From the Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, Århus C 8000, Denmark
Received for publication, October 9, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Eukaryotic DNA topoisomerase II is a dimeric
nuclear enzyme essential for DNA metabolism and chromosome dynamics. It
changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and
functionally complex enzyme passes through a cascade of conformational changes, which requires intra- and intersubunit communication. To study
the importance of ATP binding and hydrolysis in relation to DNA strand
transfer, we have purified and characterized a human topoisomerase
II DNA topoisomerase II is a multifunctional and highly complex
enzyme, which uses the energy of ATP to resolve topological problems generated during DNA metabolic processes, including DNA replication, transcription, and recombination (1, 2). Beyond these functions, topoisomerase II is an abundant component of the mitotic chromosome scaffold (3), and it alleviates constraints in the DNA during chromosome segregation and condensation (1).
To preserve the topological integrity of the genome, the dimeric
topoisomerase II enzyme strictly controls the passage of one duplex DNA
(the T-segment) through another duplex (the G-segment) coordinately
cleaved by the enzyme. ATP is required during the process to drive the
enzyme through a series of conformations, which are essential to
topoisomerase II catalysis.
During the last years, extensive research has provided valuable
information about the usage of ATP during the reaction pathway of
topoisomerase II, but still several steps in this highly complex process remain to be unraveled. A study of the ATP consumption in yeast
topoisomerase II has indicated that a tight coupling exists between ATP
utilization and DNA transport under unsaturated ATP concentrations (5).
However, a human topoisomerase II enzyme lacking amino acids 350-407
at the interface between the ATPase and the cleavage/ligation domain
was unable to perform strand passage, although ATPase and cleavage
activities were intact (6). These results indicate that correct
interdomain communication as well as signaling of ATP binding and
hydrolysis to the rest of the enzyme are essential for the coupling of
ATP consumption and DNA transport.
The binding of ATP is cooperative in the presence of DNA (5) and is
thought to be stimulated by the binding of the G-segment (7). A study
of a yeast topoisomerase II heterodimer having only one ATP binding
site has demonstrated that conformational changes are induced in the
entire enzyme upon ATP binding, because the enzyme was still able to
trap circular DNA by closing the N-terminal clamp. This indicates that
allosteric communication exists between the two enzyme subunits (8).
Together with results obtained from studies employing nonhydrolyzable
ATP analogues (9, 10), this initially led to the assumption that ATP
binding triggers the conformational changes needed for one DNA
transport event, whereas ATP hydrolysis and product release are
exclusively coupled to enzyme resetting (4).
However, recent discoveries employing rapid quench techniques and
pre-steady-state analysis of ATP hydrolysis in the yeast enzyme have
revealed that hydrolysis of the two ATP molecules occurs sequentially
with one of the two ATP molecules being rapidly hydrolyzed before the
rate determining step in the reaction cycle (11). This sequential
hydrolysis is explained in terms of a model, where hydrolysis of one
ATP precedes and accelerates transport of the T-segment, although the
reaction can occur in the absence of hydrolysis (12). Hydrolysis of the
first ATP and release of Pi and ADP are supposed to induce
conformational changes in the N-terminal ATPase region of the enzyme,
which are transmitted to the central DNA cleavage/ligation domain,
where large rotations are proposed to form a gate in the cleaved
G-segment to allow transport of the T-segment. This conformational
cascade is predicted to occur in both subunits, such that the enzyme
maintains structural symmetry, despite the different nucleotide bound
states of the two subunits (4, 12). Investigations of a yeast
topoisomerase II heterodimer, which hydrolyzes only one of the two
bound ATP molecules, have shown that this enzyme is able to perform DNA strand passage at the same rate as the wild type enzyme, although resetting of the enzyme is delayed, probably due to the bound unhydrolyzed ATP, which keeps the clamp closed. Based on these results,
it has been hypothesized that the second hydrolysis event and the
subsequent product release are linked to resetting of the enzyme (12).
However, it is still unknown to what extend binding of the second ATP
per se influences the individual steps in the catalytic cycle.
To investigate the coupling of ATP binding and hydrolysis to DNA strand
passage and enzyme resetting, a human topoisomerase II Yeast Strains and Plasmids--
The Saccharomyces
cerevisiae strain JEL1 Introduction of the G164I Point Mutation--
Two rounds of PCR
were carried out to introduce the G164I point mutation in the
human topoisomerase II Construction of Plasmids--
To construct a plasmid expressing
the G164I mutated topoisomerase II Yeast Transformation--
Yeast cells were transformed using
electroporation and transferred to SC-media plates lacking uracil to
select against the YEpWOB6-based constructs.
