by electrophoresis. If the ternary complex
is still reversible after collision, incubation with EDTA will reverse
the ternary complex, and the annealed oligonucleotide will remain
intact. However, if the ternary complex is no longer reversible after
collision, even after the incubation with EDTA, denaturation of Topo IV
will result in the formation of DSBs and the cleavage of the annealed oligonucleotide.
We analyzed the DNA products by electrophoresis through polyacrylamide
gels (Fig. 4). Collisions of a Topo
IV-Norf-DNA ternary complex with DnaB (Fig. 4, lanes
2-5), T7 gene 4 protein (Fig. 4, lanes
6-9), and SV40 T-ag (Fig. 4, lanes
10-13) had no effect on the reversibility of the ternary
complexes (Fig. 4, lanes 5, 9, and
13). These results demonstrated that, although a Topo
IV-Norf-DNA ternary complex blocked the passages of both the DnaB
helicase and a replication fork, the consequence of the collision of a ternary complex with the DnaB helicase alone was significantly different from that with the same helicase when it was part of the
replication fork. The encounter of a replicative helicase alone, unlike
that of a replication fork, was not sufficient to generate a cytotoxic
DNA lesion by converting a ternary complex to a nonreversible form.
This may be due to the distinct characteristics of the DnaB protein
when it is incorporated in a replication fork and when it functions by
itself. It has been demonstrated that the DnaB helicase moves much
faster when it establishes an interaction with the DNA polymerase III
holoenzyme at the replication fork (22). Alternatively, it is possible
that the encounter of a topoisomerase-quinolone-DNA ternary complex
with a DNA polymerase, but not a DNA helicase, causes the conversion of
the ternary complex to a nonreversible form.
Collision between a Topo IV-Norf-DNA Ternary Complex and a UvrD
Helicase Results in the Conversion of the Ternary Complex to a
Nonreversible Form--
We also assessed the effects of the encounter
of a Topo IV-Norf-DNA ternary complex with the UvrD helicase on the
reversibility of the ternary complex. In contrast to three replicative
helicases (Fig. 4), the normally reversible ternary complex became
completely nonreversible when the UvrD helicase collided with a Topo
IV-Norf-DNA ternary complex (Fig. 5). We
used two independent preparations of wild type Topo IV and UvrD and
obtained virtually identical results (data not shown). These results
were consistent with the previous observations by Howard et
al. (23), demonstrating that the collision of UvrD with a T4
topoisomerase-m-AMSA-DNA ternary complex converted the
ternary complex to a nonreversible form. In contrast, ternary complexes
formed with a quinolone-resistant Topo IV, ParC S80L Topo IV, were
still reversible (Fig. 5, lanes 9 and
10). This quinolone-resistant mutation seems to prevent Topo
IV from forming a ternary complex that can arrest the progression of a
replication fork (5) and DNA helicases (see below). We did not observed
any DNA cleavages when a catalytically inactive Topo IV, ParC Y120F
Topo IV, was used (Fig. 5, lanes 11 and
12). Thus, unlike DnaB and other hexameric helicases, the
collision between UvrD and a topoisomerase-quinolone-DNA ternary
complex disrupted the topoisomerase complex and converted a normally
reversible ternary complex to a nonreversible form.
Inhibition of Helicase Activities Does Not Require the Formation of
a Covalent Topoisomerase-DNA Complex--
We have shown here that Topo
IV-Norf-DNA ternary complex exhibits a general inhibition of DNA
helicases (Fig. 3). However, the encounter of DnaB, unlike that of a
replication fork (5), with a Topo IV-Norf-DNA ternary complex did not
affect the reversibility of the ternary complex (Fig. 4). On the other
hand, the UvrD helicase could, upon collision, convert a Topo
IV-Norf-DNA ternary complex to a nonreversible form (Fig. 5). To
determine if there are differences between the mechanisms of
replication arrest and helicase inhibition by a
topoisomerase-quinolone-DNA ternary complex, we further investigated the requirements for helicase inhibition by topoisomerase complexes. To
avoid partial inhibitions of DnaB- and SV40 T-ag-catalyzed strand
displacement activities, by Topo IV alone (Fig. 3A) and Norf
alone (Fig. 3C), respectively, we used T7 gene 4 protein and
UvrD helicases in the following experiments.
