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J. Biol. Chem., Vol. 275, Issue 44, 34780-34786, November 3, 2000
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From the Department of Pharmacology, University of Minnesota
Medical School, Minneapolis, Minnesota 55455
Received for publication, February 28, 2000, and in revised form, July 21, 2000
The ability of DNA gyrase (Gyr) to wrap the DNA
strand around itself allows Gyr to introduce negative supercoils into
DNA molecules. It has been demonstrated that the deletion of the
C-terminal DNA-binding domain of the GyrA subunit abolishes the ability
of Gyr to wrap the DNA strand and catalyze the supercoiling reaction (Kampranis, S. C., and Maxwell, A. (1996) Proc. Natl. Acad.
Sci. U. S. A. 93, 14416-14421). By using this mutant Gyr, Gyr
(A59), we have studied effects of Gyr-mediated wrapping of the DNA
strand on its replicative function and its interaction with the
quinolone antibacterial drugs. We find that Gyr (A59) can
support oriC DNA replication in vitro. However,
Gyr (A59)-catalyzed decatenation activity is not efficient enough to
complete the decatenation of replicating daughter DNA molecules. As is
the case with topoisomerase IV, the active cleavage and reunion
activity of Gyr is required for the formation of the ternary complex
that can arrest replication fork progression in vitro.
Although the quinolone drugs stimulate the covalent Gyr (A59)-DNA
complex formation, the Gyr (A59)-quinolone-DNA ternary complexes do not
arrest the progression of replication forks. Thus, the
quinolone-induced covalent topoisomerase-DNA complex formation is
necessary but not sufficient to cause the inhibition of DNA
replication. We also assess the stability of ternary complexes formed
with Gyr (A59), the wild type Gyr, or topoisomerase IV. The ternary
complexes formed with Gyr (A59) are more sensitive to salt than those
formed with either the wild type Gyr or topoisomerase IV. Furthermore,
a competition experiment demonstrates that the ternary complexes formed
with Gyr (A59) readily disassociate from the DNA, whereas the ternary
complexes formed with either the wild type Gyr or topoisomerase IV
remain stably bound. Thus, Gyr-mediated wrapping of the DNA strand is required for the formation of the stable Gyr-quinolone-DNA ternary complex that can arrest replication fork progression.
Topoisomerases are ubiquitous enzymes that alter the linking
number of DNA. As such, they play essential roles in every aspect of
DNA metabolism (1). Type II topoisomerases are well conserved throughout the evolution and form a large protein family. DNA gyrase
(Gyr)1 is unique among the
type II topoisomerases. Gyr wraps the DNA strand around itself when it
binds to the DNA, and this unique mode of DNA binding allows Gyr to
introduce the negative supercoils into DNA molecules (1).
Kampranis and Maxwell (2) have recently demonstrated that Gyr can be
converted into a conventional type II topoisomerase by deleting the C
terminal of the DNA binding domain of the GyrA subunit. This mutant
Gyr, Gyr (A59), no longer wraps the DNA strand around itself and cannot
catalyze the supercoiling reaction. However, Gyr (A59) is still capable
of catalyzing decatenation and relaxation reactions. In addition, Gyr
(A59) can partially complement the phenotype of a
parCts mutant (2). It is suggested that Gyr (A59)
could substitute for the cellular function of topoisomerase IV (Topo IV).
Topoisomerases are the cellular targets for clinically important
antibacterial and anticancer drugs (3-5). These topoisomerase inhibitors convert topoisomerases into poisons by trapping the covalent
topoisomerase-DNA complex (also called the "cleavable complex") as
a topoisomerase-drug-DNA ternary complex. In the ternary complex, the
DNA helix is broken, and the topoisomerase bridge maintains the linear
integrity of the DNA. Although the ternary complex formation is
critical for the cytotoxicity of these topoisomerase inhibitors,
ternary complexes are normally reversible. It has been proposed that an
active DNA transaction, such as the passage of replication forks, is
required for the disruption of ternary complexes and the generation of
nonreversible, cytotoxic DNA lesions (3-5).
We have studied the molecular events during the collision between a
replication fork and a Topo IV-norfloxacin (Norf)-DNA ternary complex
in vitro, using the oriC replication system
reconstituted with purified proteins (6). The active strand cleavage
and reunion activity of Topo IV are required for the formation of the
ternary complex that can arrest replication fork progression. Interestingly, the collision between a replication fork and a Topo
IV-Norf-DNA ternary complex converts the ternary complex to a
nonreversible form but does not generate a double-strand break (DSB).
An additional step is required for the generation of DSBs. Thus, the
cytotoxicity associated with this class of topoisomerase inhibitors
results from a two-step process as follows: the conversion of a
topoisomerase-quinolone-DNA ternary complex to a nonreversible form and
the generation of a DSB by subsequent denaturation of the topoisomerase
in the dead-end complex (6).
To investigate further the molecular mechanism of replication fork
arrest by the topoisomerase-quinolone-DNA ternary complex, we modeled
the collision between a replication fork and a Gyr-Norf-DNA ternary
complex, using the wild type and two mutant Gyr proteins, Gyr (A59) and
Gyr (A, Y122F). Gyr (A59) cannot wrap the DNA strand around itself (2)
and Gyr (A, Y122F) is a catalytically inactive mutant. Norf stimulated
both the wild type Gyr- and Gyr (A59)-catalyzed DNA cleavages. No DNA
cleavage was detected when Gyr (A, Y122F) was used. The ternary
complexes formed with either Gyr (A, Y122F) or Gyr (A59) did not block
the progression of replication forks in vitro. These results
demonstrated that the formation of the quinolone-induced covalent
Gyr-DNA complex was necessary but not sufficient to arrest the
replication fork progression.
