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INTRODUCTION |
Topoisomerase II (TOP2)1
catalyzes DNA topological reactions via a DNA breakage/reunion
mechanism. The DNA topological reactions allow the enzyme to segregate
interlocked chromosomal DNA at mitosis (1-3) and to remove excess DNA
supercoils generated during processes such as DNA replication, RNA
transcription, and chromosome condensation (4-7). The breakage/reunion
reaction of TOP2, which is ATP-dependent, can be
interrupted by many antitumor drugs (TOP2 poisons) resulting in
accumulation of a TOP2-DNA covalent intermediate, the cleavable complex
(8). Accumulation of TOP2 cleavable complexes causes tumor cell death
(8).
The molecular mechanism(s) by which TOP2 poisons interfere with the
breakage/reunion reaction of TOP2 is largely unknown. Several studies
have implicated a possible role of ATP in modulating the
pharmacological action of TOP2 poisons; uncouplers of oxidative phosphorylation (e.g. DNP and 2-deoxyglucose) have been
shown to enhance survival of adriamycin (doxorubicin)-treated Chinese hamster cells (9). Similarly, DNP, 2-deoxyglucose, and sodium cyanide,
all of which affect ATP metabolism, effectively protect L1210 cells
from the cytotoxic action of VM-26 and m-AMSA (10). Simultaneous cotreatment with DNP or novobiocin has been shown to
abrogate m-AMSA cytotoxicity in Chinese hamster cells (11). VP-16 (etoposide)-induced chromosome-type aberrations (mainly breaks
and exchanges) in cultured Chinese hamster lung fibroblasts are reduced
also by cotreatment with DNP (12). Whether these effects are due to a
direct effect of ATP on TOP2-mediated DNA cleavage induced by TOP2
poisons is not known.
Studies in bacteria have demonstrated also that the ATP/ADP ratio plays
a critical role in modulating the supercoiling state of chromosomal DNA
as well as cytotoxicity of quinolone antibiotics (13-15). In this
case, the role of ATP/ADP has been shown to directly affect
TOP2-mediated DNA cleavage in the presence of quinolones (13). Studies
of a drug-resistant TOP2 from mammalian cells have demonstrated also
that ATP plays a direct role in modulating TOP2-mediated DNA cleavage
in vitro (16, 17).
In the current studies, we show that the ATP-bound conformation is the
target of a class of ATP-sensitive TOP2 poisons that includes many
clinically useful TOP2-directed antitumor drugs such as doxorubicin,
mitoxantrone, VP-16 (etoposide), and m-AMSA. The ATP-bound
form of TOP2 has been shown to be a circular protein clamp based on
biochemical and x-ray crystallographic studies (18-20). Using Lac
repressor-operator complexes as roadblocks, we have demonstrated
further that the circular TOP2 protein clamp is capable of sliding on
unobstructed duplex DNA.
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EXPERIMENTAL PROCEDURES |
Chemicals and Drugs--
VM-26 was a gift from Bristol-Myers
Squibb Co. m-AMSA, mitoxantrone, and amonafide were obtained
from the Drug Synthesis and Chemistry Branch, Division of Cancer
Treatment, National Cancer Institute. Batracylin was a gift from Dr.
C. C. Cheng (University of Kansas). 5-Hydroxy-1,4-naphthoquinone
was obtained from Aldrich. All drugs were dissolved in
Me2SO (10 mM) and kept frozen in
aliquots at
20 °C. Ciprofloxacin was obtained from Bayer. ATP,
AMPPNP, and ADP were purchased from Sigma. Except for fetal bovine
serum, which was obtained from Gemini Biotech, media and other agents for tissue culture were purchased from Life Technologies, Inc. [
-32P]dATP (3000 Ci/mmol) was obtained from DuPont.
Construction of Mutant Topoisomerase II Overexpression
Plasmid--
The mutation C427A was generated by PCR-based
site-directed mutagenesis. Three rounds of PCR were carried out to
introduce the C427A into human TOP2
cDNA in YEpWob6. The
resulting plasmid is named Yhtop2
C427A. The following four primers
were used (the mutations are marked in bold, and the restriction sites
are underlined): primer A,
5'-ACGCGTCGACGAATTCGACAGGTTATC-3' SalI; primer
B, 5'-ACAAGAAGGCCTCAGCTGTA-3'; primer C,
5'-TACAGCTGAGGCCTTCTTGT-3'; and primer D,
5'-GCCTGGTACCAAACTGAC-3' KpnI.
