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J. Biol. Chem., Vol. 277, Issue 8, 5944-5951, February 22, 2002
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From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, November 29, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We have prepared full-length
Drosophila and human topoisomerase II and truncation
constructs containing the amino-terminal ATPase domain, and we have
analyzed their biochemical properties. The ATPase activity of the
truncation proteins, similar to that of the full-length proteins, is
greatly stimulated by the presence of DNA. This activity of the
truncation proteins is also sensitive to the inhibition by the drug
bisdioxopiperazine, ICRF-193, albeit at a much lower level than the
full-length protein. Therefore, bisdioxopiperazine can directly
interact with the NH2-terminal ATPase domain, but the
drug-enzyme interaction may involve other domains as well. The ATPase
activity of the ATPase domain protein showed a quadratic dependence on
enzyme concentration, suggesting that dimerization of the
NH2-terminal domain is a rate-limiting step. Using both
protein cross-linking and sedimentation equilibrium analysis, we showed
that the ATPase domain exists as a monomer in the absence of cofactors
but can readily dimerize in the presence of a nonhydrolyzable analog of
ATP, 5'-adenylyl- Type II DNA topoisomerases (topo
II)1 are ubiquitous enzymes
that catalyze DNA topological changes by transporting one double strand
DNA segment through another (1-4) (for a review, see Refs. 5 and 6).
They play essential roles in many aspects of DNA transactions in
vivo, including chromosome condensation and segregation, and
removal of the supercoils generated during replication and transcription. In addition to such essential functions in the cells,
topo II is the target of many widely used antibiotic and anti-tumor
drugs (7-11).
Recent biochemical and structural studies have provided an
understanding of the molecular mechanism for eukaryotic topo II (12-15) (also reviewed in Refs. 5, 6, and 16). Eukaryotic topo II is a
homodimer with a primary dimeric interface at its COOH terminus. The
enzyme binds to a segment of DNA (G-segment) and generates a reversible
double strand break to serve as a gateway for the strand passage. The
binding of ATP to the ATPase domain leads to the dimerization of this
domain and closure of the NH2-terminal protein clamp
(N-gate). This movement in the NH2-terminal clamp can
entrap another DNA segment (T-segment) and initiate a series of
conformational changes that include the passage of the T-segment through the protein-mediated DNA break, the religation of the DNA gate,
and release of the T-segment through the opening of its COOH-terminal
dimer interface. Recent rapid kinetic measurements have provided
further insights into this process (17, 18). The two ATP molecules
bound to each topo II homodimer are hydrolyzed at very different rates,
and strand passage of T-segment occurs after the hydrolysis of the
first ATP.
The ATPase activity of topo II is reduced by a unique class of
catalytic inhibitors, bisdioxopiperazines like ICRF-193 and ICRF-159
(7). Bisdioxopiperazines are anti-tumor agents that target eukaryotic
topo II in vivo (19). A previous study on a member of this
drug family, ICRF-193, has shown that in the presence of ATP, the drug
inhibits yeast topo II activities by trapping the enzyme in a closed
clamp form (20). Using rapid kinetic analysis, Morris et al.
(21) have shown that ICRF-193, which does not work as a competitive
inhibitor of ATP, can bind to a closed clamp complex but still allow
ATP hydrolysis at a much lower rate. Initial analysis of some of the
cell lines resistant to this drug indicates that mutations in the
ATPase domain of topo II are responsible for the resistance (22, 23). A
recent study on the ATPase domain of yeast topo II has shown that
ICRF-193 can inhibit the ATP hydrolysis, indicating a direct
interaction between the drug and ATPase domain (24). In contrast, a
study with the 52-kDa ATPase domain of human topo II did not find any significant inhibition of ATPase activity by ICRF-193 (25). Analysis of
the cytotoxicity of this drug in the yeast model system indicates that
cell killing requires more than just the irreversibly closed clamp
conformation of intracellular topo II (26). Our previous study on the
core domain of Drosophila topo II, which lacks the ATPase
domain, also raised the possibility of the involvement of other topo II
domains in the protein-drug interaction (27). Whether this class of
drugs can stimulate topo II-mediated DNA cleavage is still another
issue remaining to be clarified. Earlier works have demonstrated that
these drugs can inhibit the catalytic activities of the enzyme without
stabilizing the covalent enzyme-DNA intermediate (28, 29). Recent
experiments using a different procedure to trap the topo II-DNA
cleavable complex showed that ICRF-193 can promote DNA cleavage
mediated by human topo II (30). To further probe the action of
bisdioxopiperazines, especially on the ATPase domain of topo II, we
examined the effects of this drug on three types of topo II constructs:
the ATPase domain; the GyrB homologous domain, which includes both the
ATPase domain and the GyrB' domain; and the full-length enzyme. We have
also monitored the effect of nucleotide cofactors on the dimerization of the ATPase domain to gain insights into the effects of cofactors on
the clamp closure at the N-gate.