Human Topoisomerase II Hydrolysis of ATP by Topoisomerase II--
The ATPase assay was
based on the method of Osheroff et al. (17). Reactions
contained 150 nM topoisomerase II and 30 nM negatively supercoiled pUC19 and were carried out in 20 µl of 50 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 5 mM MgCl2, 2.5% glycerol, and 140 mM KCl containing a final concentration of 1 mM
cold ATP and 0.08 nM of [ ATP Binding Assay--
150 nM topoisomerase II Topoisomerase II-mediated DNA Relaxation--
DNA relaxation was
performed by incubating 2.2 nM topoisomerase II and 5.5 nM negatively supercoiled pUC19 in 50 mM
Tris-HCl, pH 7.9, 0.1 mM EDTA, 5 mM
MgCl2, 1 mM ATP, 2.5% glycerol, and 125 mM KCl at 37 °C. Reactions were stopped by adding 3 µl
of 0.77% SDS and 77 mM EDTA after 5 min unless otherwise
indicated. Samples were subjected to electrophoresis in 1% agarose
gels in 100 mM Tris borate, pH 8.3, 2 mM EDTA.
Gels were stained with 1 µg/ml ethidium bromide and visualized by UV
light using the Gel Doc 2000 from Bio-Rad.
Topoisomerase II-mediated DNA Cleavage--
DNA cleavage was
performed by incubating 6.6 nM topoisomerase II with 5.5 nM negatively supercoiled pUC19 in 50 mM
Tris-HCl, pH 7.9, 0.1 mM EDTA, 5 mM
MgCl2, 1 mM ATP, 2.5% glycerol, and 125 mM KCl for 10 min at 37 °C. Cleavage products were
trapped by addition of SDS to 1%, and samples were treated with 2 µl
of 0.8 mg/ml proteinase K before being subjected to electrophoresis in
1% agarose gels. Southern blotting was performed using Zeta-Probe GT
membranes (Bio-Rad) and random primed plasmids for hybridization (Roche
Molecular Biochemicals). A Molecular Imager was used for gel scanning.
Clamp Closing Assay--
Clamp closing experiments were
performed as described by Bjergbaek et al. (6). Briefly, 1 nM topoisomerase II was incubated with 2.5 nM
supercoiled and 2.5 nM linearized pUC19 DNA at 37 °C for
5 min before ATP or AMPPNP was added to a final concentration of 1 mM. Reactions were stopped by the addition of SDS or NaCl to final concentrations of 1% and 800 mM, respectively,
and after addition of 1 volume of phenol, DNA trapped by topoisomerase
II-mediated clamp closure was isolated from the phenol interphase.
Following proteinase K treatment, the trapped DNA was subjected to
electrophoresis and Southern blotting together with the DNA recovered
from the phenol water phase. A Molecular Imager was used for
quantification. The amount of enzyme-trapped circular DNA was
determined by subtracting the amount of linear DNA in the interphase
from the total amount of supercoiled circular and relaxed circular DNA
in the interphase.
Purification of a Human Topoisomerase II The Mutation G164I disrupts ATP Binding and Hydrolysis Activities
of Human Topoisomerase II
The capability of the G164I homodimer to hydrolyze ATP was subsequently
examined using thin-layer chromatography. The ATPase activity of the
enzyme was equivalent to the background levels observed in the absence
of enzyme (Fig. 2B). Taken together, these results
demonstrate that conversion of Gly-164 to isoleucine in human
topoisomerase II The G164I/wt Heterodimer Is Able to Perform DNA
Relaxation--
Yeast topoisomerase II has been shown to hydrolyze
only one ATP during transport of the T-segment, whereas the second ATP is thought to be essential for resetting of the enzyme (12). To
investigate the importance of the second ATP for human topoisomerase II The G164I/wt Heterodimer Possesses a Normal Clamp
Closing Activity--
Binding of the first ATP molecule to
topoisomerase II is presumed to induce closure of the N-terminal clamp,
and this is followed by binding of a second ATP (8). To test if the
lack of a second ATP molecule influences clamp closing, we analyzed the
heterodimer in a clamp closing assay. The assay is based on a stable
closure of the N-terminal clamp upon binding of a nonhydrolyzable ATP analogue. If the wild type enzyme is incubated with equal amounts of
linear and circular DNA before the addition of the ATP analogue, AMPPNP, only circular DNA will become trapped by the enzyme, and the
interlinked enzyme-DNA complexes can be collected from a water-phenol interphase (Fig. 4, lanes 1 and 2) (6). When G164I/wt was incubated in the presence of
AMPPNP, the amount of supercoiled DNA trapped in the interphase was
equivalent to the amount trapped by the wild type enzyme (Fig. 4,
compare lanes 1 and 2 with lanes 7 and 8) showing that the heterodimer has retained the ability to
close the N-terminal clamp. When the enzymes were incubated with ATP rather than the ATP analogue, no DNA trapping took place, demonstrating that G164I/wt like the wild type enzyme reopens the clamp upon ATP
hydrolysis (Fig. 4, lanes 6 and 12). Furthermore,
neither enzyme allowed trapping of DNA upon addition of SDS, which
disrupts the interlink between enzyme and DNA (Fig. 4, lanes
4 and 10) or in the absence of AMPPNP, where clamp
closing does not occur (data not shown). Together, the results show
that the heterodimeric N-terminal clamp operates with a mechanism
similar to the clamp in the wild type enzyme. Thus, binding and
hydrolysis of one ATP molecule is sufficient to allow both clamp
closing and reopening in agreement with the observed relaxation
activity.