Primers B and C contain the alanine codon instead of the wild-type
cysteine codon. Primers A and D contain the recognition sites for
SalI and KpnI, respectively. Two fragments, AC
and BD, which have the alanine codon, were generated during the first round PCR in the presence of YEpWob6 DNA. After denaturation and renaturation of fragments AC and BD, two cycles of a second round of
PCR without any primer were carried out to generate a small amount of
the fragment containing C427A as the template for the third round of
PCR. The third round of PCR amplified the fragment containing C427A
using primers A and D. SalI and KpnI were used to
digest both the fragment-containing C427A and YEpWob6 (partially digested with SalI), and ligation was carried out at
14 °C overnight. The mutated site was confirmed by sequence analysis.
Enzymes and Nucleic Acids--
TOP2 was purified to homogeneity
from calf thymus glands according to the published procedure (21).
Full-length human TOP2
cDNA (hTOP2
cDNA) was isolated by
reverse transcription-PCR using mRNA isolated from human U937 cells
and primers with sequences according to the published sequence of HeLa
TOP2
(22). For overexpression, hTOP2
cDNA was used to replace
the human TOP2
cDNA (hTOP2
cDNA) in YEpWob6 (23). The
resulting plasmid, YEphTOP2
, then was used to transform
protease-deficient yeast BCY123 (23). Purification of both TOP2
isozymes and mutant enzyme were performed following the published
procedure (23). Lac repressor was a kind gift from Dr. Kathleen S. Matthews (Rice University). YEpG (24) is a derivative of YEP24.
pY
YOm was constructed by inserting a 42-bp DNA oligomer containing a
21-bp essential Lac repressor binding site (25) into the
NotI site in pBR
Y (26). pY
YOd, which contains two Lac
repressor binding sites, was constructed by inserting the same 42-bp
oligomer into the XbaI site in pY
YOm. All plasmids were
purified using the Qiagen purification kit.
Preparation of End-labeled DNA Fragments--
3' end-labeling of
plasmid DNA was performed as described previously (21). Briefly, 10 µg of DNA was digested with a proper restriction enzyme followed by
labeling at its 3' ends with the large fragment of Escherichia
coli DNA polymerase I and [
-32P]dATP.
Unincorporated triphosphates were removed by two cycles of ethanol
precipitation in the presence of 2.5 M ammonium acetate.
TOP2 Cleavage Assay--
The TOP2 cleavage assay was performed
as described previously (27). The reaction mixtures (20 µl each)
containing 40 mM Tris-HCl, pH 7.5, 100 mM KCl,
10 mM MgCl2, 1.0 mM ATP, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30 µg/ml
bovine serum albumin, 20 ng of 3' end-labeled DNA, 10 ng of TOP2, and
various drugs were incubated at 37 °C for 30 min. The reactions were
terminated by the addition of 5 µl of 5% SDS and 150 µg/ml
proteinase K and incubated for an additional 60 min at 37 °C. After
the addition of sucrose (5% final concentration) and bromphenol blue
(0.05 mg/ml final concentration), DNA samples were loaded onto a 1% agarose gel in TPE (90 mM Tris phosphate, 2 mM
EDTA, pH 8.0) buffer. Gels then were dried onto Whatman No. 3MM
chromatographic paper and autoradiographed at
80 °C using Kodak
XAR-5 films.
P4 Unknotting Assay--
Knotted P4 phage DNA was used to assay
the strand-passing activity of TOP2 (28). Reactions (20 µl each)
containing 40 mM Tris-HCl, pH 7.5, 100 mM KCl,
10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30 µg/ml bovine serum albumin, calf thymus
TOP2 (titrated prior to the experiment), and various amounts of ADP
and/or ATP as indicated were incubated at 37 °C for 30 min.
Reactions were terminated by adding 5 µl of a solution containing
20% Ficoll, 5% Sarkosyl, 50 mM EDTA, and 0.05 mg/ml
bromphenol blue. Reaction products were analyzed by electrophoresis in
1% agarose gel containing TPE buffer.
