From the Departments of Biochemistry and
Medicine (Hematology/Oncology), Vanderbilt University
School of Medicine, Nashville, Tennessee 37232-0146 and
¶ BioStructures Group, deCODE Genetics,
Bainbridge Island, Washington 98110
Received for publication, November 26, 2002
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ABSTRACT |
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The widely used anticancer drug etoposide kills
cells by increasing levels of topoisomerase II-mediated DNA breaks.
While it is known that the drug acts by inhibiting the ability of
topoisomerase II to ligate cleaved DNA molecules, the precise mechanism
by which it accomplishes this action is not well understood. Because
there are two scissile bonds per enzyme-mediated double-stranded DNA break, it has been assumed that there are two sites for etoposide in
every cleavage complex. However, it is not known whether the action of
etoposide at only one scissile bond is sufficient to stabilize a
double-stranded DNA break or whether both drug sites need to be
occupied. An oligonucleotide system was utilized to address this
important issue. Results of DNA cleavage and ligation assays support a
two-drug model for the action of etoposide against human
topoisomerase II Etoposide is a highly successful anticancer drug that has been in
clinical use for nearly 20 years (1). It is employed as frontline
therapy for a variety of human malignancies, including leukemias,
lymphomas, and several solid tumors (1-3). Until the emergence of the
taxanes, etoposide was the most widely prescribed anticancer drug in
the world.
The primary cellular target for etoposide is topoisomerase II (1, 2,
4-8). This essential enzyme alters DNA topology by passing an intact
double helix through a transient double-stranded break that it
generates in a separate nucleic acid segment (4, 5, 7-11). The enzyme
is required to resolve knots and tangles in the genetic material that
are produced by normal cellular processes such as DNA recombination and
replication (4, 5, 9, 10, 12, 13). In the absence of topoisomerase II,
cells are unable to segregate daughter chromosomes and die of mitotic
failure (5, 9, 12).
Eukaryotic topoisomerase II is homodimeric in nature (14-18). Each
protomer contains an active site tyrosine (Tyr-805 in human topoisomerase II In contrast to most drugs that target specific enzymes, etoposide and
other topoisomerase II-targeted anticancer agents act in an insidious
manner. Rather than blocking the activity of this essential enzyme,
etoposide kills cells by increasing the concentration of topoisomerase
II-DNA cleavage complexes (2, 4-8). This action converts topoisomerase
II to a potent cellular toxin that fragments the genome. Consequently,
etoposide is referred to as a topoisomerase II poison to
distinguish it from drugs that inhibit the overall catalytic activity
of the enzyme (2, 4-7, 27, 28).
It has been known for more than a decade that etoposide stabilizes
topoisomerase II-associated double-stranded DNA breaks by inhibiting
the ability of the enzyme to ligate cleaved nucleic acid molecules (4,
5, 8, 29-31). However, the molecular mechanism by which it does so is unknown.
Topoisomerase II manufactures double-stranded breaks in DNA by
generating two staggered nicks (one on each strand) in the sugar-phosphate backbone (14, 20, 21). The fact that there are two
scissile bonds per double-stranded break implies that there are two
available sites for drug action in every cleavage complex. The
potential presence of two etoposide molecules raises a fundamental
question regarding drug mechanism. Does topoisomerase II require the
actions of etoposide at both scissile bonds in order to stimulate the
formation of double-stranded DNA breaks, or does the
presence of drug at either scissile bond suffice? If enzyme catalysis
at both strands is tightly coupled, the presence of etoposide at one
scissile bond may inhibit DNA ligation at the other. Conversely, if
catalysis is not highly coordinated, the presence of drug molecules at
both scissile bonds may be required.