We also assessed the stability of ternary complexes formed with either
Gyr (A59), the wild type Gyr, or Topo IV. We found that Gyr
(A59)-Norf-DNA ternary complexes were more sensitive to salt than
Gyr-Norf-DNA and Topo IV-Norf-DNA ternary complexes. A competition
experiment showed that the Gyr (A59)-Norf-DNA ternary complexes readily
disassociated from the DNA, whereas both Gyr-Norf-DNA and Topo
IV-Norf-DNA ternary complexes remained stably bound. These results
demonstrated that the ternary complexes formed with Gyr (A59) were less
stable than those formed with either the wild type Gyr or Topo IV.
Thus, Gyr-mediated wrapping of the DNA strand was required for the
formation of stable Gyr-quinolone-DNA ternary complexes that could
arrest the progression of replication forks. These results indicated
that the stability of the topoisomerase-quinolone-DNA ternary complex
might determine if the collision between a replication fork and a
topoisomerase-quinolone-DNA ternary complex would result in the
inhibition of DNA replication.
Replication Proteins--
Escherichia coli DNA
replication proteins, generous gifts of Kenneth Marians (Memorial
Sloan-Kettering Cancer Center), were as described previously
(7-9).
A truncated GyrA subunit, GyrA(59) (2), was overexpressed and
purified according to Shea and Hiasa (10) with a slight modification.
Briefly, after the chromatography on Q-Sepharose FF (Amersham Pharmacia
Biotech) and DEAE-cellulose DE52 (Whatman), the protein fraction was
loaded onto a Superose 6 HR column (Amersham Pharmacia Biotech)
equilibrated with Buffer A (25 mM Tris-HCl, pH 7.5 (4 °C), 2.5 mM dithiothreitol (DTT), 0.5 mM
EDTA, 10% glycerol, and 500 mM NaCl). The peak of the
59-kDa protein was pooled and dialyzed against Buffer B (50 mM Tris-HCl, pH 7.5 (4 °C), 5 mM DTT, 1 mM EDTA, 40% glycerol, and 100 mM NaCl). This
final preparation of GyrA(59) was greater than 95% homogeneous
for a single band on SDS-polyacrylamide gel electrophoresis (data not shown).
The active site Tyr of the GyrA subunit was replaced with Phe by the
site-directed mutagenesis of the cloned gyrA gene using the
overlap extension polymerase chain reaction
technique.2 The GyrA (Y122F)
protein was overexpressed and purified according to the same protocol
used for the purification of GyrA(59). The final preparation of
GyrA (Y122F) was greater than 95% homogeneous for a single band on
SDS-polyacrylamide gel electrophoresis (data not shown).
GyrA(59) and GyrA (Y122F) were mixed with the wild type GyrB to
reconstitute Gyr (A59) and Gyr (A, Y122F), respectively. The wild type
Gyr was also prepared by mixing the wild type GyrA and GyrB proteins.
DNAs--
Two types of oriC plasmids, pBROTB535 type
I (11) and pBROTB353 type I (12), were prepared according to Hiasa and
Marians (13).
oriC DNA Replication--
The standard oriC DNA
replication assay was performed as described previously (11, 13),
except that phosphocreatine and creatine kinase were omitted from the
reaction mixtures. Reaction mixtures (12.5 µl) containing the
oriC plasmid pBROTB535 type I DNA, DnaA, DnaB, DnaC, DnaG,
HU protein, single-stranded DNA-binding protein, the DNA polymerase III
holoenzyme, and topoisomerase were incubated at 30 °C for 10 min.
After terminating the reaction by adding EDTA to 25 mM,
nucleotide incorporation was measured, and the replication products
were analyzed by the native agarose gel electrophoresis as described by
Hiasa and Marians (11). Any changes in the reaction conditions are
indicated in the figure legends.
Staged Nascent Chain Elongation during oriC DNA
Replication--
The modified pulse-chase protocol was performed,
using pBROTB535 type I DNA as the DNA template, as described previously
(6, 14). Any changes in the reaction conditions are indicated in the
figure legends.
Topoisomerase-catalyzed DNA Cleavage Assay--
pBROTB353 type I
DNA was linearized by digesting with the EcoRI restriction
endonuclease and 3'-end-labeled by incorporation of 2 residues of
[32P]dAMP with Klenow enzyme. This 3'-end-labeled linear
plasmid DNA was used as a substrate.
Standard reaction mixtures (20 µl) containing 40 mM
HEPES-KOH (pH 7.6), 10 mM MgOAc2, 10 mM DTT, 50 µg/ml bovine serum albumin, 2 mM
ATP, 20 fmol (as molecule) of 32P-labeled linear pBROTB353
type I DNA, the indicated concentrations of Norf, and the indicated
amounts of either the wild type or a mutant Gyr were incubated at
37 °C for 10 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) and then analyzed
by electrophoresis through vertical 1% agarose (Seakem ME, FMC) gels
(14 × 10 × 0.3 cm) at 4 V/cm for 4 h or at 2 V/cm for
8 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). Gels were dried under vacuum onto GR3 papers (Whatman) and
autoradiographed with Hyperfilm MP films (Amersham Pharmacia Biotech).