ATPase Assay--
The ATPase assay was performed as described
(29) except that an 8.6-kilobase plasmid DNA, pCaSpeRhs83 (30), was
used. Radioactivities were quantified by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
Differential ATP Stimulation of TOP2-mediated DNA Cleavage Induced
by TOP2 Poisons--
The possible effect of ATP on TOP2-mediated DNA
cleavage was studied using purified TOP2 and various TOP2 poisons. As
shown in Fig. 1A, 1 mM ATP stimulated calf thymus TOP2-mediated DNA cleavage
induced by VM-26 by about 60-fold (Fig. 1A) (ATP stimulation is estimated as the -fold of increased drug concentration in the absence of ATP for achieving the same extent of cleavage in the presence of ATP). By contrast, TOP2-mediated DNA cleavage induced by
amonafide was affected only slightly (less than 3-fold) by 1 mM ATP (Fig. 1B). The nonhydrolyzable ATP
analog, AMPPNP, gave similar results as ATP (data not shown). Similar
results were obtained with recombinant human TOP2
and TOP2
(data
not shown). TOP2 poisons that are highly (about 30-100-fold)
stimulated by ATP in the TOP2-mediated DNA cleavage assay include
VM-26, VP-16, m-AMSA, doxorubicin, and mitoxantrone
(referred to as ATP-sensitive TOP2 poisons). TOP2 poisons that are
affected only slightly by ATP (less than 3-fold) include amonafide,
batracylin, and menadione (referred to as ATP-insensitive TOP2
poisons).

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Fig. 1.
ATP stimulation of TOP2-mediated DNA cleavage
induced by TOP2 poisons. TOP2 cleavage assays were performed as
described under "Experimental Procedures." The presence or absence
of ATP (1.0 mM) is indicated on top of each
lane. All reactions contained 1% Me2SO. The
concentrations of VM-26 (A) and amonafide (AM)
(B) are as indicated.
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ADP Antagonizes ATP-stimulated DNA Cleavage Activity and
ATP-dependent Strand-passing Activity of
TOP2--
Although ATP strongly stimulated TOP2-mediated DNA cleavage
induced by VM-26, ADP alone had no such effect (Fig.
2). However, in the presence of ATP, ADP
effectively antagonized the ATP stimulatory effect on TOP2-mediated DNA
cleavage induced by VM-26 (Fig. 2). The antagonistic effect of ADP on
VM-26-induced DNA cleavage was observed also with other ATP-sensitive
TOP2 poisons (data not shown). By contrast, ATP had a minimum effect on
TOP2-mediated DNA cleavage in the presence of amonafide, an
ATP-insensitive TOP2 poison. In this case, ADP had a minimal effect on
TOP2-mediated DNA cleavage induced by amonafide in the presence of ATP
(Fig. 2). These results suggest that ADP specifically antagonizes the ATP-stimulatory effect on TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons. We also had tested the effect of ADP on the
catalytic activity of TOP2 using a P4 unknotting assay. As shown in
Fig. 3, ADP also effectively antagonized
the P4 unknotting activity of TOP2.

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Fig. 2.
ADP antagonizes the cleavage-stimulatory
effect of ATP. TOP2 cleavage assays were performed using calf
thymus TOP2. The concentrations of ATP and ADP are as indicated on
top of each lane.
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Fig. 3.
ADP inhibits the P4 unknotting activity of
TOP2. The P4 unknotting assay for the catalytic activity of TOP2
was performed as described under "Experimental Procedures." Various
concentrations of ATP and ADP were present as indicated on
top of each lane. The P4 knotted DNA migrated as
a smear as indicated. The unknotted P4 DNA migrated as a band as
indicated.
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C427A Mutant TOP2
Is Cross-resistant to ATP-sensitive but Not
ATP-insensitive TOP2 Poisons--
To characterize the role of ATP in
the action of TOP2 poisons further, we generated C427A mutant TOP2
.
The cysteine 427 is located in the ATPase domain of TOP2
(31). In
the absence of ATP, C427A mutant TOP2
exhibited almost identical
sensitivity to both VM-26 and amonafide compared with wild-type TOP2
in a standard DNA cleavage assay (Fig.
4). Strikingly, in the presence of 1 mM ATP, C427A mutant TOP2
was at least 10-fold more
resistant to VM-26 as compared with the wild-type enzyme. The
resistance of C427A mutant TOP2
to VM-26 is apparently caused by a
reduced ATP-stimulatory effect on DNA cleavage as compared with the
wild-type enzyme (Fig. 4). By contrast, both mutant and wild-type
TOP2
were equally sensitive to amonafide (Fig. 4). We also tested
other ATP-sensitive drugs such as doxorubicin, m-AMSA,
mitoxantrone, and CP115,953. Similar results to VM-26 were
observed (data not shown). These results suggest that C427A mutant
TOP2
can distinguish between ATP-sensitive and -insensitive TOP2
poisons, possibly because of its altered interaction with ATP.

