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INTRODUCTION |
DNA topoisomerase II is an enzyme that is necessary for the
survival of all proliferating cells (1, 2). In addition to its normal
functions in replication and mitosis, it is the target for some of the
most widely prescribed drugs used in the treatment of human cancers
(3-8). The essential nature of topoisomerase II, as well as its role
as a target for anticancer chemotherapy, extend from its unique status
in the cell; it is the only enzyme known to create transient
double-stranded breaks in the genetic material (9-12). This ability to
cleave and religate DNA in a concerted fashion allows topoisomerase II
to disentangle topologically linked DNA molecules or alter the
supercoiled state of nucleic acids without compromising the integrity
of the genome (2). Conversely, when the cleavage/religation cycle of
the enzyme is perturbed by anticancer drugs that enhance cleavage or
inhibit religation, topoisomerase II is converted to a lethal enzyme
that generates high levels of breaks in the DNA of treated cells (3, 6,
12-14).
The reversibility of topoisomerase II-mediated DNA scission results
from the fact that the enzyme forms a proteinaceous bridge that spans
the double-stranded break and never releases its cleaved nucleic acid
intermediate (15, 16). Throughout its scission reaction, topoisomerase
II remains covalently linked to the newly generated 5' termini of the
cleaved DNA through the active-site tyrosyl residue of each of its two
identical subunits (17, 18). Although the covalent topoisomerase II-DNA
"cleavage complex" is a fleeting intermediate in the catalytic
cycle of the enzyme (9-12), it ensures resealing of the
double-stranded DNA break and prevents illegitimate recombination that
would result from ligation of DNA termini to different nucleic acid molecules.
Sites of covalent topoisomerase II-DNA cleavage complex formation on
any given nucleic acid substrate are reproducible and nonrandom, but
the basis of this DNA sequence specificity remains obscure. Consensus
DNA cleavage sequences have been determined for topoisomerase II from
several eukaryotic species, ranging from Drosophila
melanogaster to humans; however, these consensus sequences are
generally weak and vary significantly from one another (9, 19-22). In
addition, DNA sequences that contain strong sites of enzyme-mediated
scission have been identified that bear little relation to the
published consensus for topoisomerase II from that species (16, 19,
23). Thus, the predictive value of consensus DNA cleavage sequences for
the eukaryotic type II enzyme appears to be limited.
Because the nucleic acid sites at which topoisomerase II acts probably
govern (to at least some extent) the ability of the enzyme to carry out
its physiological functions (1, 2, 24-27), it is critical to
understand the basis by which topoisomerase II selects its specific
sites of action on DNA. Therefore, a systematic evolution of ligands by
exponential enrichment
(SELEX)1 approach (28) was
utilized to address this fundamental issue for topoisomerase II from
Drosophila. This approach differs from previous studies that
mapped and compared enzyme-mediated DNA cleavage sites in that it
identifies "preferred" (i.e. highly selected) rather
than consensus (i.e. average) sites of topoisomerase II
scission. Following 20 rounds of selection/evolution based on
enzyme-mediated DNA scission, a predominant 18-mer sequence emerged.
This sequence is dramatically overrepresented in the Drosophila euchromatic genome, suggesting that it may
represent a site of physiological action of topoisomerase II.
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EXPERIMENTAL PROCEDURES |
Materials--
D. melanogaster
topoisomerase II was purified from embryonic Kc cells as described by
Shelton et al. (29). Saccharomyces cerevisiae
wild-type topoisomerase II and the mutant ytop2Y783F enzyme (in which
the active-site tyrosine was replaced by a phenylalanine) were
overexpressed and purified from yeast cells as described by Elsea
et al. (30) except that the initial phosphocellulose chromatography was replaced by hydroxylapatite (29). The construct utilized for the overexpression of ytop2Y783F was the generous gift of
Dr. J. E. Lindsley (University of Utah) and has been described previously (31). The
and
isoforms of human topoisomerase II
were overexpressed in S. cerevisiae and purified as
described previously (32). Etoposide and amsacrine were purchased from Sigma, and the quinolone CP-115,953 was the generous gift of Drs. T. Gootz and P. McGuirk (Pfizer Central Research, Groton, CT). All drugs
were stored at
20 °C as 10 mM stocks in
Me2SO. Tris and urea were purchased from Sigma; SDS was
from Merck; proteinase K was from U. S. Biochemical Corp.; restriction
endonucleases, calf intestine alkaline phosphatase, and polynucleotide
kinase were from New England BioLabs; Klenow and Taq DNA
polymerases were from Promega; [
-32P]ATP (6000 Ci/mmol) and Sequenase DNA polymerase were from Amersham Pharmacia
Biotech; and Bluescript SK+ phagemid was from Stratagene. All chemicals
were analytical reagent grade.