The G164I/wt Heterodimer Is Sensitive to
ICRF-187--
The bisdioxopiperazines, ICRF-193, ICRF-187, and
ICRF-159, are catalytic inhibitors of topoisomerase II, which have been
shown to lock the enzyme on DNA by trapping it in the closed clamp form (19, 20). Several point mutations leading to resistance to these drugs
indicate that their effect is mediated through an interference with the
ATPase domain (21-24). Steady-state and rapid kinetic analyses
performed by Lindsley and coworkers have suggested that
bisdioxopiperazines bind to the enzyme-ADP complex after DNA transport
has occurred and have no effect on hydrolysis of either the first or
the second ATP. Instead the drugs inhibit dissociation of the second
ADP, thereby preventing the enzyme from catalytic turnover (25). To
test whether the heterodimer can adopt the conformation required for
bisdioxopiperazine interaction and further investigate how these drugs
perturb the mechanism of topoisomerase II, we investigated the effect
of ICRF-187 on the ability of G164I/wt to perform DNA relaxation. Fig.
5 illustrates that 100 µM
ICRF-187 decreases the relaxation activity of the heterodimer, whereas
activity is completely abolished at a concentration of 500 µM. These results predict that, when binding and
hydrolysis of only one ATP is possible, the enzyme is still able to
enter a definite conformational state, where recognition and binding of
ICRF-187 can occur, resulting in a trapping of the enzyme in the closed
clamp form.
DNA Cleavage by the G164I/wt Heterodimer Is Unaffected
by ATP--
Because G164I/wt has a reduced relaxation activity, we
investigated if the catalytic site for cleavage and ligation located in
the central domain of topoisomerase II still constitutes a full
functional domain. For this purpose cleavage experiments were performed
with G164I/wt employing the circular DNA substrate used for relaxation.
When topoisomerase II and DNA are incubated, a cleavage/ligation
equilibrium is normally established, where the amount of enzyme-DNA
cleavage complex intermediates that can be trapped, when a denaturating
agent is added, depends on both the DNA cleavage and ligation rates. If
ATP or an ATP analogue is present, strand passage is allowed, and the
equilibrium will be shifted toward cleavage, resulting in a 3- to
5-fold increase in cleavage complex formation (6, 10, 26). When
cleavage was carried out in the absence of nucleotide, cleavage complex formation by G164I/wt and the wild type enzyme was similar (Fig. 6). However, in contrast to the 4-fold
stimulation of cleavage obtained with the wild type enzyme in the
presence ATP or the ATP analogue, AMPPNP, cleavage complex formation by
G164I/wt was not influenced by nucleotide (Fig. 6), demonstrating a
role of the second ATP molecule in this stimulation. In conclusion,
these results suggest that the central domain of G164I/wt operates
normally with respect to DNA binding, cleavage, and ligation. However, the inability of ATP or the ATP analogue to stimulate cleavage indicates an impaired ability of the heterodimer to transmit
information between the N-terminal ATPase and the central
cleavage/ligation domains, which might contribute to the lower
relaxation activity observed with G164I/wt.
ATP Binding and Hydrolysis Activities of the G164I/wt
Heterodimer Are Independent of DNA--
It has previously been
presumed that the second ATP in topoisomerase II plays a role during
enzyme resetting (4, 12). However, the cleavage results presented in
Fig. 6 strongly indicate that binding of a second ATP molecule is also
essential for conformational changes initiated earlier in the catalytic
cycle. To further study this hypothesis, we examined the ability of DNA
to stimulate the ATP binding and hydrolysis activities of G164I/wt.
Because the ATP binding and hydrolysis assays require higher amounts of
enzyme than that obtained from the two-affinity tag purification
method, another purification was performed using only one affinity tag, in this case a hexahistidine tag on the G164I mutant subunit (see "Experimental Procedures"). The purified enzyme fractions, which contained both G164I/wt heterodimers and G164I homodimers, were suitable for analysis given that the ATP binding- and hydrolysis activities of the G164I homodimer were abolished (Fig. 2). The results
presented in Fig. 7A
illustrate that binding of ATP[
Earlier studies on human topoisomerase II The current mechanistic model of topoisomerase II catalysis
involves a complex sequential mechanism of ATP hydrolysis, where binding and hydrolysis of ATP drive the enzyme through a series of
conformations. Whereas hydrolysis of one ATP is thought to accelerate
transport of the T-segment through a gate formed upon cleavage of the
G-segment, it is presently unclear how the second ATP is correlated to
the remaining steps of the catalytic cycle, although a connection to
the resetting of the enzyme has been suggested (11, 12). In the present
study we have investigated the coupling between ATP binding/hydrolysis
and DNA strand passage/resetting by studying a human topoisomerase
II Investigation of the enzyme having the G164I mutation in both subunits
showed that it was unable to bind and hydrolyze ATP, demonstrating that
Gly-164 is essential for ATP interaction in human topoisomerase II The amino acid residue mutated in G164I/wt is located in the highly
conserved Walker A motif, which is a glycine-rich region found in the
ATP binding domain of all known type II topoisomerases (29). Assuming
that the ATP binding domain folds into a structure similar to the one
formed in gyrase B, Gly-164 would be located on a flexible loop of the
Walker A motif. This motif is in close contact with another loop, which
extends from an enzyme domain located further toward the
cleavage/ligation domain. Both loops contact the The ability of G164I/wt to perform clamp closure was further manifested
through its sensitivity to the bisdioxopiperazine ICRF-187. Recent data
have indicated that ATP binding is required for efficient binding of
bisdioxopiperazines to the enzyme (25). In fact, investigations
performed by Lindsley and coworkers have suggested that
bisdioxopiperazines bind to the enzyme-ADP complex after hydrolysis of
both ATPs and after dissociation of the products of the first
hydrolysis (25). The sensitivity of G164I/wt to ICRF-187 predicts that
binding and hydrolysis of only one ATP induce a specific conformation
in the N-terminal clamp, where binding of the drug is possible. This
suggests that the N-terminal conformation adopted by the G164I/wt
heterodimer after hydrolysis of the first ATP at a point becomes
similar to the conformation attained by the wild type enzyme after
hydrolysis of the second ATP.