Amounts of cleaved DNA were quantitated by scanning images by a STORM 840 PhosphorImager (Molecular Dynamics).
Stability Assay for the Topoisomerase-Quinolone-DNA Ternary
Complex--
Reaction mixtures were assembled as described in the
previous section, and 300 fmol (as tetramer) of Gyr, Gyr (A59), or Topo IV was bound to 20 fmol (as molecule) of 32P-labeled linear
pBROTB353 type I DNA in the presence of 100 µM Norf
during the first stage of incubation at 37 °C for 10 min. Then,
various concentrations of NaCl were added to the reaction mixtures, and
the reaction mixtures were incubated at 37 °C for 5 min. Reactions
were terminated by adding SDS to 1% and incubating at 37 °C for 5 min. EDTA and proteinase K were added to a final concentration of 25 mM and 100 µg/ml, respectively, and the incubation was
further continued at 37 °C for an additional 15 min. The DNA products were purified and analyzed as described in the previous section.
Decay Assay for the Topoisomerase-Quinolone-DNA Ternary
Complex--
Reaction mixtures (120 µl) containing 40 mM
HEPES-KOH (pH 7.6), 10 mM MgOAc2, 10 mM DTT, 50 µg/ml bovine serum albumin, 2 mM
ATP, 120 fmol (as molecule) of 32P-labeled linear pBROTB353
type I DNA, 1.2 pmol (as tetramer) of the wild type Gyr, Gyr (A59), or
Topo IV and 100 µM Norf were incubated at 37 °C for 10 min. A cold competitor, 9.6 µg (2.4 pmol as molecule) of pBROTB353
type I DNA, was added to the reaction mixtures, and the incubation was
continued at 37 °C. Portions (18 µl each) of the reaction mixtures
were withdrawn at indicated times, and SDS was added to 1% to
terminate the reactions. 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 and
then analyzed as described in the previous section.
The ability of Gyr to introduce negative superhelicity into DNA
molecules distinguishes this enzyme from other topoisomerases (1).
Recently, it has been demonstrated that Gyr can be converted into a
conventional type II topoisomerase, an enzyme similar to Topo IV, by
deleting the C terminus of the DNA binding domain of the GyrA subunit
(2). This mutant protein, Gyr (A59), unlike the wild type Gyr, does not
wrap the DNA strand and cannot catalyze the supercoiling reaction. By
using our preparations of the wild type Gyr and Gyr (A59), we assessed
biochemical activities of these enzymes. Gyr (A59) could catalyze
decatenation and relaxation reactions, but no supercoiling activity was
detected. The specific activity of Gyr (A59) was identical to that of
the wild type Gyr when the decatenation activity of these enzymes was
measured using kinetoplast DNA as a substrate (data not shown). Here,
we investigated effects of Gyr-mediated wrapping of the DNA strand on
its functional activities during DNA replication and its interaction
with the quinolone antibacterial drugs.
Gyr (A59) Can Support Nascent Chain Elongation, but Not
Decatenation of Replicating Daughter DNA Molecules, during oriC DNA Replication in Vitro Standard oriC replication reactions were incubated in the
presence of the various amounts of either Gyr (A59) or Topo IV, in
addition to a constant amount of the wild type Gyr, and then the DNA
products were analyzed by native agarose gel electrophoresis (Fig.
1A). In the presence of Gyr
alone, the majority of the replication products were late replicative
intermediates (LRI) and multiply linked form II-form II DNA dimers
(Fig. 1A, lane 1). The addition of Topo IV resulted in the
accumulation of form II DNA molecules (Fig. 1A, lanes 6 and
7). In contrast, the addition of Gyr (A59) did not change
the pattern of the replication products (Fig. 1A, lanes
2-5), although the linking number of DNA dimers was reduced when
Gyr (A59) was present at high concentrations. These results showed that
the decatenation activity of Gyr (A59) was not efficient enough to
complete the decatenation of replicating daughter DNA molecules and
produce the final monomer product.
We further examined if Gyr (A59) alone could support oriC
DNA replication. Standard oriC replication reactions were
incubated in the presence of various amounts of either the wild type
Gyr or Gyr (A59). Analysis of the replication products revealed the types of DNA molecules generated during the Gyr- and Gyr
(A59)-supported oriC DNA replication (Fig. 1B).
In the absence of any topoisomerase, only ERIs were accumulated (Fig.
1B, lane 1). ERI are molecules on which initiation has
occurred but no extensive nascent chain elongation has ensued (6, 14).
As described above, LRI and form II-form II DNA dimers were accumulated
during the wild type Gyr-supported oriC DNA replication
(Fig. 1B, lanes 2-5). The same patterns of the replication
products were generated when Gyr (A59) was used (Fig. 1B, lanes
6-9). We also performed the product analysis by
alkaline-denaturing agarose gel electrophoresis, and we confirmed that
both leading and lagging strands were synthesized during the
oriC DNA replication supported by either the wild type Gyr or Gyr (A59) (data not shown).
These results demonstrated that Gyr (A59) was capable of supporting the
nascent chain elongation but not decatenating daughter DNA molecules
during the oriC DNA replication in vitro. These results also demonstrated that the supercoiling activity of Gyr per se was not required to support nascent chain elongation.