Helicase activities were measured in the presence of the wild type Topo
IV, a quinolone-resistant Topo IV, ParC S80L Topo IV, and a
catalytically inactive Topo IV, ParC Y120F Topo IV, either in the
absence or presence of Norf (Fig. 6). In
the absence of Norf, neither the wild type nor mutant Topo IV affected
the activities of T7 gene 4 protein protein (Fig. 6A) and
UvrD (Fig. 6B). Formation of a ternary complex with the wild
type Topo IV inhibited activities of both helicases (Fig. 6,
A and B, lane 4). A
quinolone-resistant mutation, S80L, in the ParC subunit, significantly
reduced the inhibitory effects of the ternary complex on DNA helicases
(Fig. 6, A and B, lane 6).
Interestingly, a ternary complex formed with ParC Y120F Topo IV
inhibited the activities of T7 gene 4 protein (Fig. 6A,
lane 8) and UvrD helicases (Fig. 6B,
lane 8). These results demonstrated that the
strand cleavage and reunion activity of Topo IV was not required for
the inhibition of T7 gene 4 protein and UvrD helicases by the Topo
IV-Norf-DNA ternary complex. Note that the ParC Y120F Topo IV-Norf-DNA
ternary complex cannot arrest replication fork progression (5).
Furthermore, these results suggest that replication fork arrest may be
due to the inhibition of the DnaB helicase by the
topoisomerase-quinolone-DNA ternary complex. However, collisions
between the ternary complexes and the DnaB helicase do not result in
the disruption of the topoisomerase complexes and the formation of
DSBs.
Topoisomerases catalyze strand cleavage and rejoining reactions by
forming a covalent linkage between the enzyme and the DNA at the site
of strand breakage (1). The cleavable complex, a covalent
topoisomerase-DNA complex, is normally a fleeting catalytic intermediate. The steady-state level of the cleavable complex depends
on the cleavage religation equilibrium. If the equilibrium is shifted
to either stimulate strand cleavage or inhibit religation, the
cleavable complex that contains broken DNA strands can persist on the
DNA, as if the topoisomerase were trapped in the cleavable complex.
Thus, although topoisomerases are essential enzymes in DNA metabolism,
these enzymes pose a potential danger to the genome (2-4).
DNA gyrase was recognized, shortly after its discovery (24-26), as the
cellular target of the quinolone antibacterial drugs (24, 27). Kreuzer
and Cozzarelli (21) have shown that quinolones block DNA replication
not by depriving the cell of DNA gyrase but by converting DNA gyrase
into a poison of DNA replication. The poisoning of topoisomerases is
mediated by trapping of a cleavable complex as a topoisomerase-drug-DNA
ternary complex. Disruption of the ternary complexes leads to the
generation of DSBs and subsequent cell death (2-4). The cytotoxicity
of topoisomerase poisons is correlated with the appearance of DSBs in
the genome. Anticancer drugs that target human type II topoisomerases
also convert their targets into poisons (28).
A number of studies have investigated the molecular mechanisms of cell
killing as a result of the action of topoisomerase poisons in both
prokaryotic and eukaryotic systems. Formation of topoisomerase-drug-DNA
ternary complexes is necessary, but these complexes are normally
reversible. An additional step, an active DNA transaction, is required
to disrupt the topoisomerase complex and to generate an irreversible
cytotoxic DNA lesion (2-4). Passage of the replication machinery has
been the leading candidate for this disruptive process that triggers
lethal events. It has been proposed that when DNA replication occurs in
the presence of topoisomerase-drug-DNA ternary complexes, the collision
between a replication fork and a ternary complex results in the
disruption of topoisomerase complex and the generation of DSBs (2-4).