The ability of G164I/wt to perform more than stoichiometric relaxation
demonstrates that the enzyme is able to go through successive catalytic
cycles. Binding and hydrolysis of only one ATP molecule is therefore
sufficient for resetting of the enzyme, although we cannot exclude that
the decreased relaxation activity observed with the heterodimer to some
extent is caused by a less efficient resetting. However, the lack of
DNA-stimulated ATP binding and hydrolysis and the lack of
ATP-stimulated DNA cleavage observed in the heterodimer demonstrate
that steps in the catalytic cycle prior to the resetting event are
affected, when the second ATP is inhibited from binding. Thus, although
the clamp closing data suggest that the intersubunit communication is
retained in the N-terminal region, lack of a second ATP seems to
influence the intrasubunit communication at least between the ATP
binding region and the central cleavage/ligation domain.
Studies of a yeast topoisomerase II mutant lacking cleavage activity
have indicated that DNA stimulation of the ATP binding rate is
primarily mediated through G-segment binding, and it has been suggested
that binding of the T-segment might be required to obtain full
stimulation of ATP hydrolysis (7). Additionally, investigation of wild
type human topoisomerase II Like ATP binding, ATP hydrolysis in G164I/wt is unaffected by DNA
binding, and the enzyme does not show the normal stimulatory effect of
ATP on DNA cleavage. When topoisomerase II-mediated cleavage is
performed in the presence of ATP or an ATP analogue, the DNA
cleavage/ligation equilibrium is normally shifted toward cleavage. This
shift has been suggested to result from a delay in the ligation
reaction due to formation of a transient gate in the cleaved G-segment
that can accommodate T-segment transport (10, 31). However, the
heterodimer lacked the stimulatory effect of ATP on cleavage complex
formation, although the active site tyrosines were able to move apart
to allow transport of the T-segment. Based on these results, binding of
the second ATP molecule could be required for stabilization of a gate
already formed upon binding of the first ATP, or it could influence the
rate of cleavage per se. Alternatively, if signaling between
the ATP binding domain and the cleavage/ligation domain requires a
functional interaction between the two ATP binding sites, the
stimulatory effect of the nucleotide on cleavage could be abolished in
the heterodimer.
Cleavage might indirectly be related to a DNA-dependent
stimulation of ATP hydrolysis. This has been suggested earlier from studies with yeast topoisomerase II, where an active site mutant unable
to perform DNA cleavage was found to have a decreased rate of ATP
hydrolysis in the presence of DNA. The data were explained by an
inability of the mutant enzyme to adopt a specific cleavage conformation needed for the stimulatory effect of DNA on ATP hydrolysis or by an inability of the enzyme to interact properly with the T-segment, thus causing a decreased ATP hydrolysis (7). In the G164I/wt
heterodimer, ATP hydrolysis is unaffected by DNA, although the
heterodimer can trap a T-segment. It is therefore more likely that
binding of the second ATP molecule, either directly or indirectly, is
necessary for the ability of DNA to stimulate the rate of ATP
hydrolysis at least of the first ATP molecule. Such a coupling between
binding of the second ATP and hydrolysis of the first would seem
natural, because it would place the different events in the correct
order, ensuring that G-segment binding and cleavage have occurred
before ATP hydrolysis allows T-segment transport.
Together, our data can be explained in a model in which the catalytic
cycle begins with binding of a G-segment and one ATP molecule to the
enzyme. Binding of the first ATP will lead to clamp closure and
consequently trapping of a T-segment. After clamp closure the G-segment
will facilitate binding of another ATP molecule. This ATP could now
stimulate G-segment cleavage or stabilize the gate in the G-segment.
Either through this event or via the T-segment the second ATP will
stimulate hydrolysis of the first ATP molecule, which will force the
T-segment through the subunit interface and the gate in the G-segment.