Generating the NH2-terminal Fragments of Human and
Drosophila Topo II--
We used PCRs to construct the expression
vectors for Drosophila and human NH2-terminal
ATPase domains (DmATPD and HsATPD, respectively). The sequences of 5'
and 3' PCR primers for generating HsATPD were 5'-CCT GGT TTG TAC
AAA ATC TTT G-3' and 5'-GGC AAC CTA GGT TAA TGG TGA TGA TGA TGG TGC TTG TTT AAC TGG ACT TGG GC-3', respectively. The 5' primer contains the sequence encoding residues 79-85 of human topo II including a BsrGI digestion
site (underlined) that is unique on the yeast expression vector
containing the entire human topo II coding sequence, YEpWOB6 (31). The
3' primer contains the sequence encoding residue 419-425 of human topo
II and His6 tag (italicized) followed by a stop codon
(boldface type) and a unique AvrII site (underlined). The
sequences of 5' and 3' PCR primers for generating DmATPD are 5' CAT TCC
GGT GAC CAT GCA CAA G 3' and 5' GGC TTG CAT
GCT TAA TGG TGA TGA TGA TGG TGC TTG GCA ATG TCA
TTT TGG GCC 3', respectively. The 5' primer contains the sequence
encoding residues 106-113 of Drosophila topo II including a
BstEII site (underlined) that is unique on the yeast
expression vector containing the entire Drosophila topo II
coding sequence, YGBBX Purification of Wild-type and Truncation Topo II--
Both the
wild-type and truncation proteins were overexpressed and purified as
described previously (27, 34). Purification of the His-tagged
truncation proteins was performed as follows. The cleared lysate
prepared from an 8-liter culture was passed through a 5-ml
Ni2+-nitrilotriacetic acid column (Qiagen) in the presence
of 15 mM imidazole, pH 7.0. 250 mM NaCl was
also included in all of the buffers throughout the purification. After
washing with 50 ml of 30 mM imidazole solution, stepwise
concentrations of 60, 120, 240, and 500 mM imidazole were
used to elute the protein, with a volume of 10 ml at each step. The
His-tagged topo II protein was eluted between the 120 and 240 mM imidazole steps. The peak fractions were combined and
further purified by HQ and HE column chromatography (PerSeptive
Biosystem) using a procedure modified from our published methods (34).
The final peak fractions were dialyzed against the storage buffer
containing 10 mM Tris acetate, pH 7.8, 50 mM
KAc, 5 mM Mg(Ac)2, and 50% glycerol. Also
included in solutions used throughout the steps of purification and
storage was a mixture of protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin). Prior to the ATPase assay, the proteins were concentrated
and exchanged into a buffer of 10 mM Tris acetate, pH 7.9, using a Microcon centrifugal device (Millipore Corp., Bedford, MA).
ATPase Assay--
Two methods were used to measure the ATP
hydrolysis. The TLC method was done as described previously (34). The
coupled PEP/LDH spectrophotometric assay was performed at 30 °C on
an HP 8453 diode array spectrophotometer with a thermostat cuvette cell
according to a published procedure (35). In all of the experiments,
only the data from the linear range of ATP hydrolysis were used to calculate the reaction rate. We have tested for the optimal conditions for these proteins. Both ATPD proteins have shown the highest activity
in a buffer with an ionic strength that is less than 10 mM
in monovalent salt and 2-4 mM in divalent cation (data not shown). In most experiments, the standard reaction mixture contained 10 mM Tris acetate (pH 7.9), 5 mM KAc, 2.5 mM Mg(Ac)2, and 1.25 mM ATP. The
optimal ionic conditions for ATPase activity of DmGyrB fragment were
also determined to be 10 mM Tris acetate (pH 7.9), 25 mM KAc, 10 mM Mg(Ac)2. The ionic
strength for DmGyrB is higher than that optimal for the HsATPD and
DmATPD but still much lower than that for full-length topo II. One
apparent effect of the truncation of the carboxyl portion of topo II is
that optimal ATPase activity in these proteins occurs at a much lower
ionic strength in the reaction conditions.