The recent demonstration of the disruption of an
m-AMSA-induced T4 topoisomerase-DNA complex by the UvrD
helicase supports this model (23). However, the molecular events during
this collision and the mechanism of DSB formation are not yet clear.
We have studied the consequences of the encounter between a replication
fork and a Topo IV-Norf-DNA ternary complex using the oriC
replication system (5). Three mutant Topo IVs, ParC Y120F Topo IV,
which is catalytically inactive, ParC S80L Topo IV, which is resistant
to inhibition by quinolones such as Norf, and the double mutant, ParC
S80L,Y120F Topo IV, were used. Only the wild type enzyme was capable of
forming a ternary complex with the DNA and Norf that blocked
replication fork progression. The ternary complexes formed by a
catalytically inactive and a quinolone-resistant Topo IV could not halt
progression of the replication fork. Thus, an active strand cleavage
and reunion activity of Topo IV was required for the arrest of
replication fork progression in vitro by the Topo
IV-Norf-DNA ternary complex (5). Similar studies have been performed
demonstrating that a ternary complex formed by wild type DNA gyrase
could block transcription by bacterial RNA polymerase (29).
Here, we continued our studies on the mechanism of replication fork
arrest by topoisomerase-quinolone-DNA ternary complexes. Replication
forks are composed of a DNA helicase, DNA polymerase, and primase. The
replicative helicase is positioned at the front of the replication fork
and collides with any roadblocks on the DNA. We investigated the
effects of the Topo IV-Norf-DNA ternary complex on DNA helicases. We
found that a Topo IV-Norf-DNA ternary complex could block the passage
of DnaB, T7 gene 4 protein, SV40 T-ag, and UvrD (Fig. 3).
Interestingly, this general inhibition of DNA helicases did not require
the strand cleavage and reunion activity of Topo IV (Fig. 6).
Furthermore, we found that unlike the case with a replication fork,
collisions of Topo IV-Norf-DNA ternary complexes with replicative
helicases did not affect the reversibility of the ternary complex (Fig.
4). In contrast, the UvrD helicase could convert the ternary complex to
a nonreversible form (Fig. 5).
The DnaB protein is the replicative helicase in E. coli
(10). We showed here that a Topo IV-Norf-DNA ternary complex formed at
a defined Topo IV binding site could halt the passage of DnaB and other
hexameric replicative helicases (Fig. 3A). Inhibition of
DnaB translocation may lead to replication fork arrest. Does the
encounter of the DnaB helicase alone with a Topo IV-Norf-DNA ternary
complex result in the disruption of the topoisomerase complex and the
generation of a cytotoxic DNA lesion? This is not likely to be the
case. Because the collision between a Topo IV-Norf-DNA ternary complex
and the DnaB helicase did not affect the reversibility of the ternary
complex (Fig. 4). In contrast, the encounter of a Topo IV-Norf-DNA
ternary complex with a replication fork converted the ternary complex
to a nonreversible form (5). Thus, we concluded that, although the
inhibition of DnaB translocation may cause replication fork arrest, the
collision of DnaB alone is not sufficient to disrupt the
topoisomerase-quinolone-DNA ternary complexes and generate DSBs. We
have previously noted that the DnaB helicase changes its sensitivity to
the Tus/Ter complex depending on its interaction with other
primosomal proteins (9). Kim et al. (22) have demonstrated
that the interaction between DnaB and the DNA polymerase III holoenzyme
stimulates the rate of DnaB-catalyzed unwinding of the duplex DNA.
Observations presented here also showed that there are qualitative
differences in the characteristics of DnaB depending on its association
with other replication fork components.