Because DNA ligation is favored over cleavage in the normal
cleavage/ligation equilibrium, the gate in the G-segment might be
passively ligated following strand passage. At this step, the T-segment
needs to be expelled through the primary dimerization region, and the
enzyme subsequently resets with opening of the N-terminal clamp. Either one or both of these events might be facilitated by hydrolysis of the
second ATP. Although the precise details of some of these steps are not
yet known, it is clear from the present study that binding of a second
ATP is not essential for DNA transfer and enzyme resetting but exerts
an important function in the communication between the N-terminal
ATPase domain and the cleavage/ligation domain to promote efficient catalysis.
heterodimer with only one ATP binding site. The heterodimer was
able to relax supercoiled DNA, although less efficiently than the wild
type enzyme. It furthermore possessed a functional N-terminal clamp and
was sensitive to ICRF-187. This demonstrates that human topoisomerase
II
can pass through all the conformations required for DNA strand
passage and enzyme resetting with binding and hydrolysis of only one
ATP. However, the heterodimer lacked the normal stimulatory effect of
DNA on ATP binding and hydrolysis as well as the stimulatory effect of
ATP on DNA cleavage. The results can be explained in a model, where
efficient catalysis requires an extensive communication between the
second ATP and the DNA segment to be cleaved.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
heterodimer,
constructed to bind only one ATP, has been purified and characterized.
The results obtained with the heterodimer demonstrate that
topoisomerase II can perform strand passage and resetting with only one
ATP bound and hydrolyzed. However, the second ATP molecule facilitates
interdomain communication, especially between the N-terminal ATP
binding region and the central cleavage/ligation domain and is thus
important for efficient catalysis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
top1 (
leu2 trp1
ura3-52 prb1-1122 pep4
his3::PGAL1-GAL4
top1::LEU2) (kindly provided by J. C. Wang) was used for overexpression of topoisomerase II
constructs.
For coexpression of wild type and mutant topoisomerase II
cDNAs,
YEpWOB6, containing the human topoisomerase II
cDNA under
control of the yeast GAL1 promoter, was used as a backbone. Plasmid
pRS315 was used as a backbone for pHT212, which contains the human
topoisomerase II
cDNA. Construction of pHT212 was described in a
previous study (13).
cDNA. In the first round, primer A
(5'-GTCGTCGAAATATCTATGGAGCC-3') containing the mutation that
changes Gly-164 to isoleucine (boldface letters) and primer B
(5'-GGATCCAGCAATATCATATG-3'), which corresponds to the antisense strand and contains a NdeI restriction site
(underlined) were used. The 265-bp PCR fragment synthesized using these
primers was gel-purified and used as antisense primer in a second round of PCR together with primer C (5'-CTAGCCGACGCGTCCATGGAAGTG-3'), which is located 5' of the human topoisomerase II
cDNA in
pHT212. The final PCR fragment was used to substitute the corresponding fragment of the hTOP2
cDNA in pHT212 employing MluI
and XbaI as 5' and 3' cloning sites, respectively, resulting
in pHT212-G164I.
enzyme behind the GAL1-promoter,
a DNA fragment containing the G164I mutation was excised from
pHT212-G164I by digestion with BglII and BsrGI.
The fragment was used to replace the corresponding hTOP2
cDNA
fragment in YEpWOB6. The hTOP2
cDNA was furthermore modified
with a hexahistidine tag at the 3'-end as described by Bjergbaek
et al. (6). The resulting plasmid was termed YEpWOBG164IHT. To fuse a hemagglutinin (HA)1
tag to the C-terminal end of human topoisomerase II
for antibody detection, PCR was carried out using pHT300 as a template. The antisense primer was designed using a stretch of 51 overhanging nucleotides containing the HA tag and an NheI restriction
site, whereas the sense primer (5'-GAGAGAGTTGGACTACAC-3') for cloning purposes contained a SpeI restriction site. The synthesized
fragment was gel-purified and inserted at the 3'-end of the hTOP2
cDNA in YEpWOB6 employing the restriction sites NheI and
SpeI. A DNA fragment encoding the glutathione
S-transferase (GST) tag used for purification was
subsequently ligated to the 3'-end of the HA-tagged hTOP2
cDNA.
The GST sequence was amplified from pET-HTG (14) using a sense primer
(5'-AGCTAGCCTGGTTCCGCGTGGATCTATGTC-3') containing a NheI
restriction site and an antisense primer
(5'-CCTTTTGCGGCCTATTATTTTGGAGGATGGTCGCCACCACC-3') containing a
NotI restriction site. The PCR fragment was inserted using
the NheI and NotI restriction sites. This
resulted in plasmid YEpWOBHAGST. To construct the plasmid used for the
production of topoisomerase II
heterodimers, YEpWOBG164I/wt (Fig.
1A), the hTOP2
cDNA fused to the HA and the GST tags
together with the GAL1-promoter was excised from YEpWOBHAGST using the
restriction enzymes SalI and NotI and inserted in
YEpWOBG164IHT digested with NotI and XhoI. The
plasmids YEpWOBwt/wt and YEpWOBG164I/G164I used for expression of the
wild type and the homodimeric mutant enzyme, respectively, were
constructed in a similar way. The constructs were sequenced to verify
the presence of the mutation G164I.