Gyr (A59) Is Sensitive to the Quinolone Antibacterial
Drugs--
Next, we assessed the effect of the quinolone drugs on Gyr
(A59). By using a 3'-end-labeled linear plasmid DNA as a substrate, we
measured the stimulation of Gyr (A59)-catalyzed DNA cleavages by Norf
(Fig. 2). Norf stimulated both the wild
type Gyr- and Gyr (A59)-catalyzed cleavages in a
concentration-dependent manner. However, Gyr (A59) was less
sensitive to Norf than the wild type protein. The
[Norf]1/2 for Gyr (A59)-catalyzed and the wild type Gyr-catalyzed DNA cleavages were 4-5 and 0.1-0.2 µM,
respectively (Fig. 2B; data not shown).
Both enzymes cleaved greater than 90% of the plasmid DNA when Norf was
present at high concentrations (>50 µM). These results showed that, under these conditions, the plasmid DNA was occupied by at
least one ternary complex formed with either the wild type Gyr or Gyr (A59).
The Active Strand Cleavage and Reunion Activity of Gyr Is Required
for the Formation of the Gyr-Norf-DNA Ternary Complex That Can Arrest
Replication Fork Progression--
The active strand cleavage and
reunion activity of Topo IV are required for the formation of the Topo
IV-Norf-DNA ternary complexes that can arrest the progression of
replication forks in vitro (6). We examined if this was also
the case with Gyr, by using a catalytically inactive mutant Gyr (A,
Y122F). First, the standard DNA cleavage assay was performed and showed
no Gyr (A, Y122F)-catalyzed DNA cleavage (Fig.
3A). These results confirmed that the substitution of the active site Tyr with Phe abolished the
strand cleavage activity of Gyr.
The modified oriC pulse-chase protocol (6, 14) was employed
to assess the ability of the ternary complex formed with Gyr (A, Y122F)
to arrest replication fork progression in vitro (Fig.
3B). ERI were formed and labeled, and then the paused
replication forks were released by linearizing the DNA template with
the SmaI restriction endonuclease, which digested the DNA
template once at oriC. Linearization of the DNA template was
sufficient to release the paused replication forks and generate the
full-length product as a result of the run-off DNA replication (Fig.
3B, lane 1). Because no topoisomerase was required to
relieve topological constraint, this reaction was insensitive to the
presence of Norf (Fig. 3B, lane 2). Either the wild type Gyr
or Gyr (A, Y122F) was added, together with Norf, to the reaction
mixtures prior to the addition of the SmaI restriction
enzyme. The subsequent release of replication forks would result in
collisions between replication forks and the ternary complexes. The
ternary complexes that could arrest replication fork progression would,
therefore, manifest themselves in this assay by preventing the
appearance of the full-length DNA product.
In the absence of Norf, neither the wild type Gyr nor Gyr (A, Y122F)
affected elongation of the nascent chains in the ERI to the full-length
product (Fig. 3B, lanes 3 and 5). When Norf was
present, replication fork progression was blocked in the presence of
the wild type Gyr (Fig. 3B, lane 4) but not in the presence of Gyr (A, Y122F) (Fig. 3B, lane 6). These
results demonstrated that the active strand cleavage and reunion
activity of Gyr was required for the formation of the ternary complex
that could arrest replication fork progression.
The Covalent Gyr-DNA Complex Formation Is Not Sufficient to Arrest
Replication Fork Progression in Vitro--
We further examined if the
formation of covalent Gyr-DNA complexes was not only necessary but also
sufficient to arrest replication fork progression. If the covalent
Gyr-DNA complex formation is sufficient to inhibit DNA replication, the
Gyr (A59)-Norf-DNA ternary complex would block the replication fork progression.
The occupancy of the topoisomerase on the DNA is one of the determining
factors of the probability of collisions between replication forks and
topoisomerase-DNA complexes. The amounts of topoisomerase-catalyzed DNA
cleavages represent the formation of the covalent topoisomerase-DNA complexes on the DNA. As shown in Fig. 2, the occupancies of the plasmid DNA template by the ternary complexes formed with either the
wild type Gyr or Gyr (A59) were similar when Norf was present at high
concentrations. Thus, under these conditions, we expected the
collisions between replication forks and the ternary complexes formed
with either the wild type Gyr or Gyr (A59) would take place at a
similar frequency.
The modified oriC pulse-chase protocol (6, 14) was employed
again to model the events during the collision between a replication
fork and a Gyr (A59)-Norf-DNA ternary complex (Fig. 4). In the absence of Norf, the presence
of either the wild type Gyr or Gyr (A59) had no effect on the
replication fork progression (Fig. 4, lanes 3 and
5). When the Gyr-Norf-DNA ternary complex was formed,
replication fork progression was inhibited (Fig. 4, lane 4).
Interestingly, the ternary complexes formed with Gyr (A59) did not
arrest the progression of replication forks (Fig. 4, lane
6). We also performed the same assay using ciprofloxacin as a
quinolone drug and obtained identical results (data not shown). These
results demonstrated that the ternary complexes formed with Gyr (A59)
could not arrest replication fork progression. Thus, the
quinolone-induced covalent topoisomerase-DNA complex formation was
necessary but not sufficient for the inhibition of DNA replication.