What are the differences between the DnaB helicase and a replication
fork? Because of the functional interaction between DnaB and the DNA
polymerase III holoenzyme, the replication fork progresses much faster
than DnaB alone (22). In addition, the mass of a replication fork is
larger than that of a DnaB hexamer. These may be sufficient to account
for the abilities of the replication fork to move through a ternary
complex formed with a catalytically inactive Topo IV and to convert the
wild type Topo IV-Norf-DNA ternary complex to a nonreversible form upon
its collision (5). Another possibility is that DNA polymerase is the
active agent that converts the topoisomerase-quinolone-DNA ternary
complex to a nonreversible form. If this is the case, when a
replication fork encounters a ternary complex, the translocation and
unwinding of DnaB is presumably halted, and the helicase must
disassociate from the DNA. DNA polymerase III continues its progression
until it physically contacts the topoisomerase in the ternary complex, and it is this collision that disrupts the topoisomerase complex and
converts the ternary complex to a nonreversible form.
We used a catalytically inactive Topo IV, ParC Y120F Topo IV, in the
helicase assays and found that the strand cleavage and reunion activity
of Topo IV was not required for the inhibition of DNA helicases. The
inhibition of DNA helicases by ParC Y120F Topo IV was still dependent
on the presence of Norf (Fig. 6). These results support the previous
observations demonstrating that quinolones can act on a topoisomerase
and form a ternary complex with the enzyme and the DNA prior to the
strand cleavage reaction (6, 29, 30). It will be interesting to
investigate if interactions between the topoisomerase and the drug are
identical before and after the strand cleavage reaction.
UvrD was unique among the DNA helicases we examined. Only the encounter
of UvrD with a Topo IV-Norf-DNA ternary complex resulted in the
conversion of the ternary complex to a nonreversible form (Fig. 5). It
is not clear, at this point, what makes UvrD different from other
helicases. One possible explanation for this difference is the distinct
modes of helicase action. UvrD is thought to function as a dimer (31),
whereas the three other helicases we used in these studies are
hexameric helicases (14-16). Thus, UvrD, but not the hexameric DNA
helicases, may reach closer to the topoisomerase in the ternary complex
and disrupt its conformation. Another possibility, although it is also
related to the molecular mechanisms of helicase action during the
unwinding of duplex DNA, is that the mode of DNA binding and the
stability of the DNA helicase on the DNA are different among helicases.
Some DNA helicases, such as UvrD, may interact with both
single-stranded and double-stranded DNAs at the junction between the
unwound single-stranded DNA and the parental double-stranded DNA (31).
On the other hand, other DNA helicases, such as DnaB and T7 gene 4 protein, may interact with only single-stranded DNA at the junction
(14, 16, 32).
Both a replication fork assembled at oriC and a repair
helicase UvrD can convert a Topo IV-Norf-DNA ternary complex to a
nonreversible form upon their collisions. It is not clear if both
affect the ternary complex in the same manner. We suspect that dead end
topoisomerase complexes generated by replication forks may be different
from those generated by the UvrD helicase. Collisions between
topoisomerase-quinolone-DNA ternary complexes and replication forks do
not generate any strand breaks (5). Denaturation of the topoisomerase
was required for the generation of DSBs. In contrast, the collision of
UvrD and a T4 topoisomerase-m-AMSA-DNA ternary complex
results in the generation of a single strand break (SSB) and leads to
the release of a single-stranded DNA from the frozen cleavable complex
(23). We also detected a short DNA strand released from nonreversible Topo IV complexes without denaturing the
topoisomerase.3 However, the
generation of SSBs was observed only at a portion (less than 25%) of
the frozen cleavable complexes of Topo IV, whereas the conversion of a
Topo IV-Norf-DNA ternary complex to a nonreversible form was nearly
complete (Fig. 5, lanes 7 and 8).
Thus, it is not clear if the conversion of a Topo IV-Norf-DNA ternary
complex to a dead end complex is due to the SSB formation at the
ternary complex. Alternatively, differences in stability of the ternary
complexes formed with different topoisomerases and drugs may be
attributable to the efficiency of the generation of SSBs when UvrDs
collide with the topoisomerase-quinolone-DNA ternary complexes.