Induction, Overexpression, and
Purification--
Expression of the recombinant hTOP2
enzymes in
the JEL1
top1 yeast strain was induced by the addition of
galactose to glucose-free medium (15). Yeast cells were extracted with
1 volume of 50 mM Tris-HCl, pH 7.8, 1 M NaCl
and 1 volume of acid-washed glass beads (425-600 µm, Sigma). Further
preparation of yeast extracts was done according to the procedure of
Jensen et al. (13). The initial purification step using a
6-ml Ni2+-nitrilotriacetic acid-agarose column was
performed as described previously by Biersack et al. (16).
For further purification of the heterodimeric enzymes the fractions
pooled from the Ni2+ column was loaded onto a 1-ml GST
column (Amersham Biosciences), and elution was performed in a buffer
containing 100 mM Tris-HCl, pH 7.5, 500 mM
NaCl, 10% glycerol, 1 mM dithiothreitol, 1 mM
EDTA, and 25 mM glutathione. The fractions containing
topoisomerase II
enzyme were pooled, and, after addition of glycerol
to a concentration of 40%, the enzymes were stored in liquid nitrogen.
After each step of purification the eluted fractions were tested for
topoisomerase II
heterodimers on a silver-stained 6% polyacrylamide
gel. Ruby Protein Gel staining (Molecular Probes) of 3-8% Tris
acetate gradient gels (Novex Pre-Cast Gels) was performed to quantify
the purified topoisomerase II heterodimers. The same procedure was used
for purification of the wild type and homodimeric mutant enzymes, which
were used as controls in the enzyme activity assays. All three enzymes
were further purified according to the procedure described in Bjergbaek
et al. (6) involving only Ni2+-nitrilotriacetic
acid chromatography.
-32P]ATP (3000 Ci/mmol, Amersham Biosciences). Mixtures were incubated at 37 °C,
and 2.5-µl aliquots were taken at various time points and spotted
onto thin-layer cellulose plates impregnated with poly(ethyleneimine)
(TLC plastic sheets, PEI Cellulose F, Merck). Chromatography was
performed using freshly made 0.4 M
NH4HCO3. Levels of free Pi were
quantified by phosphorimaging (Molecular Imager, Bio-Rad).
The concentration of ATP hydrolyzed was determined by dividing the
Pi counts by the total number of counts per lane and
multiplying that fraction by the starting ATP concentration. The rate
of ATP hydrolysis was calculated from at least three different time courses.
was incubated with 0.3 nM [35S]ATP (600 Ci/mmol, Amersham Biosciences) and 5.5 nM negatively
supercoiled pUC19 in 50 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 5 mM MgCl2, 2.5% glycerol, and 140 mM KCl. Samples were incubated for 5 min
at 37 °C and loaded onto a nitrocellulose filter using a slot blot apparatus (PR 648 Slot Blot Manifold, Amersham Biosciences). Each sample was washed 10 times in a buffer containing 50 mM
Tris-HCl, pH 7.9, 0.1 mM EDTA, 5 mM
MgCl2, 2.5% glycerol, and 800 mM KCl. Loading
and washing of samples were performed at a rate of 1 ml/min. A
Molecular Imager was used for quantification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Heterodimer with Only
One ATP Binding Site--
To investigate the coupling of ATP binding
and hydrolysis to DNA strand passage and resetting in human
topoisomerase II
, we characterized a heterodimeric enzyme having
only one intact ATP binding site. ATP binding was inhibited by changing
Gly-164 located within the highly conserved Walker A motif to
isoleucine. Gly-164 in human topoisomerase II
corresponds to Gly-117
and Gly-144 in gyrase and yeast topoisomerase II, respectively, where Gly-144 has been demonstrated to be essential for ATP binding (8, 18).
Human topoisomerase II
heterodimers, G164I/wt, consisting of one
wild type subunit and one subunit with the G164I mutation, were
produced in yeast after transformation of yeast cells with a vector
containing both the wild type and mutant cDNAs (Fig.
1A). The wild type and mutant
enzyme subunits were expressed as fusion proteins containing a
hexahistidine tag and a GST tag, respectively. By
nickel-nitrilotriacetic acid and GST chromatography, heterodimers were
purified to homogeneity as demonstrated by the equal intensity of the
two bands corresponding to the wild type and the mutant subunit in the
silver stain presented in Fig. 1B.
View larger version (25K):
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Fig. 1.
Purification of G164I/wt heterodimers.
A, expression vector showing the organization of
YepWOBG164I/wt, which permits galactose-inducible coexpression of
His-tagged G164I mutant and GST-tagged wild type human topoisomerase
II . B, extracts from cells coexpressing His-tagged mutant
and GST-tagged wild type topoisomerase II were submitted to
nickel-nitrilotriacetic acid and GST column chromatography. The silver
stain shows the purified G164I/wt heterodimers eluted from the GST
column. The two bands denoted wt GST and G164I
6xhis represent the GST-tagged wild type subunit and the
His-tagged mutant subunit, respectively. The fraction numbers are
indicated above each lane.