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Fig. 4.
C427A mutant TOP2 is
resistant to VM-26 but not amonafide. TOP2 cleavage assays were
performed as described under "Experimental Procedures." The
concentrations of VM-26 and amonafide (AM) are as indicated
on top of each lane. 1 mM ATP was
used in the +ATP lanes.
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C427A Mutant Enzyme Exhibits Reduced ATPase Activity and Increased
ATP Requirement for Its Catalytic Activity--
Previous studies on
another multidrug-resistant mutant TOP2
mutant have shown that the
mutant enzyme exhibits an increased ATP requirement for catalysis (16).
To test whether C427A behaves similarly, the catalytic activity of the
C427A mutant TOP2
was measured by a P4 unknotting assay in the
presence of two different concentrations of ATP. As shown in Fig.
5, C427A mutant TOP2
was about 5-fold
less active than the wild-type enzyme in the presence of 1 mM ATP. However, in the presence of 50 µM
ATP, C427A was at least 25-fold less active than the wild-type enzyme.
This result is similar to the result from the experiment performed on
another mutant TOP2
enzyme, R450Q TOP2
, which is cross-resistant to ATP-sensitive TOP2 poisons and exhibits an increased ATP requirement for enzyme catalysis (17).

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Fig. 5.
The P4 unknotting activity of C427A mutant
TOP2 exhibits an increased requirement for
ATP. The P4 unknotting assay for the catalytic activity of C427A
mutant TOP2 and wild-type TOP2 were performed as described under
"Experimental Procedures." The concentrations of TOP2 and ATP were
as indicated on top of the figure.
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The DNA-stimulated ATPase activity of C427A mutant TOP2
also was
measured and shown to be much reduced relative to the wild-type enzyme.
The Vmax was reduced from 60 to 11 mM min
1, and
Km was increased from 0.78 to 3.2 mM.
Ciprofloxacin Antagonizes TOP2-mediated DNA Cleavage Induced by
ATP-sensitive but Not ATP-insensitive TOP2 Poisons--
Ciprofloxacin
is known to interact with TOP2 but does not induce significant
TOP2-mediated DNA cleavage (32). Consequently, ciprofloxacin has been
used to compete with other TOP2 poisons in a standard DNA cleavage
assay to assess possible overlap of interaction domains on TOP2 (32).
Based on this kind of ciprofloxacin competition assay, it has been
suggested that various TOP2 poisons including etoposide,
m-AMSA, genistein, and the antineoplastic quinolone,
CP-115,953, share a common interaction domain with ciprofloxacin on
TOP2 (32). To test whether ATP-sensitive and -insensitive TOP2 poisons
may interact with different domains on TOP2, we performed the
ciprofloxacin competition assay (32). As shown in Fig.
6, ciprofloxacin reduced TOP2-mediated
DNA cleavage induced by VM-26 as evidenced by the gradual increase in
band intensity of the uncleaved DNA bands (see the arrow)
with increasing ciprofloxacin concentrations (see lanes
labeled 0.1 µM VM). By contrast,
ciprofloxacin had little effect on TOP2-mediated DNA cleavage induced
by amonafide (see the intensity of the uncleaved DNA bands in
lanes labeled 1.0 µM
AM). Similar ciprofloxacin competition assays were performed
with other TOP2 poisons. All ATP-sensitive but not ATP-insensitive TOP2
poisons were antagonized by ciprofloxacin in this DNA cleavage assay
(data not shown).

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Fig. 6.
Ciprofloxacin antagonizes TOP2-mediated DNA
cleavage induced by VM-26. TOP2 cleavage assays were performed as
described under "Experimental Procedures." 0.1 µM
VM-26 (lanes labeled 0.1 µM
VM) and 1.0 µM amonafide (lanes
labeled 1.0 µM AM) were used in the
cleavage assays with increasing concentrations of ciprofloxacin (0, 125, and 250 µM). The control experiment in the presence
of 1% Me2SO (solvent control for VM-26 and amonafide) and
increasing concentrations of ciprofloxacin is shown in lanes
labeled 1% DMSO. All drugs were present in the
reaction mixture prior to the addition of TOP2. The arrow
points to the uncleaved DNA bands.