Let us assume that the mechanisms of disruption of the
topoisomerase-quinolone-DNA ternary complexes by the replication fork and UvrD are distinct. This might reflect two different biological processes in the cells. It is interesting to speculate that the encounter of a topoisomerase-quinolone-DNA ternary complex with the
UvrD helicase on the chromosome may occur during the removal of the
topoisomerase and repair of DNA damage, whereas that with the
replication fork may trigger the drug-induced lethal event. A
UvrD-directed repair system may recognize the
topoisomerase-quinolone-DNA ternary complexes on the chromosome and
remove them to prevent the replication fork from colliding with these
complexes. If this is the case, the distinct effects of a
uvrD mutant on the lethality of DNA gyrase- and Topo
IV-targeted quinolone drug action in E. coli (33) may be
explained by the time given for the operation of a UvrD-directed repair
system and the positioning of topoisomerases relative to the advancing
replication forks. It has been suggested that DNA gyrase acts ahead of
the advancing replication forks and that Topo IV acts behind them in
E. coli (34, 35). Thus, ternary complexes formed with Topo
IV have a greater chance of being removed by repair machinery that
includes UvrD before their collisions with replication forks than those
with DNA gyrase.
We thank Dr. Kenneth Marians for gifts of
purified proteins, continuous discussions, and critical comments on
this manuscript. We also thank Drs. Jerard Hurwitz, Smita Patel, and
Steve Matson for gifts of purified DNA helicases.
*
These studies were supported by National Institutes of
Health Grant GM59465-01, American Cancer Society Grant
IRG-58-001-40-IRG-15, and the Minnesota Medical Foundation.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
H. Hiasa and K. J. Marians, unpublished data.
3
M. E. Shea and H. Hiasa, unpublished data.
The abbreviations used are:
Topo, topoisomerase;
bp, base pair(s);
BSA, bovine serum albumin;
DSB, double strand break;
DTT, dithiothreitol;
nt, nucleotide(s);
SSB, single strand break;
Norf, norfloxacin;
T-ag, T-antigen;
m-AMSA, 4'-(9-acridinylamino)methanesalfon-manisidide.
Interactions between DNA Helicases and Frozen Topoisomerase
IV-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
-32P]ATP (ICN) and then hybridized to the M13-T440
single-stranded DNA to prepare the 5'-end-labeled partial duplex DNAs.
These substrates were referred to as 5'-T440 and 5'-T440T, respectively
(Fig. 1B).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (8K):
[in a new window]
Fig. 1.
Schematic presentations of partial duplex
DNAs. Annealed oligonucleotide was either 3'-end-labeled by Klenow
enzyme (A) or 5'-end-labeled by T4 polynucleotide kinase
(B). Closed circular and
dotted lines represent 32P-nucleotide
and nonhybridized tail, respectively. The 40-nt defined Topo IV binding
site located in the middle of the duplex region is shown as an
open rectangle. For details, see "Materials
and Methods."
View larger version (14K):
[in a new window]
Fig. 2.
Norf-induced, Topo IV-catalyzed cleavage of a
partial duplex DNA. A, DNA cleavage assays using wild
type Topo IV, substrate 3'-T440, and the indicated concentrations of
Norf were performed and analyzed as described under "Materials and
Methods." B, the extent of the cleavage as a function of
the Norf concentration was quantitated using a PhosphorImager.
3' direction (10,
14-16).
View larger version (46K):
[in a new window]
Fig. 3.
Topo IV-Norf-DNA ternary complexes inhibit
strand displacement activities of DnaB, T7 gene 4 protein, SV40 T-ag,
and UvrD helicases. Helicase assays for DnaB (A), T7
gene 4 protein (B), SV40 T-ag (C), and UvrD
(D) in the presence and absence of Topo IV and Norf, as
indicated, were performed, and the DNA products were analyzed as
described under "Materials and Methods." Lane
1 in all panels shows heat-denatured substrate.