An oligonucleotide system was utilized to address this unresolved
issue. Results support a two-drug model for the action of etoposide against human topoisomerase II Enzymes and Materials--
Wild-type human topoisomerase II Preparation of Oligonucleotides--
A 47-base oligonucleotide
corresponding to residues 80-126 of pBR322 and its complement were
prepared on an Applied Biosystems DNA synthesizer. The sequences of the
top and bottom strands were 5'-CCGTGTATGAAATCTAACAATG DNA Cleavage--
DNA cleavage assays were carried out as
described previously (33, 36). Reaction mixtures contained 200 nM wild-type human topoisomerase II
For reactions containing pBR322 DNA, cleavage intermediates were
trapped and digested as above (except that 5% SDS was used) and
subjected to electrophoresis in a 1% agarose gel in 40 mM Tris acetate, pH 8.3, 2 mM EDTA containing 0.5 µg/ml
ethidium bromide. DNA bands were visualized by ultraviolet light and
quantified using an Alpha Innotech digital imaging system. Relative
levels of single- versus double-stranded breaks were
determined directly by quantifying the fluorescence of linear and
nicked plasmid molecules, respectively (ethidium bromide displays
identical binding and fluorescence properties for both topological
forms of DNA (37)). These values were converted to absolute cleavage
levels by normalizing data to the total percent DNA cleavage, which was
calculated from the loss of supercoiled plasmid DNA substrate.
DNA Ligation--
DNA ligation reactions were carried out
according to Bromberg et al. (34). Briefly, assays contained
200 nM wild-type or Y805F human topoisomerase II Although etoposide kills cells by stabilizing topoisomerase II-DNA
cleavage complexes (2, 4-8), the precise mechanism by which it
accomplishes this action remains enigmatic. Because this drug is in
wide use for the treatment of human cancers (1-3), it is critical to
understand the molecular basis for its effects on the enzyme.
Because there are two scissile bonds per topoisomerase II-mediated
double-stranded DNA break (14, 20, 21), it has been assumed that there
are two sites for etoposide in every cleavage complex (38). However, it
is not known whether the presence of etoposide at only one scissile
bond is sufficient to stabilize a double-stranded DNA break or whether
both drug sites need to be occupied. This is a fundamental issue that
has important implications for topoisomerase II-based cancer
chemotherapy, including drug design and the cellular processing of
drug-stabilized cleavage complexes.
Two potential models for etoposide action can be envisioned. In the
first, or one-drug model, the action of etoposide at either of the two scissile bonds is sufficient to produce enzyme-mediated double-stranded DNA breaks. This model postulates strong molecular communication between the active site of each topoisomerase II protomer, such that drug-induced inhibition of ligation of one DNA
strand also impairs ligation of the other strand.
In the second, or two-drug model, the action of etoposide at
both of the two scissile bonds is required to generate double-stranded DNA breaks. This model postulates that there is little or no molecular communication between the two protomer active sites of topoisomerase II. Each drug acts independently, inhibits DNA ligation only at a
single scissile bond, and therefore induces only a single-stranded DNA break.
Although these hypotheses have not been tested for any eukaryotic type
II topoisomerase, results of a study that examined drug effects on
bacteriophage T4 topoisomerase II (38) were consistent with the
one-drug model. Under all conditions examined in this work, primarily
double-stranded DNA breaks were induced by etoposide. However, because
treatment of eukaryotic type II topoisomerases with anticancer drugs
often produces cleavage complexes containing DNA nicks (29, 39-41),
the one-drug model may not extend to the eukaryotic enzymes.
Treatment of Human Topoisomerase II Etoposide Acts Independently at the Two Scissile Bonds--
A
postulate of the two-drug model is that the action of etoposide at one
scissile bond does not influence the DNA cleavage/ligation equilibrium
at the other. This implies that there is little or no communication
between the protomer active sites of topoisomerase II, at least in the
presence of the drug.
This prediction cannot be tested using a plasmid-based DNA cleavage or
ligation assay, because it is impossible to control the distribution of
sites utilized by the enzyme in large nucleic acid substrates.
Consequently, an oligonucleotide system was established that allowed
the actions of etoposide at each of the two scissile bonds to be
manipulated individually (see Fig. 2).