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ATP-bound TOP2 Is a Sliding Protein Clamp--
Previous studies
have suggested that yeast TOP2 when bound to AMPPNP can undergo a
conformational change into a circular protein clamp (19, 20), which is
consistent with results from x-ray crystallographic studies (18). To
test whether calf thymus TOP2 also can form a circular protein clamp in
its ATP-bound form, we have designed a more stringent assay requiring
the TOP2 protein clamp to slide on unobstructed DNA under physiological
conditions. As shown in Fig. 7, a linear
DNA (8310-bp, 32P end-labeled) with two internally bound
Lac repressor molecules at their respective Lac operator sites was used
to demonstrate entry and sliding of AMPPNP-bound TOP2. Calf thymus TOP2
was reacted first with AMPPNP to form a circular protein clamp and then
incubated with the linear DNA bound by Lac repressors. VM-26 or
amonafide was used subsequently to locate the TOP2 sliding clamps on
DNA by inducing TOP2-mediated DNA cleavage. As shown in Fig. 7, in the
presence of ATP, TOP2-mediated DNA cleavage sites induced by VM-26
(lane 3) scattered all over the linear DNA (a similar result
was obtained in the absence of ATP; data not shown). However, in the
presence of AMPPNP, TOP2-mediated DNA cleavage induced by VM-26
(lane 5) occurred primarily near the two ends of the linear
DNA and extended up to the Lac repressor binding sites. Similar results
were obtained in the absence of VM-26 (compare background DNA cleavage
in lanes 2 and 4 with VM-26-induced DNA cleavage
in lanes 3 and 5, respectively). The lower part
of the gel was overexposed to reveal cleavage sites from the other end (Fig. 7). The results from this experiment support the previous claim
that AMPPNP-bound TOP2 is in the form of a circular protein clamp. In
addition, this experiment has demonstrated that AMPPNP-bound TOP2 is a
protein clamp capable of entering DNA ends and sliding on unobstructed
duplex DNA while retaining sensitivity to VM-26.

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Fig. 7.
AMPPNP-bound TOP2 is a sliding protein
clamp. pY YOd DNA, containing two Lac repressor binding sites,
was digested with HindIII and 3' end-labeled with
[ -32P]dATP by the Klenow polymerase. The labeled DNA
then was digested with ScaI, resulting in two
one-end-labeled DNA of the sizes of 8310 and 546 bp, respectively. The
two Lac repressor binding sites are located at 808-829 and 4527-4548
bp from the labeled end of the 8310-bp fragment. Demonstration of entry
and sliding of AMPPNP-bound TOP2 was performed in three sequential
steps: (a) calf thymus TOP2 (30 ng each) was incubated with
2 mM AMPPNP or ATP at 37 °C for 10 min; (b)
3 × 10 13 M labeled DNA was incubated
with 2 × 10 11 M Lac repressor protein
in 40 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol,
0.5 mM EDTA, and 30 µg/ml bovine serum albumin at room
temperature for 7 min; (c) AMPPNP-bound TOP2 and
repressor-bound DNA were mixed together followed by incubation at
37 °C for 7 min in the presence of 0.5 µM VM-26. After
treatment with SDS (final concentration 1%) and proteinase K (final
concentration 200 µg/ml) for 1 h at 37 °C, the samples were
subjected to electrophoresis in 1% agarose gel. Lane 1, DNA
alone; lane 2, contained Lac repressor and TOP2; lane
3, contained Lac repressor, TOP2, and VM-26. Lanes 4 and 5 were identical to lanes 2 and 3,
respectively, except that AMPPNP rather than ATP was present in each
reaction. Lane 6, DNA size markers. To the right
of the panel, the 8310-bp DNA, which is 3' end-labeled with
32P (see the asterisk), is aligned schematically
with the gel to indicate the cleavage sites relative to the Lac
repressor binding sites on DNA. In the presence of AMPPNP, TOP2 is
shown as a protein doughnut that can enter linear DNA only through DNA
ends. Drug-induced (also background) cleavage sites mark the accessible
regions of DNA to the TOP2 sliding clamp.
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DISCUSSION |
Our results have demonstrated that different TOP2-mediated DNA
cleavage induced by various TOP2 poisons exhibits a different degree of
ATP dependence. The differences in ATP dependence among various TOP2
poisons may reflect differences in their interaction with TOP2 and/or
TOP2-DNA complexes. Based on our current results, there seems to be two
distinct classes of TOP2 poisons, ATP-sensitive and
ATP-insensitive.