DnaB (as hexamer), T7 gene 4 protein (as hexamer), SV40 T-ag (as
hexamer), UvrD (as dimer), and Topo IV (as tetramer) were present at
25-, 25-, 50-, 12.5-, and 30-fold molar excess over substrate,
respectively. Norf was at 400 µM when present.
3' direction (17,
18). Thus, biochemically, structurally, and functionally, DnaB and T7
gene 4 protein are homologous to each other.
5'
direction would encounter the covalent linkage between the
topoisomerase and the DNA at the DSB in the complex. On the other hand,
DNA helicases that translocate in the 5' 3' direction would reach
an end of the DNA strand. Thus, it is possible that helicase inhibition
by the Topo IV-Norf-DNA ternary complex is dependent on the direction
in which each DNA helicase approaches the topoisomerase in the
ternary complex (i.e. a topoisomerase-quinolone-DNA ternary
complex could block DNA helicases that approach from one direction but
not from the other). To test this directly, the effect of a Topo
IV-Norf-DNA ternary complex on SV40 T-ag was examined. SV40 T-ag also
functions in a hexameric form like DnaB and T7 gene 4 protein, but this
helicase translocates in the opposite direction (3' 5') on a DNA
strand (19, 20).
View larger version (47K):
[in a new window]
Fig. 4.
Collisions between a Topo IV-Norf-DNA ternary
complex and replicative helicases do not convert the ternary complex to
a nonreversible form. Reversal assays for Topo IV-Norf-DNA ternary
complexes in the absence and presence of DnaB (lanes
2-5), T7 gene 4 protein (lanes 6-9),
and SV40 T-ag (lanes 10-13), as indicated, were
performed and analyzed as described under "Materials and Methods."
Reaction mixtures shown in lanes 1-9 contained
5'-T440T as a substrate and were analyzed through 10% polyacrylamide
gels, and those in lanes 10-13 contained 3'-T440
and were analyzed through 8% polyacrylamide gels. DnaB (as hexamer),
T7 gene 4 protein (as hexamer), SV40 T-ag (as hexamer), and Topo IV (as
tetramer) were present at 25-, 25-, 50-, and 30-fold molar excess over
substrate, respectively. Norf was present at 400 µM.
Lane 1 shows heat-denatured substrate.
E and S represent the reactions terminated by the
addition of EDTA and SDS, respectively.
View larger version (63K):
[in a new window]
Fig. 5.
UvrD converts a Topo IV-Norf-DNA ternary
complex to a nonreversible form upon its collision with the ternary
complex. Reversal assays for the wild type and mutant Topo IV
complexes in the absence and presence of Norf and the UvrD helicase, as
indicated, were performed and analyzed as described under "Materials
and Methods." Lane 1 in all panels was
heat-denatured substrate. UvrD (as dimer) and Topo IV proteins (as
tetramer) were present at 12.5- and 30-fold molar excess over
substrate, respectively. Norf was at 400µM when present.
wt, wild type Topo IV; S80L, ParC S80L Topo IV;
Y120F, ParC Y120F Topo IV. E and S are
as in the legend to Fig. 4.
View larger version (36K):
[in a new window]
Fig. 6.
Helicase inhibition by the Topo IV-Norf-DNA
ternary complex does not require the cleavage and reunion activity of
Topo IV. Helicase assays for T7 gene 4 protein (A) and
UvrD (B) in the presence and absence of the wild type and
mutant Topo IV and Norf, as indicated, were performed, and the DNA
products were analyzed as described under "Materials and Methods."
Lane 1 in all panels was heat-denatured
substrate. T7 gene 4 protein (as hexamer), UvrD (as dimer), and Topo IV
(as tetramer) were present at 25-, 12.5-, and 30-fold molar excess over
substrate, respectively. Norf was at 400 µM when present.
Abbreviations are as in the legend to Fig. 5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Member of the University of Minnesota Cancer Center.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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