--
To verify that ATP binding was
completely blocked by the G164I point mutation, a homodimeric enzyme
containing the mutation in both subunits was tested for its ability to
bind and hydrolyze ATP. ATP binding was investigated using a
nitrocellulose filter binding assay. The enzyme was incubated with
plasmid DNA and a labeled ATP analogue, ATP[
35S], to
avoid hydrolysis and product release. After incubation at 37 °C, the
reaction mixture was loaded onto a nitrocellulose filter followed by
several washes in high salt to minimize nonspecific binding of the ATP
analogue to the enzyme. The level of ATP[
35S] binding
observed with the homodimeric G164I enzyme was similar to the level
obtained with heat-inactivated wild type or mutant enzyme, strongly
indicating that the observed activity was caused by residual unspecific
binding (Fig. 2A).
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Fig. 2.
The mutation G164I in human topoisomerase
II disrupts ATP binding and hydrolysis
activities. A, the ATP binding activity of human
topoisomerase II
was measured using a nitrocellulose filter binding
assay, where various amounts of wild type enzyme (wt), G164I
homodimer (G164I), heat-inactivated G164I homodimer
(HI G164I), or heat-inactivated wild type enzyme (HI
wt) were incubated with 0.3 nM [35S]ATP
in the presence of 5.5 nM plasmid DNA. Levels of ATP
binding are relative to the amount of binding observed in the presence
of 120 nM wild type enzyme (set to 1). B, the
ATPase activity of human topoisomerase II
was measured by thin-layer
chromatography. 150 nM enzyme was incubated with 1 mM cold ATP and 0.08 nM
[
-32P]ATP in the presence of 30 nM plasmid
DNA. Aliquots were withdrawn at the indicated time points and loaded
onto thin-layer cellulose plates. Quantification was done using a
PhosphorImager. Error bars represent the standard deviations
from three independent experiments.
eliminates the enzymatic ATP binding and hydrolysis
activities. Based on these findings, only the wild type subunit of the
G164I/wt heterodimer is expected to bind and hydrolyze ATP.
catalysis, we investigated the relaxation activity of G164I/wt. Interestingly, the enzyme was found to relax supercoiled DNA, although
less efficiently than the wild type enzyme (Fig.
3). As anticipated, the G164I homodimer
was not able to perform relaxation. Because the enzyme:DNA molar ratio
was only 1:2.5, the level of relaxation obtained by the heterodimer
clearly demonstrates that each enzyme molecule performs more than one
DNA strand passage event. The data illustrate that human topoisomerase
II
can go through all the conformational changes required for DNA
strand passage and resetting of the enzyme, with binding and hydrolysis of ATP taking place in only one subunit of the enzyme.
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Fig. 3.
The G164I/wt heterodimer is able to perform
DNA relaxation. Samples containing 5.5 nM supercoiled
plasmid DNA and 2.2 nM either wild type enzyme, G164I/wt
heterodimer, or G164I homodimer were incubated at 37 °C and stopped
with SDS at the time points indicated above each lane.
N, RC, and SC indicate the position of
nicked, relaxed circular, and supercoiled circular DNA,
respectively.
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Fig. 4.
The G164I/wt heterodimer is able to perform
N-terminal clamp closure. Upper panel, Southern blot
showing a clamp closing experiment, where 1 nM enzyme was
incubated with 2.5 nM supercoiled and 2.5 nM
linearized plasmid DNA. ATP or AMPPNP was added to a final
concentration of 1 mM after preincubation of enzyme and
DNA. Reactions were stopped by addition of NaCl or SDS to final
concentrations of 800 mM or 1%, respectively. w
and i indicate phenol-water and phenol interphases,
respectively. RC, L, and SC indicate
relaxed circular, linear, and supercoiled circular DNA, respectively.
Lower panel, histogram of the clamp closing experiment
described in A. Levels of supercoiled plasmid DNA trapped in
the interphase were quantified using a PhosphorImager and are
represented in arbitrary units relative to the amount of DNA trapped by
the wild type enzyme in the presence of AMPPNP and NaCl. The
error bars represent the standard deviations from three
independent experiments. Southern blotting was performed using random
primed plasmids as probes.
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Fig. 5.
The G164I/wt heterodimer is sensitive to
ICRF-187. A relaxation assay was performed, where samples
containing 5.5 nM supercoiled plasmid DNA and 2.2 nM of either wild type enzyme or G164I/wt heterodimer were
incubated at 37 °C in the absence or presence of ICRF-187 as
indicated above each lane and subsequently stopped with SDS.
N, RC, and SC indicate nicked, relaxed
circular, and supercoiled circular DNA, respectively.
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Fig. 6.
DNA cleavage mediated by the G164I/wt
heterodimer is unaffected by ATP and AMPPNP. Upper
panel, 6.6 nM of either wild type enzyme or G164I/wt
heterodimer was incubated with 5.5 nM supercoiled plasmid
DNA at 37 °C in the presence or absence of 1 mM ATP or
AMPPNP as indicated. Samples were stopped after 10 min by the addition
of SDS to 1%, and following proteinase K treatment they were subjected
to electrophoresis in a 1% agarose gel containing ethidium bromide.