Sedimentation Equilibrium--
Samples of HsATPD were dialyzed
against 10 mM Tris acetate, pH 7.8, 30 mM
potassium phosphate, and 5 mM Mg(Ac)2. 75-µl
samples (1 mg/ml) were successively brought to sedimentation
equilibrium at 9,000, 12,000, and 15,000 rpm over a period of 2 days at
20 °C in an analytical ultracentrifuge (XL-A ultracentrifuge;
Beckman Instruments). To establish a base line, the samples were
subjected then to an overspeed at 50,000 rpm, and, where needed, the
data were base line-corrected. Cofactors like ATP, AMPPNP, and ICRF-193 were included in some samples at the concentrations indicated. In each
case, the same concentration of cofactor was included both in the
sample and in the reference solvent. Absorbance measurements were made
routinely at 290 nm, where the absorbance due to the cofactors is low
compared with the protein. The data were fitted by the
Ideal-1 program (Beckman Instruments) using a value of
To calculate the dimerization constants, we used two software programs
provided by Dr. Allen Minton to process and analyze the sedimentation
equilibria data. The best fit weight average molecular weight data from
all three speeds were obtained first by using the program
MWAVCALC6. The dimerization constants were then obtained by
using the program F-MWN50-N to globally fit the molecular
weight data from all three speeds. This program uses Marquardt-Levenberg Data Analysis of the Dimerization Equilibria of ATPD Induced by
ADP--
We assume that the following three equilibria best describe
the reactions of HsATPD in the presence of ADP under the conditions of
sedimentation equilibrium experiments.
The apparent dimerization constant
K Protein Cross-linking Assay--
The assay was performed as
described in Ref. 25 with the following modifications. The protein was
dialyzed into 50 mM HEPES, pH 8.5, 5 mM KCl, 4 mM dithiothreitol, and 4 mM
Mg(Ac)2. After incubation for 1 h at 25 °C,
dimethylsuberimidate was added to a final concentration of 0.2 mg/ml
followed by an additional 1-h incubation at 25 °C. The reactions
were quenched by adding Tris-glycine, pH 8.0, to 50 mM and
analyzed by 6% SDS-PAGE (36).
Construction and Purification of NH2-terminal Fragments
of Drosophila and Human Topo II--
To study the functions of the
ATPase domain and the GyrB homologous domain of eukaryotic topo II, we
have constructed two NH2-terminal truncation fragments of
Drosophila topo II and one from human topo II. The
truncation end points are based on the domain structures of
Drosophila topo II, as determined by protease-sensitive site
and linker insertion analysis (Fig.
1A) (33). The domain structure
of eukaryotic topo II is conserved as shown by similar analysis with
yeast topo II (37, 38) and x-ray crystallography (12). For the
Drosophila truncation constructs, the 47-kDa fragment of the
ATPase domain (DmATPD) contains the first 406 amino acid residues plus
a hexahistidine tag, whereas the 77-kDa fragment of the GyrB homologous
domain (DmGyrB) contains the first 668 amino acid residues plus a
hexahistidine tag (Fig. 1, B and C). The 46-kDa
fragment of ATPase domain of human topo II, HsATPD, contains the first
6 amino acid residues of Saccharomyces cerevisiae topo II
followed by Ser-29 to Lys-425 of human topo II and a hexahistidine tag
(Fig. 1D). All three constructs were overexpressed in a
protease-deficient yeast strain BCY123 and purified to over 95% of
homogeneity (Fig. 2).
ATPase Activity of HsATPD, DmATPD, and DmGyrB--
For both DmATPD
and HsATPD, the presence of DNA greatly stimulates their ATPase
activity (comparing A and B of Fig.