Preparation of Oligonucleotides--
Oligonucleotides were
synthesized on an Applied Biosystems DNA synthesizer, followed by
purification on 14% denaturing polyacrylamide gels. The initial
substrate was constructed as a single-stranded 60-mer oligonucleotide
with the following sequence:
5'-TAGTGGATCCGCTAACGCAGN20CAGTTAAATTGAATTCGATA-3', where the central 20 bp contained random sequence (N) based on equal
incorporation of A, G, C, and T at each position. The complementary strand was synthesized by annealing an oligonucleotide primer with the
sequence 5'-TATCGAATTCAATTTAACTG-3' (primer 1) followed by incubation
at 37 °C for 20 min with Sequenase in 40 mM Tris, pH
7.5, 5 mM MgCl2, 5 mM
dithiothreitol, 50 mM NaCl, and 50 µg/ml bovine serum
albumin in the presence of 1 mM each TTP, dCTP, dGTP, and
dATP. The double-stranded DNA product was gel-purified on an 8%
nondenaturing polyacrylamide gel. Additional oligonucleotides used in
this study were: 5'-TAGTGGATCCGCTAACGCAG-3' (primer 2), 5'-TAGTGGATCCGCTAACGCAGTATATATACATATATATACAGTTAAATTGAATTCGATA-3' (clone 1, top),
5'-TATCGAATTCAATTTAACTGTATATATATGTATATATACTGCGTTAGCGGATCCACTA-3' (clone
1, bottom),
5'-TAGTGGATCCGCTAACGCAGTATATATACATGTATATACAGTTAAATTGAATTCGATA-3' (clone
17, top), and
5'-TATCGAATTCAATTTAACTGTATATATCATGTATATATACTGCGTTAGCGGATCCACTA-3' (clone 17, bottom). Annealing of oligonucleotides was
accomplished by mixing equimolar amounts of each strand, heating at
70 °C for 20 min, and then gradually cooling to room temperature
over at least 1 h.
Selection Protocol--
Cleavage complexes were established at
30 °C by incubating 200 ng of oligonucleotide substrate (100 nM) with 2 µg of Drosophila topoisomerase II
(100 nM) for 10 min in 50 µl of cleavage buffer (10 mM Tris-Cl (pH 7.9), 50 mM NaCl, 50 mM KCl, 0.1 mM EDTA, and 2.5% glycerol)
containing 5 mM CaCl2. CaCl2,
rather than MgCl2, was employed because topoisomerase II
generates significantly higher levels of single-stranded DNA breaks in
the presence of Ca2+ (33). This is important because the
subsequent amplification of the cleavage products requires an intact
template DNA strand. Cleavage complexes were trapped by the addition of
50 µl of 4% SDS, followed by 50 µl of 4 mM EDTA, and
were precipitated by the addition of 50 µl of 400 mM
Tris-Cl, pH 7.9, 250 mM KCl, and 5 µg/ml tRNA. After
incubation on ice for 10 min, precipitates were collected by
centrifugation at 14,000 × g for 15 min at 4 °C.
Samples were resuspended in 200 µl of 10 mM Tris, pH 8.0, 100 mM KCl, 1 mM EDTA, and 5 µg/ml tRNA for
10 min at 45 °C, reprecipitated by shifting the temperature from
45 °C to ice for 10 min, and collected by centrifugation as above.
Precipitates were resuspended at 45 °C for 10 min in 200 µl
of water containing 5 µg/ml tRNA, ethanol-precipitated twice, washed
with 95% ethanol, and dried under partial vacuum at room temperature.