These two classes of TOP2 poisons show quite different responses to ATP
stimulation in the standard DNA cleavage assay. The specific
antagonistic effect of ADP against ATP-sensitive but not -insensitive
TOP2 poisons has demonstrated further the differences between these two
classes of TOP2 poisons. The fact that ADP also strongly inhibits
ATP-dependent catalytic activity of TOP2 suggests that ADP
may compete with ATP both in enzyme catalysis and cleavable complex
formation by the same mechanism. Studies in bacteria have established
that the ATP/ADP ratio is a critical determinant for the supercoiling
state in cells probably because of the sensitivity of DNA gyrase to the
ATP/ADP ratio (14, 15). More recent studies have demonstrated also that
quinolone-induced DNA cleavage depends strongly on the ATP/ADP ratio,
both in cells and using purified gyrase (13). These results suggest
that the ATP/ADP ratio may be a common determinant for
sensitivity/resistance to both antibiotics and antitumor drugs directed
against TOP2. Although ATP-sensitive TOP2 poisons used in this work
have very disparate structures, their interaction domains with TOP2
have been suggested to overlap (32).
Previous studies have demonstrated that a multidrug-resistant mutant
TOP2
is cross-resistant to all ATP-sensitive TOP2 poisons (17). This
multidrug-resistant mutant TOP2
was shown to exhibit an increased
requirement of ATP for catalysis and cleavage. It has been suggested
that the R450Q mutation on this mutant TOP2
is responsible for
altered ATP utilization and cross-resistance to ATP-sensitive TOP2
poisons (17). This mutation is located in a Walker consensus motif
(17). In the current study, we have created another mutation C427A on
TOP2
. Like the R450Q mutant TOP2
, C427A mutant TOP2
also
exhibits multidrug resistance to all ATP-sensitive poisons.
Interestingly, C427A mutant TOP2
retains the same sensitivity to
ATP-insensitive TOP2 poisons as compared with the wild-type enzyme.
C427A mutant TOP2
exhibits reduced ATPase activity and an increased
requirement of ATP for catalysis. Taken together, these results suggest
that ATP-sensitive and -insensitive TOP2 poisons interfere with the
breakage/reunion reaction of TOP2 by distinct mechanisms and that
ATP-sensitive TOP2 poisons may interfere specifically with a step in
TOP2 catalysis requiring ATP utilization.
Results from the ciprofloxacin competition experiment have suggested
that the ATP-insensitive TOP2 poisons do not share the same interaction
domain on TOP2 with ATP-sensitive TOP2 poisons. This result suggests
that ATP-sensitive TOP2 poisons may target TOP2 with a distinct
conformation compared with ATP-insensitive poisons.
Based on our results, it seems plausible that ATP-sensitive TOP2
poisons may specifically target an ATP-bound conformation of TOP2. Our
current studies have suggested that AMPPNP-bound TOP2 is capable of
entering duplex DNA only from its ends, consistent with the closed
circular clamp conformation of ATP-bound TOP2 proposed previously on
the basis of studies of yeast TOP2 (19). Our results also indicate that
upon entry, AMPPNP-bound TOP2 can slide on unobstructed DNA under
physiological conditions. Previous studies on Drosophila
TOP2 and yeast TOP2 have implicated linear diffusion in high salt
conditions that presumably weaken protein-DNA interactions to allow
mobility of the protein circular clamp (19, 33). Our results, however,
show that AMPPNP-bound mammalian TOP2 is able to linearly diffuse under
physiological conditions. The ability of TOP2 to slide on DNA under
physiological conditions may imply a role of limited linear diffusion,
dictated by ATP binding and hydrolysis, in its strand-passing reaction.
Our limited understanding of the role of ATP in TOP2 catalysis
precludes us from any meaningful speculation on the mechanistic and/or
functional implications of the sliding action of ATP-bound TOP2. The
resistance of TOP2 poisons has been studied in cells under stress
conditions (e.g. hypoxia and glucose deprivation) that are
associated often with solid tumors (9-12, 34-38). Reduced TOP2
levels in stressed cells have been found and suggested to be
responsible in part for the observed resistance (38-40). Our results
raise the possibility that alteration in ATP/ADP ratios, which is known
to occur in hypoxic and nutrient-depleted cells (41), may contribute to the overall resistance mechanisms through its modulation on
TOP2-mediated DNA cleavage. Thus, it seems plausible that
ATP-insensitive TOP2 poisons may be useful particularly for treating
hypoxic tumors that have compromised ATP/ADP ratios.