The Ternary Complexes Formed with Gyr (A59) Are More Sensitive to
Salt Than Those Formed with Either the Wild Type Gyr or Topo
IV--
It was not clear why the ternary complexes formed with Gyr
(A59) could not arrest replication fork progression. One possible explanation was that the ternary complexes formed with Gyr (A59), unlike those formed with either the wild type Gyr or Topo IV, were not
stable enough to arrest replication fork progression. To examine this
possibility, we assessed salt sensitivity of the ternary complexes
formed with Gyr (A59), the wild type Gyr, or Topo IV. The ternary
complexes formed with 32P-labeled linear pBROTB353 type I
DNA, Norf, and Gyr (A59), the wild type Gyr, or Topo IV were incubated
in the presence of various concentrations of NaCl at 37 °C for 5 min, followed by the denaturation of topoisomerases by SDS. In this
assay, unstable ternary complexes would be reversed and DNA strands
would be religated during the incubation in the presence of salt. On
the other hand, stable ternary complexes would remain on the DNA, which
would result in the generation of DSBs upon the denaturation of topoisomerases.
The DNA products were analyzed by native agarose gel electrophoresis to
measure the extent of DNA cleavages (Fig.
5). In the absence of salt, about 90% of
the linear plasmid DNA was cleaved, at least once, by either one of
these topoisomerases (Fig. 5, lanes 2, 7, and
12). The ternary complexes formed with Gyr (A59) (Fig. 5,
lanes 7-11) were more sensitive to salt than those formed with either the wild type Gyr (Fig. 5, lanes 2-6) or Topo
IV (Fig. 5, lanes 12-16). After a 5-min incubation in the
presence of 0.5 M NaCl, 64, 25, and 21% of the plasmid DNA
was recovered as the full-length linear molecule as a result of the
reversal of Gyr (A59)-Norf-DNA, Gyr-Norf-DNA, and Topo IV-Norf-DNA
ternary complexes, respectively. These results demonstrated that the
ternary complexes formed with Gyr (A59) were less stable than those
formed with either the wild type Gyr or Topo IV.
Gyr (A59)-Norf-DNA Ternary Complexes Are Less Stable Than
Gyr-Norf-DNA and Topo IV-Norf-DNA Ternary Complexes--
We also
performed a competition experiment to assess the stability of
Gyr-Norf-DNA, Gyr (A59)-Norf-DNA, and Topo IV-Norf-DNA ternary
complexes. The ternary complexes were formed with
32P-labeled linear pBROTB353 type I DNA, Norf, and the wild
type Gyr, Gyr (A59), or Topo IV. After a 10-min incubation at 37 °C, an excess amount of pBROTB353 type I DNA was added to the reaction mixtures as a cold competitor. The incubation was continued at 37 °C, and portions of the reaction mixtures were withdrawn at indicated times. After the denaturation of topoisomerases, the DNA
products were purified and analyzed as described under "Material and Methods."
The amount of the intact linear plasmid DNA in the reaction mixture in
the absence of any topoisomerase at 0 min (Fig.
6, lane 1) was used as a
standard (100%). After a 60-min incubation in the absence of any
topoisomerase, the relative amounts of the intact DNA remained constant
(97%; data not shown). At 0 min, the relative amounts of the intact
linear plasmid DNA were 3.9, 4.4, and 3.7% in the reaction mixtures
containing Gyr-Norf-DNA, Gyr (A59)-Norf-DNA, and Topo IV-Norf-DNA
ternary complexes, respectively (Fig. 6, lanes 2, 8, and
14). Relative amounts of the intact DNA remained essentially
constant during the period of incubation when either Gyr-Norf-DNA
(5.5% at 60 min; Fig. 6, lanes 2-7) or Topo IV-Norf-DNA
ternary complexes (5.6% at 60 min; Fig. 6, lanes 14-19)
were present in the reaction mixtures, demonstrating that the ternary
complexes formed with the wild type Gyr or Topo IV did not disassociate
from the DNA. In contrast, the amounts of the intact DNA increased with
time when the reaction mixtures contained Gyr (A59)-Norf-DNA ternary
complexes (Fig. 6, lanes 8-13), and nearly 40%
of the plasmid DNA was recovered as the full-length product at 60 min.
These results showed that Gyr (A59)-Norf-DNA ternary complexes readily
disassociated from the DNA, whereas both Gyr-Norf-DNA and Topo
IV-Norf-DNA ternary complexes remained stably bound.
Results described in this section and in previous sections clearly
demonstrated that ternary complexes formed with Gyr (A59) were less
stable than those formed with the wild type Gyr or Topo IV. Thus,
Gyr-mediated wrapping of the DNA strand was required for the formation
of stable Gyr-Norf-DNA ternary complexes that could arrest replication
fork progression. These results suggested that the stability of the
topoisomerase-quinolone-DNA ternary complex would determine if the
collision between a replication fork and a ternary complex would result
in the inhibition of DNA replication. We also showed that ternary
complexes formed with Topo IV were as stable as those formed with Gyr.
These results suggested that Gyr and Topo IV interacted differently
with the DNA and/or the quinolone drug. Topo IV could form stable
ternary complexes without wrapping the DNA strand, whereas Gyr required the wrapping of the DNA strand around itself to form stable ternary complexes.