N, RC, L, and SC indicate
nicked, relaxed circular, linear, and supercoiled circular DNA,
respectively. Lower panel, histogram of the cleavage
experiment described in A. Levels of DNA cleavage obtained
in the absence or presence of AMPPNP were quantified using a
PhosphorImager and are represented in arbitrary units relative to the
cleavage obtained with the wild type enzyme in the absence of
nucleotide. The error bars represent the standard deviations
from three independent experiments.
35S] to G164I/wt was
independent of DNA, whereas binding of ATP[
35S] to the
wild type enzyme was stimulated ~3-fold, when DNA was present.
View larger version (17K):
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Fig. 7.
The ATP binding and hydrolysis activities of
the G164I/wt heterodimer are independent of DNA. A, the
ATP binding activity was measured using a nitrocellulose filter binding
assay, where 150 nM of either wild type enzyme or G164I/wt
heterodimer was incubated with 0.3 nM
[35S]ATP in the absence or presence of 30 nM
plasmid DNA. Levels of ATP binding in the presence of DNA are relative
to the amount of binding observed in the absence of DNA (set to 1).
B, the ATPase activity was measured by thin-layer
chromatography. 150 nM enzyme was incubated with 1 mM cold ATP and 0.08 nM
[ -32P]ATP in the absence or presence of 30 nM plasmid DNA. Aliquots were taken at various time points
and loaded onto thin-layer cellulose plates. Quantification was done
using a PhosphorImager. Error bars represent the standard
deviations from three independent experiments.
have shown that DNA
stimulates the intrinsic ATPase activity of the enzyme (5, 17, 27, 28).
In the present study, the ATPase activity of the wild type
topoisomerase II
enzyme was stimulated about 10-fold in the presence
of supercoiled plasmid DNA (Fig. 7B, left panel). No DNA stimulation was observed with G164I/wt, demonstrating that ATP
hydrolysis is unaffected by DNA, when only one subunit is capable of
binding ATP (Fig. 7B, right panel). Thus, binding
of only one ATP molecule to the enzyme conceivably affects the
communication between the domains responsible for binding of both DNA
and ATP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
heterodimer, G164I/wt, constructed to bind ATP in only one of the
two subunits.
.
Based on this, we assume that the heterodimer only binds and hydrolyzes
one ATP molecule, although we cannot exclude the possibility that a
second ATP molecule binds to the wild type subunit in the heterodimer
upon hydrolysis of the first ATP. In this case a second ATP could
contribute to the driving force for one catalytic cycle.
-phosphate of the
bound nucleotide as revealed from the crystal structure of gyrase B
(18), and in this way information regarding the nucleotide bound state
of the enzyme could be transmitted to the central domain. In a recent
report on yeast topoisomerase II, it has been suggested that release of
the
-phosphate from the first ATP hydrolysis event triggers
conformational changes associated with DNA transport. Such a model
predicts that conformational changes in the two subunits rely on a
signal initiated in only one subunit (30). Our results demonstrate that
human topoisomerase II
still mediates DNA relaxation with only one
ATP bound and hydrolyzed, in support of this model. In agreement with
the relaxation data, our G164I/wt heterodimer was found to close the
N-terminal clamp properly, demonstrating that binding of one ATP
induces a conformational change in both subunits leading to
dimerization of the N-terminal arms. These findings correlate with
results obtained with the yeast homologue, which also possessed a
normal clamp-closing activity, although ATP binding only occurred in one subunit (8).
using DNA fragments of different lengths
have revealed that stimulation of the ATPase rate may be dependent upon
both G- and T-segment binding (28). According to these results, an
impaired ability to bind either of the two DNA duplexes could cause the
DNA-independent ATP binding and hydrolysis activities observed by
G164I/wt. However, the cleavage results showed that the mutation at
Gly-164 did not affect the intrinsic cleavage activity, indicating that
the mutant binds the G-segment normally. The relaxation data further
demonstrated that the enzyme is able to capture a T-segment. Thus, it
seems unlikely that the inability of DNA to stimulate the ATP binding and hydrolysis activities of the heterodimer are caused by impaired DNA
interactions. It is, however, more likely that the stimulatory effect
of DNA on ATP binding is normally exerted through binding of the second
ATP after clamp closure, which would correlate with the earlier
observation that ATP binding in topoisomerase II is cooperative only in
the presence of DNA (5). In that case we would not expect any DNA
stimulation of ATP binding in G164I/wt.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Prof. Ole Westergaard for valuable discussions and to Kirsten Andersen and Maria Vinther for skillful technical assistance.
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FOOTNOTES |
---|
* This work was supported by the Danish Cancer Society, The Danish Medical Research Council, and The Danish Natural Science Research Council.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.
Present address: Dept. of Molecular Biology, University of Geneva,
Geneva CH-1211, Switzerland.
§ Present address: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark.
¶ To whom correspondence should be addressed. Tel.: 45-8942-2600; Fax: 45-8942-2612; E-mail: aha@mb.au.dk.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210332200
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ABBREVIATIONS |
---|
The abbreviations used are:
HA, hemagglutinin;
GST, glutathione S-transferase;
AMPPNP, 5'-adenylyl-,
-imidodiphosphate.
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REFERENCES |
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