Selection reactions were carried out in quadruplicate and pooled prior
to the second ethanol precipitation.
Amplification of Selected DNA--
Selected DNA molecules
(i.e. molecules isolated from cleavage complexes) were
amplified under mutagenic conditions (34) as follows. The dried pellets
from the selection procedure were resuspended in 198 µl of 10 mM Tris-Cl, pH 8.3, 50 mM KCl, 7 mM MgCl2, 0.5 mM MnCl2, 1 mM dCTP, 1 mM TTP, 0.2 mM dATP, 0.2 mM dGTP, and 0.01% gelatin containing 2 ng/µl each of
primers 1 and 2. Amplification was initiated by the addition of 2 µl
(10 units) of Taq polymerase. Samples were overlaid with 100 µl of light mineral oil and cycled in an Ericomp TwinBlock thermal
cycler, using the following program: 5 min at 94 °C; followed by 20 cycles each consisting of 1 min at 94 °C, followed by 1 min at
50 °C and 1 min at 72 °C. After the last cycle, 2 µg of each
primer was added, reactions were incubated at 94 °C for 5 min, and
one additional cycle was run as above. Since the template DNA molecules for this procedure were heterogeneous, this last annealing and extension in the presence of excess primer was important to ensure that
both strands of the DNA substrate used for the next round of selection
were properly base-paired and exactly complementary for any individual
DNA molecule.
Experiments with a control oligonucleotide of known sequence indicated
that the rate of misincorporation was ~0.4 bp/oligonucleotide molecule/round (which included 20 cycles of amplification) of SELEX.
Therefore, the products of the final cycle of amplification for any
given round of SELEX should have contained no more than a single base
pair mismatch for every 50 oligonucleotide molecules. Since there is no
mechanism by which mismatched base pairs can be specifically maintained
from round to round of the SELEX protocol, it is unlikely that the
potential existence of a low level of mismatches in the DNA affected
the overall selection process.
Following amplification, samples were ethanol-precipitated, resuspended
in 40 µl of water and 4 µl of loading buffer (10 mM Tris, pH 7.9, 60% sucrose), and subjected to electrophoresis in an 8%
nondenaturing polyacrylamide gel at 10 watts for ~2 h. Amplification products were located by shadowing with ultraviolet light, and the DNA
band was excised from the gel and eluted overnight in 400 µl of 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA. The eluted DNA was ethanol-precipitated and
resuspended in 50 µl of water, and its concentration was determined
by measuring its absorbance at 260 nm. This DNA was then used as the
substrate for a new round of selection.
To control for the possibility of contamination during the SELEX
protocol, samples with no added DNA were run alongside the normal
samples for every step of the selection and amplification procedures.
When the DNA from the amplification reactions was gel-purified, a
corresponding gel slice from the contamination control reaction lane
was also excised and incubated overnight in the elution buffer. After
ethanol precipitation and resuspension, the same volume of the control
sample was used as the "DNA" for the contamination control reaction
of the next round of selection and amplification. None of the
contamination controls produced a band upon UV shadowing of the gel.
K+/SDS DNA Cleavage and Religation
Assays--
Levels of cleavage complex formation were monitored by the
K+/SDS precipitation assay (35, 36). Oligonucleotide pools
(from each round that was assayed) were digested with EcoRI
and BamHI restriction endonucleases, and the ends were
filled in using Klenow fragment and [
-32P]dATP in the
presence of nonradioactive nucleotides (37). Labeled oligonucleotides
were gel-purified as described above. DNA cleavage reactions contained
100 nM Drosophila topoisomerase II and 5 nM oligonucleotide substrate and were carried out for 10 min at 30 °C in 50 µl of cleavage buffer containing either 5 mM CaCl2 or MgCl2. For reactions
that utilized yeast topoisomerase II, no KCl was used, and the NaCl
concentration was 100 mM. For reactions that utilized human
topoisomerase II
or
, the cleavage buffer was the same except
that no NaCl was used and the KCl concentration was 100 mM.
When DNA cleavage reactions were carried out in the presence of drugs,
100 µM drug was included such that the final Me2SO concentration was 1%. Unless stated otherwise, DNA
cleavage reactions were always carried out in the presence of 5 mM CaCl2.