Among type II topoisomerases, Gyr is unique because of its ability
to supercoil DNA molecules. Gyr wraps the DNA strand around itself upon
its binding to the DNA strand and catalyzes the supercoiling reaction
(1). It has been demonstrated that the deletion of the C-terminal DNA
binding domain of the GyrA subunit converts Gyr into a conventional
type II topoisomerase (2). This mutant Gyr, Gyr (A59), does not wrap
the DNA strand around itself. Gyr (A59) can catalyze the decatenation
and relaxation but not the supercoiling reactions.
Both Gyr and Topo IV contribute to DNA unlinking during DNA
replication. However, these topoisomerases function in a distinct manner. Gyr prefers to remove positive supercoils ahead of the advancing replication forks, whereas Topo IV prefers to decatenate precatenanes behind the replication forks (17). Both Gyr and Topo IV
can support the elongation of DNA replication but only Topo IV can
decatenate replicating daughter DNA molecules. Gyr introduces negative
superhelicity into the DNA, which is required for the initiation of DNA
replication at oriC (18). It has been thought, however, that
the supercoiling activity of Gyr per se is not essential for
replication fork progression. In eukaryotes, no topoisomerase can
catalyze the supercoiling reaction but topoisomerases complete the DNA
unlinking during DNA replication. Here, we assessed the functional
activities of Gyr (A59) during oriC DNA replication in
vitro. Gyr (A59) alone could support oriC DNA
replication (Fig. 1B), demonstrating that the supercoiling
activity of Gyr is not required to support the nascent chain
elongation. However, Gyr (A59) could not decatenate replicating
daughter DNA molecules. As a result, the majority of the replication
products were LRI and DNA dimers (Fig. 1A). Thus, the
replicative function of Gyr (A59) is similar to that of the wild type
Gyr (14, 15), except that Gyr (A59) cannot introduce negative
supercoils into DNA molecules (2).
Topo IV is responsible for the decatenation of daughter chromosomes
(19, 20). In direct comparison, Topo IV is a much better decatenating
enzyme, with a turnover number nearly 100-fold greater than that of Gyr
both in vivo and in vitro (17, 21). Kampranis and
Maxwell (2) have shown that Gyr (A59) can partially complement the
phenotype of a parCts mutant in vivo,
indicating that Gyr (A59) can substitute the Topo IV function during
chromosome segregation in E. coli. However, we found that
Gyr (A59) could not decatenate replicating daughter DNA molecules
during the oriC DNA replication in vitro (Fig.
1). It is not clear what causes this apparent paradox. One possible explanation is that the high copy number plasmid-carried
gyrA(59) gene used in the in vivo
studies (2) provides an elevated level of Gyr (A59) protein, which may
be sufficient to support cell growth of the parC1215 strain
at 42 °C. Another possibility is that there might be a protein that
interacts with Gyr (A59) in the cell, and this protein-protein
interaction could stimulate Gyr (A59)-catalyzed decatenation of
daughter chromosomes in vivo. For instance, the
dnaX gene, encoding the We showed here, using mutant Gyr proteins, Gyr (A59) and Gyr (A,
Y122F), that the formation of the covalent Gyr-DNA complex was
necessary but not sufficient to arrest replication fork progression (Figs. 3 and 4). We found that the ternary complexes formed with Gyr
(A59) were less stable than those formed with either the wild type Gyr
or Topo IV (Figs. 5 and 6). Thus, the Gyr-mediated wrapping of the DNA
strand is required for the formation of the stable Gyr-Norf-DNA ternary
complex that can arrest replication fork progression. These results
suggest that the stability of the quinolone-induced covalent
topoisomerase-DNA complex is likely to determine, when a replication
fork and a ternary complex collide, if the inhibition of DNA
replication occurs.
Gyr and Topo IV are homologous to each other (1). Gyr (A59) does not
wrap the DNA strand around itself and thus this mutant Gyr seems to
bind the DNA in the same manner as Topo IV. However, we showed that
Topo IV-Norf-DNA ternary complexes were more stable than Gyr
(A59)-Norf-DNA ternary complexes. These results indicate that, despite
their similarities, there are critical differences between Gyr and Topo
IV in their interactions with the DNA and/or the quinolone drug, which
affect the stability of the ternary complexes. Alternatively, it is
possible that the deletion of the C-terminal DNA-binding domain of the
GyrA subunit not only abolishes the ability of Gyr to wrap the DNA
strand but also causes a conformational change of the GyrA subunit,
which could reduce the affinity of the Gyr (A59)-DNA interaction. The
requirement of high concentrations of Norf for the formation of Gyr
(A59)-Norf-DNA ternary complexes (Fig. 2) supports this possibility.
The mechanism by which the Gyr (A59)-quinolone-DNA ternary complex is
reversed upon its collision with a replication fork is not clear. It is
possible that the replication fork actively forces Gyr (A59) to
religate the DNA strands and disassociate from the DNA. Alternatively,
replication forks simply pause when they collide with ternary complexes
formed with either the wild type Gyr or Gyr (A59). The half-life of Gyr
(A59)-Norf-DNA ternary complexes is shorter than that of the paused
replication forks, whereas the half-life of Gyr-Norf-DNA and Topo
IV-Norf-DNA ternary complexes is longer than that of the replication
forks. As a result, replication forks can progress in the presence of
the Gyr (A59)-Norf-DNA ternary complexes, but replication fork
progression is arrested when the ternary complexes formed with either
the wild type Gyr or Topo IV are present.