The substrate contained a single, well characterized cleavage site for
topoisomerase II that was derived from pBR322 plasmid DNA (14, 42, 43). The cleavage site did not fit the "consensus sequence" for
etoposide (C at the
As a first step toward manipulating the efficacy of etoposide at the
individual scissile bonds, the nucleotide preference of the drug for
the base at the
The nucleotide preference for inhibition of DNA ligation by etoposide
was assessed by monitoring the ability of human topoisomerase II
The effects of nucleotide sequence on etoposide-induced inhibition of
DNA ligation were striking (Fig. 3, B and D).
When the
A previous study that examined interactions between etoposide and yeast
topoisomerase II determined that the affinity of the drug for the
enzyme decreased ~3-fold when the active site tyrosine was replaced
by a phenylalanine (46). Therefore, the above DNA ligation experiments
were repeated using wild-type human topoisomerase II
The DNA cleavage and ligation data presented above provide a framework
in which the effect of etoposide on each scissile bond can be
manipulated individually. Therefore, two sets of experiments were
carried out to determine whether the action of etoposide at one
scissile bond alters the DNA cleavage/ligation equilibrium at the
other. The first monitored enzyme-mediated DNA cleavage and ligation at
a scissile bond containing a weak etoposide site (
It should be noted that wild-type human topoisomerase II
In the first set of experiments, DNA cleavage and ligation mediated by
wild-type human topoisomerase II
In the second set of experiments, DNA cleavage and ligation were
monitored at a scissile bond containing a strong etoposide site (
These results indicate that the action of etoposide at one scissile
bond has little or no influence on the DNA cleavage/ligation equilibrium at the other scissile bond. They further suggest that the
actions of etoposide at the two scissile bonds of a topoisomerase II-DNA cleavage complex are independent of one another and provide strong evidence for a two-drug model in which there is little or no
communication between the two protomer active sites of topoisomerase II.
Enhanced Formation of Double-stranded DNA Breaks Requires Strong
Etoposide Sites on Both DNA Strands--
If the proposed model for
etoposide action on human topoisomerase II
Results support the proposed two-drug model (Fig.
6). When a strong etoposide site ( A Two-drug Model for Etoposide Action--
On the basis of the
data presented above, we propose a two-drug model for the actions of
the anticancer drug etoposide against human topoisomerase II
The two-drug model for etoposide is supported by several lines of
evidence. First, etoposide treatment generated a high ratio of
single-stranded to double-stranded DNA breaks at low drug
concentrations, and this ratio fell as drug concentration (and
presumably etoposide occupancy in the cleavage complex) rose. Second,
the action of etoposide on one strand of a cleavage complex had no
appreciable effect on the DNA cleavage/ligation equilibrium of the
other strand. Third, in order for etoposide to induce high levels of
topoisomerase II-mediated double-stranded (as opposed to
single-stranded) breaks, the cleavage sequence had to contain strong
drug sites on both DNA strands.
Implications of the Two-drug Model--
The two-drug model for
etoposide action has important implications for topoisomerase II-based
cancer chemotherapy. For example, the development of tethered
etoposide-based drugs might lead to more efficacious treatment by
increasing the probability that both scissile bonds within a
topoisomerase II-DNA cleavage complex would be occupied.
In addition, it may be necessary to modify our paradigm for how
transient topoisomerase II-linked DNA breaks are converted to permanent
double-stranded breaks in vivo. Most cellular models start
with the assumption that processing begins when a DNA tracking system
collides with a topoisomerase II-DNA cleavage complex that contains a
double-stranded break (2, 4-8, 26). However, results of the present
study, together with previous cellular experiments (47-49), suggest
that most etoposide-stabilized cleavage complexes contain
single-stranded breaks at clinically relevant drug concentrations.
Thus, at least in some cases, models similar to those proposed for the
conversion of transient single-stranded topoisomerase I-associated DNA
breaks to permanent double-stranded breaks in
camptothecin-treated cells (6, 50-52) may be more appropriate.