As a prelude to determining rates of DNA religation, cleavage complexes
were established in cleavage buffer containing CaCl2 and
trapped by the addition of EDTA (5 mM final concentration). Following incubation at 30 °C for 2 min, NaCl was added (250 mM final concentration of additional salt), and samples
were equilibrated at 30 °C for 1 min. DNA religation was initiated
by the addition of MgCl2 (10 µM final
concentration) and terminated by the addition of SDS (2% final
concentration) at various time points. Levels of DNA cleavage complex
remaining were determined by the K+/SDS precipitation
protocol described above.
DNA Binding Assays--
Binding of the mutant yeast
topoisomerase II, ytop2Y783F, or the wild-type Drosophila
enzyme to oligonucleotide pools was determined using a nitrocellulose
filter assay (38). Reactions were carried out in 10 µl of the
appropriate cleavage buffer in the presence (yeast) or absence
(Drosophila) of 5 mM MgCl2.
Concentrations of oligonucleotide (radioactively labeled) and enzyme
were as described above for the K+/SDS assays. After
incubation at 30 °C for 10 min, reactions were spotted onto
nitrocellulose filters (Millipore; presoaked in assay buffer), and the
filters were washed three times with 1 ml of cold cleavage buffer.
Filters were dried, and the radioactivity retained was determined by
scintillation counting.
Cloning and Sequencing of in Vitro Selection Evolution
Products--
The oligonucleotide pool from round 20 of the SELEX
procedure was reselected as above and amplified under nonmutagenic
conditions: 30 cycles as above, but with 20 fmol of round 20 DNA as the
template in 10 mM Tris-Cl (pH 9.0); 50 mM KCl;
0.1% Triton X-100; 1 mM each TTP, dATP, dGTP, and dCTP;
and 1 µg each of primers 1 and 2. Oligonucleotide products were
digested with BamHI and EcoRI restriction
endonucleases and ligated into linearized Bluescript SK+ phagemid.
Ligation was performed using the Boehringer Mannheim Rapid DNA Ligation
Kit. Ligation products were used to transform Escherichia
coli, and colonies were selected that contained the phagemid plus
insert as described by the manufacturer. Phagemid from the individual
clones was purified using the Boehringer Mannheim High Pure Plasmid
Isolation kit, and the sequences of the inserts were determined using
the Amersham Pharmacia Biotech Sequenase kit and a sequencing primer
that was complementary to phagemid sequence near the insert
(5'-AAAGCTGGAGCTCCACCGCG-3').
Mapping of Cleavage Sites--
Sites of DNA cleavage were
determined as described previously (39). The composition of cleavage
reactions was as above for the K+/SDS assays except that
the concentration of oligonucleotide was 80 nM.
Double-stranded DNA Cleavage--
In order to verify that
scission of the selected sequences was double-stranded, DNA cleavage
reactions were carried out as above for the K+/SDS assays
(in Mg2+-containing buffer), terminated with SDS, digested
with proteinase K (0.8 mg/ml) for 30 min at 37 °C, and subjected to
electrophoresis in a 14% nondenaturing polyacrylamide gel (cooled to
10 °C) at 10 watts for ~2 h. Radioactive DNA cleavage products
were visualized by PhosphorImager (Molecular Dynamics) analysis.
Genome Searches--
Searches of the European
Drosophila Genome Project data bases were conducted using
the blastn search program (40) with the threshold set at 1000.
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RESULTS |
SELEX Scheme and Rationale--
Topoisomerase II interacts with
~28 bp on its DNA cleavage helix (41, 42) and requires a minimum of
16 bp for efficient DNA scission (43). In an effort to define the
mechanism by which the enzyme recognizes its site of action on nucleic
acid substrates, previous studies have mapped sequences at which
topoisomerase II cleaves DNA. This approach has resulted in a series of
weak consensus cleavage sequences for several eukaryotic type II
enzymes including Drosophila, chicken, mouse, and the
and
isoforms of human (19-22). However, these consensus sites
exhibit little sequence agreement among themselves and even disagree
regarding the position of preferred bases relative to the point of
cleavage. The variability between reported sequences notwithstanding,
topoisomerase II displayed at least some level of specificity for 6-10
sequence positions in each of these studies (9, 19-22). Assuming an
average of eight base-specific points of contact between topoisomerase II and its DNA cleavage site, it would be necessary to determine every
site of action for the enzyme in ~65,000 bp of random DNA in order to
generate all possible sequences (i.e. 48
combinations) at these positions. Since the size of DNA substrates utilized for the generation of consensus sequences generally ranged between 1000 and 10,000 bp (19-22), it is clear that (on average) only
a small fraction of the necessary cleavage sites have been sampled in
order to confidently define the intrinsic specificity of topoisomerase II.