Quinolone resistance-conferring mutations rapidly arise and are
particularly clustered within a small region (between amino acids 67 and 106) of the gyrA gene (often referred to as the
"quinolone resistance-determining region") (23). Homologous
mutations in the parC gene also confer quinolone resistance
to Topo IV (6, 21). These mutations seem to alter the
topoisomerase-quinolone interaction and reduce the probability of the
formation of topoisomerase-quinolone-DNA ternary complexes (24). Based
on the results presented here, it is interesting to speculate that
there is another mechanism of acquiring drug resistance-conferring
mutations. Mutations in Gyr and Topo IV could affect the stability of
the topoisomerase-drug-DNA ternary complexes without changing the
probability of the ternary complex formation. In this scenario, ternary
complexes formed with a mutant topoisomerase are not stable enough to
arrest replication fork progression. Thus, collisions between
topoisomerase-drug-DNA ternary complexes and replication forks do not
result in the inhibition of DNA replication. Some of the quinolone
resistance-conferring mutations mapped in the regions outside of the
quinolone resistance-determining region of the gyrA gene and
the gyrB gene could be of this type. Of course, this
possibility needs to be tested.
The covalent topoisomerase-DNA complex is normally a fleeting catalytic
intermediate during topoisomerization. Thus, advancing replication
forks seldom collide with the covalent topoisomerase-DNA complexes
during the chromosomal DNA replication. However, at very low
frequencies, collisions do take place. If any covalent topoisomerase-DNA complex can arrest replication fork progression, every collision between an advancing replication fork and a covalent topoisomerase-DNA complex triggers the cytotoxic events. We showed here
that the formation of the covalent topoisomerase-DNA complex was
necessary but not sufficient to arrest replication fork progression. A
certain stability of the topoisomerase-quinolone-DNA ternary complex
was required for the replication fork arrest. Thus, it seems reasonable
to assume that the normal covalent topoisomerase-DNA complex, a
catalytic intermediate, is not stable enough to block the progression
of a replication fork. The replication machinery is likely to have an
ability to complete the chromosomal replication even in the presence of
these unstable topoisomerase-DNA complexes. Thus, the covalent
topoisomerase-DNA complex becomes a cellular poison only when it is
frozen as a stable topoisomerase-drug-DNA ternary complex by a
topoisomerase inhibitor.
We thank Dr. Kenneth Marians for the generous
gifts of replication proteins and critical comments on this manuscript.
*
This work was supported by National Institutes of Health
Grant GM59465.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, August 16, 2000, DOI 10.1074/jbc.M001608200
2
H. Hiasa, D. O. Yousef, and K. J. Marians, unpublished results.
The abbreviations used are:
Gyr, DNA gyrase;
DSB, double-strand break;
DTT, dithiothreitol;
ERI, early replicative
intermediates;
form II, nicked or gapped DNA molecule(s);
LRI, late
replicative intermediates;
Norf, norfloxacin;
Topo, topoisomerase.
DNA Gyrase-mediated Wrapping of the DNA Strand Is Required for
the Replication Fork Arrest by the DNA Gyrase-Quinolone-DNA Ternary
Complex*
and
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
The wild type Gyr is incapable of decatenating replicating daughter DNA molecules (15). In the oriC
replication system reconstituted with purified proteins, little monomer
product is generated when only Gyr is present as a topoisomerase.
Topoisomerase III or Topo IV is required for the production of the
final monomer product (15, 16). Kampranis and Maxwell (2) have
demonstrated that gyrA(59) can partially complement the
phenotype of a parCts mutant, indicating that Gyr
(A59) could decatenate replicating daughter chromosomes in the cell. We
assessed the decatenation activity of Gyr (A59) during oriC
DNA replication in vitro.
View larger version (60K):
[in a new window]
Fig. 1.
Gyr (A59) cannot decatenate replicating
daughter DNA molecules during oriC DNA replication
in vitro. A, standard oriC DNA
replication reactions containing 140 fmol (as tetramer) of the wild
type Gyr and the indicated amounts (as tetramer) of either Gyr (A59) or
Topo IV were incubated at 30 °C for 10 min, and the DNA products
were analyzed by the electrophoresis through 0.8% native agarose gels
(11). Total DNA synthesis (as nucleotides): lane 1, 186 pmol; lane 2, 218 pmol; lane 3, 201 pmol;
lane 4, 220 pmol; lane 5, 180 pmol; lane
6, 235 pmol; lane 7, 200 pmol. B, standard
oriC DNA replication reactions were incubated in the
presence of the indicated amounts (as tetramer) of either the wild type
Gyr or Gyr (A59). The replication products were analyzed by native
agarose gel electrophoresis (11). Total DNA synthesis (as nucleotides):
lane 1, 38 pmol; lane 2, 175 pmol; lane
3, 220 pmol; lane 4, 262 pmol; lane 5, 236 pmol; lane 6, 76 pmol; lane 7, 115 pmol;
lane 8, 164 pmol; lane 9, 234 pmol.
wt, the wild type Gyr; A59, Gyr (A59); IV, Topo IV;
LRI, late replicative intermediates; ERI, early
replicative intermediates; II:II, form II-form II DNA
dimers.
View larger version (35K):
[in a new window]
Fig. 2.
Norf stimulates Gyr (A59)-catalyzed DNA
cleavages. Stimulation of Gyr-catalyzed DNA cleavages by Norf was
measured as described under "Materials and Methods." Reaction
mixtures contained 20 fmol (as molecule) 32P-labeled linear
pBROTB353 type I DNA, 0.3 pmol (as tetramer) of either the wild type
Gyr or Gyr (A59), and the indicated concentrations of Norf.