Beyond the role of topoisomerase II in cancer chemotherapy, there is
substantial evidence suggesting that maternal consumption of
topoisomerase II-active agents triggers chromosomal translocations in
the MLL oncogene (chromosomal band 11q23) that lead to
specific de novo infant leukemias (25, 53-57). Although the
chromosomal breakpoints in these malignancies are located near
topoisomerase II-DNA cleavage sites, the genomic rearrangements in the
MLL oncogene are complex and often contain large insertions
or deletions. It has been proposed that these translocations are
initiated by the formation of multiple topoisomerase II-DNA cleavage
complexes that generate nicks (rather than double-stranded breaks) on
opposite strands of the double helix (56). Although the precise
mechanism by which leukemic chromosomal translocations are generated
has yet to be delineated, the two-drug model is compatible with
this proposal.
At the present time, the two-drug model is being proposed specifically
for etoposide. While there is a presumption (albeit untested) that it
will hold for other drugs that poison topoisomerase II by
inhibiting enzyme-mediated DNA ligation, it is not clear whether the
model can be applied to agents that act by stimulating the forward rate
of DNA scission. It has been proposed that drugs in this latter
category enhance topoisomerase II-mediated DNA breaks by distorting the
double helix proximal to the scissile bond (8, 43, 58, 59).
Consequently, it is possible that the presence of a single drug
molecule will be sufficient to induce this distortion and stimulate the
formation of a topoisomerase II-associated double-stranded DNA break.
Finally, the fact that drug-induced changes in the DNA
cleavage/ligation equilibrium at one scissile bond are not communicated to the other implies that the two protomer active sites of eukaryotic topoisomerase II work in a fashion that is less coordinated than previously assumed. Recently, it has been noted that type IA and eukaryotic type II topoisomerases share structural elements in their
catalytic domains (60). This observation, coupled with biochemical
studies (61, 62), has led to the proposal that these enzymes share a
common ancestral DNA nicking-closing mechanism (60, 62). The potential
independence between the two active sites of eukaryotic
topoisomerase II is consistent with such an hypothesis and merits
further investigation.
. This model postulates that drug interactions at
both scissile bonds are required in order to increase enzyme-mediated double-stranded DNA breaks. Etoposide actions at either of the two
scissile bonds appear to be independent of one another, with each
individual drug molecule stabilizing a strand-specific nick rather than
a double-stranded DNA break. This finding suggests (at least in the
presence of drug) that there is little or no communication between the
two promoter active sites of topoisomerase II. The two-drug model has
implications for cancer chemotherapy, the cellular processing of
etoposide-stabilized enzyme-DNA cleavage complexes, and the catalytic
mechanism of eukaryotic topoisomerase II.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(19)) that is responsible for cleaving one strand
of the double helix (14, 20, 21). In order to maintain the integrity of
the genetic material during the double-stranded DNA passage reaction of
topoisomerase II, each active site residue forms a covalent
phosphotyrosyl bond with one newly generated 5' terminus (14, 20, 21).
This covalent topoisomerase II-cleaved DNA complex is known as the
cleavage complex. Despite the physiological importance of
topoisomerase II, the cleavage complex is potentially toxic. When
DNA-tracking enzymes such as polymerases or helicases attempt to
traverse the topoisomerase II roadblock on the genetic material,
transient cleavage complexes can be converted to permanent double-stranded DNA breaks (4-6, 8, 22). Normally, cleavage complexes
are present at very low levels and are tolerated by the cell. However,
conditions that increase the cellular concentration of topoisomerase
II-associated nucleic acid breaks lead to DNA recombination and
mutagenesis (4-6, 23-26). When these breaks overwhelm the cell, they
trigger programmed death pathways (6, 26).
. This model postulates that
drug interactions at both scissile bonds are required in order to
increase enzyme-mediated double-stranded DNA breaks. Etoposide actions
at either of the two scissile bonds appear to be independent of one
another, with each individual drug molecule stabilizing a
strand-specific nick rather than a double-stranded break. This finding
suggests (at least in the presence of drug) that there is little or no
communication between the two protomer active sites of topoisomerase
II. The two-drug model has implications for future drug design and the
cellular processing of etoposide-stabilized cleavage complexes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and a mutant human enzyme containing an active site Phe in place of
Tyr-805 (Y805F) were expressed in Saccharomyces cerevisiae
and purified as described previously (32-34). Etoposide (Sigma) was
prepared as a 20 mM stock solution in 100%
Me2SO and stored at 4 °C.