3). In the absence of DNA,
both proteins had only a low level of ATPase activity, with a reduction
of about 20-fold when compared with the conditions in the presence of
DNA and at the identical protein concentrations. The DNA dependence of
the proteins has been explored over a range of concentrations from 0 to
300 µM in base pairs. The maximal DNA stimulation
for DmATPD and HsATPD happens at a concentration of 100 and 30 µM bp, respectively (data not shown). The optimal DNA
concentration in the stimulation of ATPase activity for DmGyrB protein
is also estimated to be around 100 µM bp. Interestingly, when the ATPase activities were measured at different protein concentrations, both the DNA-independent and the
DNA-dependent ATPase activity of DmATPD and HsATPD have
shown a parabolic dependence on the enzyme concentration, suggesting
that the dimerization of the subunits is the rate-limiting step in ATP
hydrolysis under such conditions (Fig. 3). In contrast, the
concentration dependence for the DmGyrB proteins follows an expected
linear relationship, just like that for the full-length proteins of
Drosophila topo II or human topo II
While all the truncation proteins, similar to the full-length enzymes,
exhibited ATPase activities that can be further stimulated by the
presence of DNA, their ATPase activities are much reduced in comparison
with the holoenzymes. To gain quantitative insight into these rate
differences, we have analyzed the ATP hydrolysis rates as a function of
substrate concentrations. For HsATPD and DmATPD, these rate curves can
be fit with a Michaelis-Menten kinetic equation (Fig.
4, A and B). For
the holoenzymes and DmGyrB, their rate data can also fit
Michaelis-Menten kinetics. While the apparent Km
values for these constructs are comparable, ranging between 0.4 and 0.6 mM (data not shown), there is a 10-fold difference in
apparent kcat between the full-length proteins
and the ATPase domain fragments (Table
I). These data suggest that substrate binding may remain unaffected whether the ATPase domain is part of the
holoenzyme or exists by itself, assuming that the apparent Km values in this case can give an estimate of the
ATP binding affinities. However, protein domains other than the ATPase part can have a major role for the ATP hydrolysis activity.
The Effect of Bisdioxopiperazine on the ATPase Domain of Drosophila
and Human Topo II--
The anti-cancer agent bisdioxopiperazine is a
topo II inhibitor and can lock up the N-gate in the presence of ATP
(20). It remains unclear, however, whether bisdioxopiperazine only
targets the ATPase domain and thereby inhibits its ATPase activity.
Whereas an NH2-terminal ATPD of yeast topo II (residues
1-409) expressed in yeast cells can be inhibited by ICRF-193 (24),
another drug in this family, ICRF-159, cannot inhibit the ATPase
activity from a similar NH2-terminal fragment (residues
1-439) of human topo II purified from the renatured fraction of a
bacterial expression system (25). The difference could be due to
different expression/purification systems and ATPD from different
organisms. To further investigate the inhibition of eukaryotic topo II
by bisdioxopiperazine drugs, we studied the effect of ICRF-193 on the
ATPase activities of both human and Drosophila topo II
holoenzymes, DmGyrB, and both human and Drosophila ATPase
domain fragments. As shown in Fig. 5, the
ATPase activity of both holoenzymes is exquisitely sensitive to the
drug, with the IC50 of the human and Drosophila
proteins being around 0.1 and 0.3 µM, respectively. The
inhibition of the ATPase domains by ICRF-193 could also be detected,
although at much higher drug concentrations, with the IC50
of HsATPD and DmATPD being 60 and 30 µM, respectively.
For DmGyrB protein, the sensitivity of its ATPase activity to the drug
is between those of the holoenzyme and the ATPase domain, with an
IC50 of 3 µM. Comparing the result shown in
Fig. 5 and the ATPase activity of the different constructs listed in
Table I, we have found that the sensitivity of topo II proteins to
ICRF-193 correlates with the ATPase activity of these proteins. For the
topo II constructs with a higher ATPase activity, there is a
concomitant increase in the sensitivity toward bisdioxopiperazine.
These results also suggest the bisdioxopiperazine drugs can directly
effect the ATPase domain of eukaryotic topo II to inhibit its ATP
hydrolysis activity. However, we cannot rule out the possible
involvement of the other domains of the enzyme in the action of these
drugs, since the drug sensitivity increases when other domains of the
holoenzyme are also included in the protein constructs.
Dimerization of ATPase Domain--
The closure of the N-gate
through dimerization of the NH2-terminal domain plays a
critical role in the initiation of the catalytic cycle of topo II
reaction. Furthermore, since the mechanism of action of
bisdioxopiperazine drugs involves the closure of the N-gate, they may
affect the dimerization of ATPD. We have examined the effects of
cofactors on the dimerization of the ATPD by monitoring the change in
the molecular mass by chemical cross-linking and by sedimentation
equilibrium. In the cross-linking experiments, dimethylsuberimidate was
used to covalently bridge the dimerized protein (Fig.