In light of the above, we have utilized an alternative approach to
address this fundamental issue of topoisomerase II specificity. Rather
than attempting to generate a consensus sequence for enzyme action
based on mapping sites in a larger fragment of DNA, a SELEX protocol (28) was employed to select/evolve preferred sites of
DNA cleavage mediated by Drosophila topoisomerase II from a pool of ~1012 potential sequences.
The scheme utilized for the present study is shown in Fig.
1. The initial DNA substrate employed for
the SELEX protocol was a 60-mer oligonucleotide that incorporated two
critical features: 1) it included 20 bp of defined flanking sequences
(derived from pBR322) at each end that were devoid of topoisomerase II
cleavage sites (16, 44) and contained the indicated restriction
endonuclease recognition sites for eventual cloning; and 2) it included
a 20-bp core of random DNA sequence that was synthesized using an
equimolar ratio of all four bases. This random portion of the substrate allowed the type II enzyme to select/evolve preferred sites of DNA
cleavage from among as many as 420 sequences, affording
topoisomerase II the opportunity to sample pools of potential cleavage
sites many orders of magnitude larger than those used in previous
studies.

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Fig. 1.
General scheme for the SELEX protocol.
Preferred topoisomerase II DNA cleavage sites were selected/evolved
using the following procedure: 1) covalent topoisomerase II-DNA
cleavage complexes were formed in the presence of Ca2+; 2)
cleavage complexes were trapped by the addition of SDS, precipitated
with KCl, washed extensively, and redissolved; 3) primers were annealed
to the oligonucleotides obtained from redissolved cleavage complexes;
and 4) the enriched pool of DNA cleavage sequences was amplified under
mutagenic conditions to provide substrate DNA for the next round of
SELEX. The initial oligonucleotide cleavage substrate is shown at the
top. It included a 20-bp core of random DNA flanked by
constant sequences that were devoid of topoisomerase II cleavage sites
(16, 44) and contained sites for restriction endonucleases.
Topoisomerase II (Topo II) was modeled after the crystal
structure reported by Berger et al. (58).
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Drosophila topoisomerase II was incubated with DNA
substrate, and cleavage complexes were established in the presence of
CaCl2 (rather than the physiological divalent cation
MgCl2 (45)). Although the DNA cleavage site specificity of
topoisomerase II appears to be the same in the presence of either
divalent cation (23, 33), the use of CaCl2 provided two
important advantages. First, levels of DNA cleavage generated in the
presence of Ca2+ are significantly higher than those
generated in the presence of Mg2+ (33). Second,
topoisomerase II generates considerably more single-stranded DNA breaks
in the presence of Ca2+ than it does in
Mg2+-containing reactions (18, 33). The stimulation of
single-stranded DNA scission by Ca2+ is especially
important, because it leaves one intact strand of DNA in the cleavage
complex, which can then act as a template for the geometric
amplification of the selected nucleic acid molecules.
Covalent topoisomerase II-DNA cleavage complexes formed in the presence
of Ca2+ were trapped by the addition of SDS and isolated by
precipitation of the enzyme in the presence of KCl (35, 36). DNA
molecules that were not covalently attached to topoisomerase II
(through at least one of the two strands) remained in solution.
Following a series of washes, oligonucleotides that co-precipitated
with the denatured enzyme were amplified to provide the substrate DNA for the next round of SELEX. As demonstrated below (see Fig.
2), the DNA pool was enriched for sites
of DNA cleavage by topoisomerase II with each successive round.

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Fig. 2.