CGCTCATCGTCATCCTCGGCACCGT-3' and
5'-ACGGTGCCGAGGATGACGATG
AGCGCATTGTTAGATTTCATACACGG-3', respectively. Points of topoisomerase II-mediated DNA cleavage are
denoted by arrows. Oligonucleotides spanning the 5' terminus to the
point of topoisomerase II scission on each strand also were
synthesized. Single-stranded oligonucleotides were labeled on their 5'
termini with [
-32P]phosphate and purified as described
(35). Oligonucleotides extending from the point of scission to the 3'
terminus of each strand were synthesized and 5'-activated with
p-nitrophenol according to Bromberg et al. (34).
Equimolar amounts of complementary oligonucleotides were annealed by
incubating at 70 °C for 10 min and cooling to 25 °C.
and 10 nM double-stranded oligonucleotide or 50 nM
enzyme and 10 nM negatively supercoiled pBR322 DNA in 20 µl of 10 mM Tris-HCl, pH 7.9, 135 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 2.5%
glycerol in the absence or presence of etoposide. Reactions were
incubated at 37 °C for 15 min. For mixtures containing
oligonucleotide substrates, cleavage intermediates were trapped by
adding 2 µl of 10% SDS followed by 1 µl of 375 mM
EDTA, pH 8.0. Samples were digested with proteinase K and
ethanol-precipitated. To monitor strand-specific single-stranded DNA
breaks, cleavage products were resolved by electrophoresis in 7 M urea, 14% polyacrylamide gels in 100 mM Tris
borate, pH 8.3, 2 mM EDTA. To monitor double-stranded DNA
breaks, products were resolved in 12% nondenaturing polyacrylamide gels. In all cases, DNA cleavage products were visualized and quantified on a Bio-Rad Molecular Imager.
and 10 nM activated nicked oligonucleotide in a total of 20 µl
of 10 mM Tris-HCl, pH 7.9, 135 mM KCl, 7.5 mM CaCl2, 0.1 mM EDTA, and 2.5%
glycerol in the absence or presence of etoposide. Reaction mixtures
were incubated at 37 °C for 48 h, and ligation was stopped by
the addition of 2 µl of 10% SDS followed by 1 µl of 375 mM EDTA, pH 8.0. Samples were treated, resolved in
denaturing polyacrylamide gels, and analyzed as described above for
oligonucleotide cleavage reactions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
with Etoposide Stimulates
the Formation of Single-stranded DNA Breaks--
The one-drug model of
etoposide action predicts that drug treatment will produce
enzyme-associated DNA breaks that are primarily double-stranded.
Conversely, the two-drug model predicts that etoposide treatment will
generate primarily single-stranded breaks at subsaturating drug
concentrations. In addition, the ratio of single-stranded to
double-stranded DNA breaks will drop with rising drug concentration,
because the probability that both protomer active sites of
topoisomerase II are occupied by drug will increase at higher levels of
etoposide. The latter two predictions appear to be true for human
topoisomerase II
(Fig. 1). At the
lowest drug concentration examined (50 µM), the ratio of
single-stranded to double-stranded DNA breaks was
~2.6:1.1 Furthermore, this
ratio dropped to approximate parity at 500 µM etoposide.
Although the experiment shown in Fig. 1 was carried out in the absence
of a high energy cofactor, similar results were observed when ATP or a
nonhydrolyzable ATP analog was included in DNA cleavage assays (not
shown). Taken together, these findings are inconsistent with the
one-drug model and provide support for the two-drug model discussed
above.
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Fig. 1.