6). For ATPD from both
Drosophila and human topo II, no dimerized species were
detected in the absence of any cofactor (Fig. 6A,
lanes 2 and 7) or in the presence of
ICRF-193 alone (Fig. 6A, lanes 4 and
9). In the presence of ATP, a significant fraction of the
protein is in the dimeric form as revealed by the multiple bands of
cross-linked species (lanes 1 and 6).
However, the addition of ICRF-193 did not increase the amount of the
cross-linked protein (compare lanes 3 and
8 with lanes 1 and 6),
while the presence of AMPPNP increased the amount of the cross-linked
protein species (lanes 5 and 10).
Therefore, ATPD can indeed dimerize in the presence of ATP or its
nonhydrolyzable analog. However, under the experimental conditions
tested here, the effect of ICRF drugs on the cofactor-induced
dimerization appears to be minimal.
The observation of the dimerized species of the ATPase domain in the
presence of ATP is interesting, since under the conditions of
cross-linking experiments, a significant fraction of ATP will be
converted into ADP. It is therefore interesting to examine whether ADP
can also promote the dimerization of the ATPase domain fragments. As
shown in Fig. 6B, in the presence of 1 mM ADP, a similar amount of cross-linked HsATPD species was detected as compared
with that formed in the presence of 1 mM ATP. Furthermore, the similar effect of ADP in promoting the dimerization has also been
observed with DmATPD (data not shown). Therefore, ADP can induce the
dimerization of ATPase domain, suggesting that it may play a role in
closing the N-gate clamp.
To gain a more quantitative picture of the nucleotide cofactor-induced
dimerization of ATPD, we carried out sedimentation equilibrium analysis
to monitor the molecular mass of HsATPD under various conditions (Fig.
7). In the absence of any cofactor, the best fit molecular mass of the protein was determined to be 48.9 kDa, very close to the calculated molecular mass 46,743 Da, indicating that the protein is almost exclusively in its monomeric form (Fig. 7A). In the presence of 1 mM ATP, the best fit
molecular mass increases to 71.7 kDa, indicating significant
dimerization of the protein under this condition (Fig. 7B).
However, the addition of 100 µM ICRF-193 does not cause
any change in the oligomerization state of HsATPD, as indicated by the
similar molecular mass under this condition (Fig. 7C).
HsATPD also stays as monomer in the presence of ICRF-193 alone (data
not shown). Interestingly, while AMP cannot promote the dimerization of
HsATPD (Fig. 7E), ADP can induce the dimerization to a
similar extent as ATP (Fig. 7D). In the presence of a
nonhydrolyzable ATP analog, AMPPNP, the best fit molecular mass of
HsATPD further increases to 93.4 kDa, suggesting that the protein is
predominantly in dimeric form under this condition (Fig.
7F). This result is in interesting contrast to the earlier study on the 43-kDa ATPase domain from E. coli gyrase, which
exhibits monomeric molecular weight in the presence of either ATP or
ADP (39). On the other hand, a human topo II ATPase domain (residues 1-439) that was purified from the bacterial expression system exists
as dimer regardless of the presence of cofactors (25). However, the
quadratic dependence of the ATPase activity on the concentrations of
ATPase domains from bacterial gyrase and topo II from yeast,
Drosophila, and humans would suggest that there is a dynamic
equilibrium for monomer/dimer interconversion in the presence of the
nucleoside triphosphates (see Refs. 39 and 24 and this work).
The sedimentation equilibrium experiments provide us an opportunity to
estimate the quantitative effects of the cofactors on the dimerization
of ATPase domain. The apparent dimerization constants were determined
based on the global fitting of the average molecular weight data from
the equilibria established in three separate centrifugation speeds, and
they were plotted as a function of ADP concentrations (Fig.