Preferred sites of topoisomerase II-mediated
DNA cleavage were produced by the SELEX protocol. Progress of the
selection/evolution of topoisomerase II-DNA cleavage sites was
monitored by the K+/SDS precipitation assay in the presence
of Ca2+. Qualitatively similar results were obtained when
levels of cleavage complex formation were determined in the presence of
Mg2+ (inset). Data represent the averages of two
(Mg2+) or three (Ca2+) independent experiments,
and the S.E. is denoted by the error bars.
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Although the random portion of the initial oligonucleotide substrate
contained (in theory) all possible 20-mer sequences, amplification was
performed under mutagenic conditions (34) to generate a slight
"drift" in the sequences selected for each round. This was done to
offset any bias in the initial pool or the loss of any important
sequences early in the selection process, since mutagenic amplification
has the potential to regenerate such lost sequences. As discussed under
"Experimental Procedures," the DNA amplification protocol employed
generated ~0.4 mutations/oligonucleotide/round of SELEX.
SELEX Generates Oligonucleotides That Contain Preferred Sequences
for Topoisomerase II-mediated DNA Cleavage--
The SELEX procedure
described above was employed for 20 cycles, with progress being
monitored by K+/SDS precipitation of cleavage complexes
containing radiolabeled DNA (Fig. 2). While only 3% of the initial
random oligonucleotide pool (round 0) was cleaved by topoisomerase II,
the emergence of preferred cleavage sequences was evident as early as
round 4. By round 20, nearly 20% of the selected oligonucleotide pool was cleaved by the Drosophila type II enzyme. In light of
the fact that the DNA cleavage/religation equilibrium of topoisomerase II normally lies far toward religation (9-13), 20% represents an
unusually high level of enzyme-mediated DNA scission. Four more rounds
of SELEX were carried out, but the levels of DNA cleavage appeared to
plateau by round 20 (data not shown).
Since the SELEX protocol employed Ca2+ as the divalent
cation for DNA cleavage reactions (see above), a control experiment was performed that monitored cleavage complex formation of the SELEX pools
in the presence of Mg2+ (Fig. 2, inset). As
expected (18, 33), levels of topoisomerase II-mediated DNA scission
were lower in Mg2+ than those generated in the presence of
Ca2+. This difference notwithstanding, the emergence of
preferred DNA sequences mirrored the trend observed in the
Ca2+-containing reactions. This finding supports the
previous observation that the DNA cleavage site specificity of
topoisomerase II is not dictated by the nature of its divalent cation
cofactor (18, 23, 33).
Topoisomerase II-mediated Cleavage of Oligonucleotides Generated by
SELEX Is Stimulated by Anticancer Drugs--
Beyond its critical
physiological functions, topoisomerase II is the target for a number of
anticancer drugs that are in wide clinical use (5, 7, 8). These agents
act by increasing levels of topoisomerase II-mediated DNA cleavage (3,
6, 12-14). Consensus sequences reported for drug-stimulated DNA
scission generally differ from those reported for drug-free reactions
and show specificity at fewer positions (4, 8). Consequently, it has
been questioned whether drug-stimulated scission takes place primarily
at a subpopulation of "intrinsic" topoisomerase II DNA cleavage
sites or rather is induced at a novel population of drug-specific sites.
In order to address this issue, the effects of three structurally
diverse topoisomerase II-targeted drugs on enzyme-mediated DNA cleavage
of the round 0 and 20 SELEX pools were determined (Fig.
3). These experiments were performed in
the presence of Mg2+ and 100 µM etoposide,
amsacrine, or CP-115,953 (or 1% Me2SO as a solvent
control). While cleavage of the round 0 (random) substrate displayed
little sensitivity (<2-fold stimulation) to the three drugs examined,
cleavage of the round 20 oligonucleotide pool was stimulated between 4- and 6-fold. This result indicates that oligonucleotide substrates that
are enriched for "intrinsic" topoisomerase II cleavage sites are
also enriched for drug-inducible sites. It further supports the
hypothesis that drug-induced DNA cleavage complexes are formed
primarily at sites intrinsic to the enzyme rather than at a novel
population of drug-specific sequences (12, 46).