Treatment of human topoisomerase
II with etoposide generates single-stranded
and double-stranded breaks in plasmid DNA. An agarose gel of a
typical DNA cleavage assay is shown at top. Single-stranded
(SS) and double-stranded (DS) DNA cleavage
convert negatively supercoiled plasmid DNA (form I,
FI) to nicked circular (form II, FII)
and linear molecules (form III, FIII),
respectively. The ratio of single-stranded to double-stranded DNA
breaks is quantified at bottom. The absolute percentage of
single-stranded (SS breaks) and double-stranded (DS
breaks) DNA breaks is shown in the inset. Error
bars represent the mean ± S.E. for two independent
experiments.
1 position relative to the point of scission)
(44, 45), but rather contained a
1 G on both the top and bottom strands. However, equilibrium levels of scission on each individual strand increased nearly 30-fold at 500 µM drug (Fig.
3, A and C).
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Fig. 2.
Oligonucleotides used to examine etoposide
action. The central sequence of the duplex substrate (residues
80-126 of pBR322) used to monitor DNA cleavage (A and
C) by wild-type human topoisomerase II or DNA
ligation (B and D) by either the Y805F or the
wild-type enzyme is shown. Points of topoisomerase II-mediated DNA
scission are denoted by the arrows. Asterisks
indicate the radiolabeled DNA strands on which strand-specific DNA
cleavage and ligation were monitored. Substrates for DNA ligation
contained a nick at the point of scission for topoisomerase II, and the
terminal 5'-phosphate at the nick was activated for DNA ligation by the
covalent attachment of p-nitrophenol (34). Oligonucleotides
A-D represent the substrates utilized in the corresponding
(panels) (A-D) of Figs. 3-5. When appropriate,
the G at the
1 position (in bold) relative to the point of
scission on the top or bottom strand was changed to an A, T, or
C.
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Fig. 3.
Sequence specificity of etoposide. DNA
substrates used in A-D are as shown in Fig. 2. DNA cleavage
mediated by wild-type human topoisomerase II (A) and
ligation by the Y805F mutant enzyme (B) were monitored on
the top strand. DNA cleavage mediated by wild-type human topoisomerase
II
(C) and ligation by the Y805F mutant enzyme
(D) were monitored on the bottom strand. The
1 base
(relative to the point of scission for topoisomerase II) on the strand
being cleaved/ligated was changed as indicated. For all substrates, the
amount of DNA cleavage observed in the absence of etoposide was set to
1 (A and C), and the amount DNA ligation observed
in the absence of drug was set to 100% (B and
D). All substrates contained a weak drug site (
1 G) on
opposite strand. Error bars indicate the standard deviation
of three independent experiments.
1 position was characterized on both strands of the
oligonucleotide substrate. The
1 G of the top strand was changed to
an A, T, or C (a corresponding change was made in the complementary
strand to maintain base-pairing), and the level of single-stranded
breaks in this strand was monitored. As predicted from previous
sequence analysis (44, 45), the greatest drug-induced stimulation of
DNA cleavage of the top strand (~70-fold at 500 µM
drug) was observed when the base at the
1 position was a C (Fig.
3A). A similar increase in the level of single-stranded
breaks in the bottom strand was seen when the
1 G was changed to a C
(Fig. 3C).
to
seal a nicked oligonucleotide whose 5'-phosphate terminus was activated
by covalent attachment to the tyrosine mimic, p-nitrophenol (34). To ensure that results were not influenced by potential drug-induced cleavage on the strand opposite to that being monitored, a
Y805F active site mutant of human topoisomerase II
(that was incapable of mediating DNA scission) (34) was used for these initial experiments.
1 G on either strand was converted to a C, the
IC50 for etoposide-induced inhibition of DNA ligation on
that strand decreased 20-25-fold (Table
I). These results supply further evidence
that etoposide raises the concentration of topoisomerase II-DNA
cleavage complexes primarily by inhibiting DNA ligation.
IC50 values for etoposide-induced inhibition of
topoisomerase II-mediated DNA ligation
in place of the
Y805F mutant enzyme. Qualitatively, results were the same (Table I).