8). These apparent dimerization data points were fitted with an equation assuming that the major dimer species are either the unliganded dimer or fully liganded dimer (see
"Experimental Procedures"). The results from the curve fitting indicate that the ADP binding increases the dimerization constant by
60-fold (K2 = 2.5 × 105
versus K0 = 4.0 × 103 M The wealth of information on the domain structure of
bacteria and eukaryotic topo II allows us to make truncation mutants of
the protein to study the individual domains of the protein. We
constructed NH2-terminal fragments from both
Drosophila and human topo II and studied the ATPase activity
associated with these fragments. In the presence of DNA, the ATPase
activity can be stimulated by about 20-fold for these constructs. The
DNA-stimulated ATPase activity of the ATPase domains is intriguing,
since the constructs lack the structure responsible for the binding and opening of the G-segment of DNA. However, these
NH2-terminal constructs still retain the binding sites for
the T-segment of DNA, and such binding may promote the ATPase activity
in the ATPD. The mechanistic significance of this stimulation of ATPase
activity in the ATPD remains to be determined. In the absence of DNA,
we have observed a nearly parabolic dependence of ATPase activity on
the enzyme concentration. These results are consistent with the
previous studies using the corresponding fragments from
Escherichia coli GyrB and yeast topo II (39, 24), suggesting
that there exists a monomer-dimer equilibrium of the ATPD fragments,
with the dimer form being the catalytically active species. In the
presence of DNA, Drosophila and human ATPD still retain the
quadratic dependence on concentration, indicating that the addition of
DNA does not significantly shift the equilibrium toward dimer
formation. In contrast, under a similar condition, yeast ATPD shows a
linear dependence on the concentration (24). Therefore, DNA may have a
different effect on the monomer/dimer equilibrium for ATPD from different species.
The mechanism of action of the anti-tumor agent
bisdioxopiperazine clearly involves locking up the N-gate that is
closed in the presence of ATP (20). Recent rapid kinetic analysis
suggests that in the closed clamp trapped by bisdioxopiperazine, one
subunit in the dimer can still continue to hydrolyze ATP at a reduced rate (21). Data from earlier works with the yeast ATPD (24) and those
presented here for Drosophila and human ATPD indicate that
the drug can directly act on this part of the topo II protein. However,
the data presented here also demonstrate that the sensitivity toward
this drug varies greatly among the various constructs for topo II;
those having a higher ATPase activity also display a higher drug
sensitivity. It is possible that the major effects of these additional
domains are mediated through their influence on the dimerization of the
ATPD, thus affecting their sensitivity toward the drug. However, we
cannot rule out the possible involvement of other domains in the effect
of ICRF-193 on topo II activities. Furthermore, while the
bisdioxopiperazines, just like AMPPNP, can promote the formation
of the salt-stable clamp in the holoenzyme (20, 27), they have
little effect on the dimerization of ATPD beyond that promoted by ATP
or ADP (see Figs. 6 and 7). Therefore, the presence of these
extra-ATPase domains is necessary for the formation of very stable
clamp complex in the presence of ATP/ICRF-193, and they may have
additional roles in the dimerization of ATPD and in the ICRF-193
sensitivity. While the exact biochemical basis of these additional
roles remains unclear, it is interesting that our earlier work showed
that bisdioxopiperazine can affect the binding of DNA to the core
domain of topo II, the topo II construct without ATPD (27), and recent
data have indicated that this drug can promote the DNA cleavage by topo
II (30).
The catalytic cycle of topo II starts with the binding of ATP to the
NH2-terminal ATPase domain and the closure of the
NH2-terminal gate to entrap the T-segment of DNA (13). The
transient kinetic experiments suggest that the DNA transport event in
which the T-segment passes through the G-segment happens after one of
the two ATP molecules bound to the homodimer is hydrolyzed (17, 18).
The fate of the closed N-gate following the ATP hydrolysis remains to
be addressed. Using both sedimentation equilibrium and a cross-linking
assay, we have tested the dimerization of the ATPase domains under
different conditions. Our data demonstrate that the presence of ADP can
enhance the dimerization equilibrium constant by about 2 orders of
magnitude. These data suggest that the N-gate may remain closed
following the ATP hydrolysis and will only reopen upon the dissociation
of ADP. Therefore, the topo II catalytic cycle utilizes the binding and
hydrolysis of ATP as well as the dissociation of ADP to drive the key
conformational changes necessary for the DNA transport event. As a
molecular machine, topo II is fueled by ATP through a series of
coordinated conformational changes that are controlled by the binding
and dissociation of nucleotide cofactors.