A, the DNA products were analyzed by the electrophoresis
through 1% native agarose gels. Abbreviations are the same as in Fig.
1 legend. B, relative amounts of the full-length plasmid DNA
were measured. Experiments were repeated twice, and the error range was
±4%. Representative results are shown. 
, the wild type Gyr; 
,
Gyr (A59).
View larger version (36K):
[in a new window]
Fig. 3.
The cleavage and reunion activity of Gyr is
required for the formation of the Gyr-Norf-DNA ternary complex that can
arrest replication fork progression. A, the strand DNA
cleavage reaction mixtures containing 20 fmol (as molecule) of
32P-labeled linear pBROTB353 type I DNA, 100 µM Norf, and the indicated amounts (as tetramer) of
either the wild type Gyr or Gyr (A, Y122F) were incubated, and the DNA
products were analyzed as described under "Materials and Methods."
B, the ability of the ternary complex formed with Gyr (A,
Y122F) to arrest the replication fork progression was assessed. The
modified pulse-chase protocol was performed, and the DNA products were
analyzed according to Hiasa et al. (6). Either the wild type
Gyr or Gyr (A, Y122F) at a molar ratio of 6:1 to the DNA template and
100 µM Norf was added to the reactions as indicated.
wt, the wild type Gyr; Y122F, Gyr (A, Y122F).
Size markers were 3'-end-labeled, HindIII-digested 
DNA.
kb, kilobase pairs.
View larger version (72K):
[in a new window]
Fig. 4.
Gyr (A59)-Norf-DNA ternary complex cannot
arrest replication fork progression in vitro. The
ability of the Gyr (A59)-Norf-DNA ternary complex to arrest the
replication fork progression was assessed. The modified pulse-chase
analysis was performed, and the DNA products were analyzed according to
Hiasa et al. (6). Either the wild type Gyr or Gyr (A59) at a
molar ratio of 6:1 to the DNA template and 100 µM Norf
were added to the reaction mixtures as indicated. Abbreviations are the
same as in Fig. 1 legend, and size markers are the same as in Fig. 3
legend. kb, kilobase pairs.
View larger version (67K):
[in a new window]
Fig. 5.
Ternary complexes formed with Gyr (A59) are
more sensitive to salt than those formed with either the wild type Gyr
or Topo IV. Salt sensitivity of the ternary complexes formed with
either Gyr (A59), the wild type Gyr, or Topo IV was measured as
described under "Materials and Methods." Reaction mixtures
contained 20 fmol (as molecule) of 32P-labeled linear
pBROTB535 type I DNA, 100 µM Norf, and 0.3 pmol (as
tetramer) of Gyr (A59), the wild type Gyr, or Topo IV. The DNA products
were analyzed by the electrophoresis through 1% native agarose gels.
Relative amounts of the full-length DNA product are as follows:
lane 1, 100% (control); lane 2, 7.5%;
lane 3, 7.3%; lane 4, 7.8%; lane 5,
18.6%; lane 6, 24.4%; lane 7, 12.8%;
lane 8, 35.7%; lane 9, 42.5%; lane
10, 52.5%; lane 11, 63.8%; lane 12, 7.9%;
lane 13, 3.6%; lane 14, 8.5%; lane
15, 13.8%; lane 16, 20.7%. Experiments were repeated
twice, and the representative results are shown. Abbreviations are the
same as in Fig. 1 legend.
View larger version (57K):
[in a new window]
Fig. 6.
Gyr (A59)-Norf-DNA ternary complexes readily
disassociate from the DNA. A competition experiment was performed
as described under "Materials and Methods." Ternary complexes were
formed in the presence of 120 fmol (as molecule) of
32P-labeled linear pBROTB535 type I DNA, 100 µM Norf, and 1.2 pmol (as tetramer) of either the wild
type Gyr, Gyr (A59), or Topo IV, and 2.4 pmol (as molecule) of
pBROTB535 type I DNA was added as a cold competitor. Aliquots were
withdrawn at the indicated times after the addition of the competitor
DNA, and the DNA products were analyzed by electrophoresis through 1%
native agarose gels. Relative amounts of the full-length DNA product
are as follows: lane 1, 100% (control); lane 2,
3.9%; lane 3, 4.0%; lane 4, 5.4%; lane
5, 5.8%; lane 6, 6.2%; lane 7, 5.5%;
lane 8, 4.4%; lane 9, 5.6%; lane 10,
8.8%; lane 11, 10.6%; lane 12, 18.0%;
lane 13, 39.2%; lane 14, 3.7%; lane
15, 4.9%; lane 16, 5.4%; lane 17, 5.8%;
lane 18, 5.2%; lane 19, 5.6%. Experiments were
repeated twice, and the representative results are shown. Abbreviations
are the same as in Fig. 1 legend.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and 
subunits of DNA
polymerase III holoenzyme, has been identified as a high copy suppressor of the phenotype of a parEts mutant
(22).
ACKNOWLEDGEMENT
FOOTNOTES
Member of the University of Minnesota Comprehensive Cancer Center.
To whom correspondence should be addressed: 6-120 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455. Tel.: 612-626-3101; Fax:
612-625-8408; E-mail: hiasa001@tc.umn.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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