The IC50 value for etoposide-induced inhibition of DNA
ligation mediated by wild-type topoisomerase II
on either strand
decreased ~30-fold when the
1 G on the strand being monitored was
converted to a C. However, as predicted by the previous drug-enzyme
binding data, DNA ligation mediated by the wild-type enzyme was always
2-5 times more sensitive to inhibition by etoposide than the Y805F
mutant (Table I).
1 G), and the
second monitored these reactions at a scissile bond containing a strong
drug site (
1 C). If etoposide action at one scissile bond is
communicated to the other, introduction of a strong drug site on the
opposite strand should have a significant effect on DNA cleavage and
ligation of the strand being monitored. Conversely, if etoposide acts
independently at the two scissile bonds, introduction of a strong
etoposide site on the opposite strand should have little or no effect
on the DNA strand being monitored.
rather than
the Y805F mutant enzyme was used to examine potential etoposide-induced
interstrand communication during DNA ligation. Because the Y805F mutant
enzyme cannot cleave DNA, it is not clear whether etoposide is capable
of acting on the strand opposite the activated nick in ligation assays
that employ this enzyme. Wild-type topoisomerase II
, however,
establishes a DNA cleavage/ligation equilibrium on the nonactivated
strand at the same time that it catalyzes a unidirectional DNA ligation
reaction on the activated strand. Under the conditions examined,
etoposide stimulation of DNA cleavage on the nonactivated strand of a
ligation substrate (see oligonucleotides B and D
in Fig. 2) was similar to that observed for the corresponding strand of
an intact DNA cleavage substrate (see oligonucleotides A and
C in Fig. 2) (data not shown).
were monitored at a scissile bond
containing a weak etoposide site (
1 G) when the opposite
strand contained either a weak (
1 G) or a strong (
1 C) site for
drug action. As seen in Fig. 4,
A and B, enhancement of DNA cleavage and
inhibition of DNA ligation at the weak etoposide site on the top strand
were unaffected when the bottom strand was converted to a strong drug
site. Although a marginal increase in cleavage of the bottom strand
(Fig. 4C) was observed when the top strand was converted to
a strong drug site, there was no effect on the inhibition of
ligation (Fig. 4D).
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Fig. 4.
Effect of a strong etoposide site on the DNA
cleavage/ligation equilibrium of a weak drug site on the opposite
strand. DNA substrates used in A-D are as shown in
Fig. 2. A and B, DNA cleavage and ligation
mediated by wild-type human topoisomerase II were monitored on the
top strand. C and D, DNA cleavage and ligation by
wild-type human topoisomerase II
were monitored on the bottom
strand. All substrates contained a weak drug site (
1 G) on the
cleaved/ligated strand. The
1 base on the opposite strand was changed
from a G to a C as indicated to create a strong etoposide site. For all
substrates, the amount of DNA cleavage observed in the absence of
etoposide was set to 1 (A and C), and the amount
DNA ligation observed in the absence of drug was set to 100%
(B and D). Error bars indicate the
S.D. of three independent experiments.
1 C) when the opposite strand contained either a weak or a
strong drug site. In all cases, the introduction of a strong drug site
on the opposite strand had (at best) a marginal effect on the ability
of etoposide to increase cleavage at the strong site being monitored
(Fig. 5, A and C).
Furthermore, no effect on the inhibition of ligation was observed (Fig.
5, B and D).
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Fig. 5.