,
-imidodiphosphate. More interestingly,
both ATP and ADP can also promote protein dimerization. This result
thus suggests that the protein clamp, mediated through the dimerization
of ATPase domain, remains closed after ATP hydrolysis and opens upon
the dissociation of ADP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
22 (32). The 3' primer contains the sequence
encoding residues 397-403 of Drosophila topo II and
His6 tag (italicized), followed by a stop codon (boldface
type) and a unique SphI site (underlined). The PCR fragments
were cloned back to the YEpWOB6 and YGBBX22 vector using the above
restriction enzymes. To generate the DmGyrB construct, we used TM10,
which is a linker insertion construct containing a 4-residue
(His-Ala-Cys-Lys) insertion between residues 668 and 669 of
Drosophila topo II (33). This insertion site is located at
the junction of GyrB and GyrA domains, and there is a unique
MluI site in the linker sequence. To generate a truncation
at this site, the following two oligonucleotides were synthesized:
5'-CGC GCA TCA TCA TCA TCA CTA GCT
GAC ATG-3' and 5'-TCA GCT AGT GAT GAT GAT GAT
G-3'. These sequences contain a 5' MluI cohesive end
and a 3' SphI cohesive end (underlined), a 5-histidine tag
(italicized), and a stop codon (boldface type). They were annealed to
form the adapter and inserted into TM10 between the MluI
site and SphI site to generate the DmGyrB construct. The
sequence 5' to that coding for pentahistidine, derived from part of the
linker insertion sequence, codes for His-Ala. Together, they generate
His-Ala-His5 at the carboxyl terminus of the DmGyrB protein construct.
20 = 1.006 (solvent density), measured by picnometry. Achievement of
sedimentation equilibrium was confirmed by time lapse scans and, more
importantly, by the agreement of calculated best fit molecular weights
from sequential sedimentation scans at each speed.
2 minimization in a nonlinear least
squares model to globally fit the sedimentation data. The S.E. in all
of the dimerization constants calculated from such analysis is usually
smaller than 10%.
(Eq. 1)
(Eq. 2)
N and NA represent the unliganded and liganded monomer of ATPD,
respectively, and A represents the cofactor ADP.
K1 defines the association constant of ADP with
ATPD. K0 and K2 are the
dimerization constants for unliganded and liganded monomer,
respectively. Inclusion of the equilibrium involving the association of
heterotypic monomer, N and NA, does not alter the curve-fitting
described below, suggesting that the above equilibria are parsimonious
with respect to the data analysis presented here.
(Eq. 3)
Substituting Equations 1-3 into Equation 4, we obtain the
following.
(Eq. 4)
By curve-fitting of the plot
K
(Eq. 5)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 1.
Schematic diagram of wild-type
(A) and truncated topo II, DmGyrB
(B), DmATPD (C), and HsATPD
(D). A coordinate of the Drosophila
topo II peptide is shown at the top. The boxed
region represents the sequences highly conserved among topo
II proteins. The regions homologous to ATPase domain, B' fragment, and
GyrA subunits of bacterial gyrase are represented by
open, shaded, and dotted
boxes, respectively. The nonconserved COOH-terminal region
that contains a number of charged residues (denoted by + +/ 
) is
represented by a thick line. The
arrowheads mark two trypsin cleavage sites determined in a
previous study (33). The solid box in the diagram
for the full-length enzyme (A) refers to the ATP binding
site, and Y indicates the location of active site
tyrosine.
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Fig. 2.
Purification of the truncated topo II. 2 µg each of molecular weight size markers and three truncation topo II
proteins were run in a 7% SDS-PAGE and stained with Coomassie
Brilliant Blue. DmATPD, HsATPD, and DmGyrB are shown in
lanes 2, 3, and 4,
respectively.
(data not shown).
Therefore, dimerization of the GyrB domain of topo II is favored at
equilibrium, and this process is not rate-limiting for the ATPase
reaction.
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Fig. 3.
ATPase activity of HsATPD and DmATPD in the
absence (A) and presence (B) of
DNA. The rate of ATP hydrolysis is plotted as a function of enzyme
concentration (as monomers). The diamonds and
circles are for DmATPD and HsATPD, respectively. The
curves drawn through the data points were based on the
quadratic dependence on the enzyme concentrations.
View larger version (9K):
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Fig. 4.
Dependence of the ATPase rate of the HsATPD
(A) and DmATPD (B) on ATP
concentrations. Rates are initial velocities measured in the
presence of DNA at a monomeric protein concentration of 2.2 µM. The curve was drawn based on the
Michaelis-Menten kinetics with Vmax = 27.3 µM/min and K 
Apparent kcat for the topo II proteins
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Fig. 5.