Effect of a strong etoposide site on the DNA
cleavage/ligation equilibrium of a strong drug site on the opposite
strand. DNA substrates used in A-D are as shown in
Fig. 2. A and B, DNA cleavage and ligation
mediated by wild-type human topoisomerase II were monitored on the
top strand. C and D, DNA cleavage and ligation by
wild-type human topoisomerase II
were monitored on the bottom
strand. All substrates contained a strong drug site (
1 C) on the
cleaved/ligated strand. The
1 base on the opposite strand was changed
from a G to a C as indicated to create the strong etoposide site. The
insets in B and D expand the data
shown for enzyme-mediated DNA ligation carried out in the presence of
low concentrations (0-10 µM) of etoposide. For all
substrates, the amount of DNA cleavage observed in the absence of
etoposide was set to 1 (A and C), and the amount
DNA ligation observed in the absence of drug was set to 100%
(B and D). Error bars represent the
S.D. of three independent assays.
is correct, etoposide
should increase levels of enzyme-mediated double-stranded DNA breaks
only when both scissile bonds are occupied with drug. To test this
critical aspect of the model, topoisomerase II-generated
double-stranded DNA breaks were monitored by resolving reaction
products in nondenaturing gels. The following conditions were examined:
both strands of the DNA cleavage sequence contained a weak etoposide
site (G at the
1 position); only one of the two strands (either the
top or the bottom) contained a strong drug site (C at the
1
position); or both strands contained a strong drug site. All
experiments utilized the oligonucleotide system described in the
previous section. Results are shown when reactions were carried out
in the presence of 50 or 500 µM etoposide.
1 C)
was introduced on either the top or bottom DNA strand at either drug
concentration, levels of double-stranded breaks increased slightly
(<60% on average). However, when strong sites were introduced on both
strands, levels of double-stranded DNA breaks rose dramatically,
~5.5-fold at 50 µM etoposide and ~3.5-fold at 500 µM drug.
View larger version (27K):
[in a new window]
Fig. 6.
Enhanced formation of double-stranded DNA
breaks requires strong etoposide sites on both DNA strands. DNA
cleavage reactions contained either 50 (left) or 500 µM (right) etoposide (Etop).
Double-stranded DNA breaks (dsDNA breaks) were monitored
when both strands of the oligonucleotide substrate contained weak drugs
sites (Both 1 G), only one of the two strands contained a
strong drug site (Top
1 C or Bottom
1 C), or
both strands contained strong drug sites (Both
1 C). In
order to quantify double-stranded DNA breaks, cleavage products were
resolved under nondenaturing conditions. Error bars
represent the S.D. of three to four independent assays.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
This model, which is depicted in Fig. 7,
postulates that separate drug molecules must act at each of the two
scissile bonds of a cleavage complex in order to increase enzyme-mediated double-stranded DNA breaks. Etoposide actions at either
of the two scissile bonds appear to be independent of one another, with
each individual drug molecule stabilizing a strand-specific nick rather
than a double-stranded DNA break. A final implication of this model is
that there is little or no communication between the two protomer
active sites of topoisomerase II, at least in the presence of
etoposide.
View larger version (41K):
[in a new window]
Fig. 7.
Two-drug model for etoposide action
against human topoisomerase II . In order to induce
double-stranded DNA breaks, etoposide (red) interactions are
required at both scissile bonds of a topoisomerase II-DNA cleavage
complex (bottom). Etoposide actions at either of the two
scissile bonds (arrows) are independent of one another, with
each individual drug molecule stabilizing a strand-specific nick
(left or right) rather than a double-stranded
break. There appears to be little or no molecular communication between
the two protomer active sites of topoisomerase II (green
ovals) in the presence of etoposide. See text for further
details.
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ACKNOWLEDGEMENTS |
---|
We are grateful to R. Vélez-Cruz and J. S. Dickey for critical reading of the manuscript.
![]() |
FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants GM33944 (to N. O.) and GM58596 (to A. B. B.).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.
§ Trainee under National Institutes of Health Grant 5 T32 CA09385.
** To whom correspondence should be addressed: Dept. of Biochemistry, 654 Robinson Research Bldg., Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-322-4338; Fax: 615-343-1166; E-mail: osheron@ctrvax.vanderbilt.edu.
Published, JBC Papers in Press, December 8, 2002, DOI 10.1074/jbc.M212056200
1 Because the agarose gel system employed to resolve pBR322 topoisomers cannot distinguish between molecules that contain single or multiple nicks, the ratio of single-stranded to double-stranded DNA breaks probably is underestimated.
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