Inhibition of the ATPase activity of
wild-type and truncated topo II by the bisdioxopiperazine
ICRF-193. DNA-dependent ATPase activity was measured
at various ICRF-193 concentrations and normalized against the ATPase
activity measured in the absence of the drug. Filled
circle, human wild type; filled
triangle, Drosophila wild type; open
circle, HsATPD; open triangle, DmATPD;
open diamond, DmGyrB. Notice that while the
ATPase activities differ greatly among the various protein constructs
(Table I), only the relative ATPase activities are shown here by
normalizing against the activity under the conditions without
inhibitors.
View larger version (52K):
[in a new window]
Fig. 6.
Cross-linking of the HsATPD and DmATPD with
dimethylsuberimidate. A, Coomassie staining of
SDS-polyacrylamide gels showing cross-linking of the HsATPD and DmATPD
in the absence of any cofactor (lanes 2 and 7) or
in the presence of 1 mM ATP (lanes 1 and
6), 100 µM ICRF-193 (lanes 4 and
9), 1 mM ATP and 100 µM ICRF-193
(lanes 3 and 8), and 1 mM AMPPNP
(lanes 5 and 10), respectively. B,
cross-linking of the HsATPD in the absence of any cofactor, in the
presence of 1 mM ATP, and in the presence of 1 mM ADP, respectively.
View larger version (25K):
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Fig. 7.
Sedimentation equilibrium measurement of the
molecular weight of HsATPD. The UV absorbances of HsATPD were
measured as a function of the distance from the center of rotation in
the centrifuge (radius). Cofactors added to the solutions were as
follows: none (A), 1 mM ATP (B), 1 mM ATP and 100 µM ICRF-193 (C), 1 mM ADP (D), 1 mM AMP (E),
and 1 mM AMPPNP (F). We used the
Ideal-1 program for curve fitting to obtain the average
molecular weight under each condition. The circles are the
measured absorbances, and the curve is based on the best fit
molecular weight shown in each graph. The residuals of each fit are
also shown above the graph. The run shown here was carried
out with a speed of 12,000 rpm. Data obtained with centrifugation
speeds of 9,000 and 15,000 rpm from the identical samples yielded the
same results in the calculated molecular weights.
1), and at the saturating
concentration of ADP, the dimer dissociation constant approaches 4 µM (dimer association K2 = 2.5 × 105 M1). The
dissociation constant for ADP is in the range of 0.13 mM (ADP association constant K1 = 7.7 × 103 M1), comparable with the
Ki of ADP being 0.12 mM for the inhibition of relaxation activity by Drosophila topo II
(40). Therefore, ADP can bind ATPD, and this binding can also induce dimerization of ATPD, suggesting that ADP may maintain the N-gate in a
closed form. The result presented here indicates that the release of
ADP from the ATPase domain is essential for the dimerized proteins to
come apart and to initiate a new round of catalytic cycle. While ADP
can induce the dimerization of ATPD, the dimer conformation and its
interface may be different from that present in the dimer generated by
the other cofactors. A similar analysis with the nonhydrolyzable
cofactor AMPPNP indicates that it can induce the dimer formation with
an equilibrium constant higher than that seen in the presence of ADP by
at least 2 orders of magnitude (K2 > 5 × 107 M1; data not shown). The
ability of the protein domains to adopt differential conformation
states upon binding with different cofactors may be a key feature for
the functions of topo II enzyme.
View larger version (12K):
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Fig. 8.
The calculated apparent dimerization
constants of HsATPD as a function of ADP concentrations. The
apparent dimerization constants were calculated using a global fitting
program for the measured molecular weight data obtained from three
sedimentation speeds (see "Experimental Procedures" for details).
The curve drawn through these data points was based on
Equation 5, with K1, K2,
and K0 being 7.7 × 103,
2.5 × 105, and 4.0 × 103
M 1, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENT |
|---|
We are grateful to Dr. Allen Minton (NIDDK, National Institutes of Health, Bethesda, MD) for the generous gift of the computer software for data analysis of sedimentation equilibrium experiments.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant GM29006.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 919-684-8885;
E-mail: Hsieh@biochem.duke.edu.
Published, JBC Papers in Press, December 7, 2001, DOI 10.1074/jbc.M111394200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
topo II, topoisomerase(s) II;
ATPD, NH2-terminal ATPase domain;
DmATPD, Drosophila melanogaster ATPD;
HsATPD, Homo sapiens ATPD;
DmGyrB, D. melanogaster
GyrB domain;
AMPPNP, 5'-adenylyl-,-imidodiphosphate.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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