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Fig. 3.
Topoisomerase II selected/evolved sites of
DNA scission that were more sensitive to cleavage-enhancing drugs than
was the initial DNA substrate pool. The effects of topoisomerase
II-targeted drugs on DNA cleavage complex formation in
Mg2+-containing buffer were determined by the
K+/SDS protocol. Levels of cleavage complex formed in the
presence of drug solvent (Me2SO; DMSO), or 100 µM etoposide, amsacrine, or CP-115,953 are shown for the
initial oligonucleotide substrate pool (Round 0,
solid bars) and the final SELEX DNA pool (Round
20, open bars). Data are the average of two
independent experiments, and the S.E. is denoted by the
error bars.
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Oligonucleotide Cleavage Substrates Selected/Evolved by Drosophila
Topoisomerase II Are Not Universally Preferred Substrates for Type II
Enzymes from Other Species--
Previous studies indicate that type II
topoisomerases, even from diverse eukaryotic organisms, will often
cleave a given DNA substrate at a similar array of sites (20, 22, 23,
47, 48). However, since consensus DNA cleavage sequences differ considerably for enzymes from different species (9, 19-22), it is
obvious that generalizations from enzyme to enzyme may not be
appropriate. To determine whether DNA cleavage substrates selected by
Drosophila topoisomerase II are also preferred substrates
for enzymes from other species, the ability of yeast (S. cerevisiae) topoisomerase II as well as the
and
isoforms
of the human enzyme to cleave the round 0 and 20 SELEX pools was
determined. As seen in Fig. 4, a
significant enhancement of cleavage (~3-fold) for the round 20 pool
over the round 0 substrate was observed for yeast topoisomerase II.
However, neither of the human isoforms displayed any appreciable
specificity for the round 20 SELEX pool. These data provide further
evidence that the specificity of type II topoisomerases from different
species is not necessarily conserved for any given preferred
sequence.

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Fig. 4.
Preference for cleavage of the
selected/evolved sites is not evolutionarily conserved. Cleavage
of the round 0 and round 20 DNA pools by yeast topoisomerase II and the
and isoforms of the human enzyme was determined by the
K+/SDS protocol in Ca2+-containing buffer. Data
are the average of two independent experiments, and the S.E. is denoted
by the error bars.
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Mechanism of DNA Cleavage Enhancement--
Higher levels of
topoisomerase II-DNA cleavage complex formation can result from
increased binding between the enzyme and its substrate DNA, from
increased cleavage within the noncovalent topoisomerase II-DNA complex,
or from both (3, 9, 12). As a first step toward determining the
mechanistic basis for the evolution of preferred cleavage sequences,
the binding of topoisomerase II to the round 0 and 20 DNA pools was
characterized. In this experiment, enzyme-DNA binding was monitored by
a nitrocellulose filter protocol (38). A mutant yeast topoisomerase II
in which the active-site tyrosine was replaced with a phenylalanine
(ytop2Y783F) was utilized for this study (31). The use of this mutant
enzyme allowed binding to be monitored in the absence of DNA cleavage, even when Mg2+ was present in assay mixtures. As seen in
Fig. 5, ytop2Y783F displayed a similar
binding affinity for the round 0 and 20 SELEX pools.

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Fig. 5.
Topoisomerase II binds the round 0 and round
20 DNA pools with similar affinity. A nitrocellulose filter
protocol was used to determine the binding of topoisomerase II
(Topo II) to the DNA pools in Mg2+-containing
buffer. The enzyme used was ytop2Y783F, a mutant yeast type II
topoisomerase in which the active-site tyrosine is replaced with a
phenylalanine. Alternatively, binding assays were performed with
wild-type Drosophila (D. mel) topoisomerase II in
the absence of Mg2+ (inset). Inset
axes are as in the main figure. Data are the
average of two independent experiments, and the S.E. is denoted by the
error bars.
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Previous studies indicate that topoisomerase II will bind DNA in the
absence of a divalent cation, albeit with a decreased affinity (45,
49). Therefore, to extend the above results to the
Drosophila enzyme under conditions that did not allow DNA cleavage within the noncovalent complex, the binding of
Drosophila topoisomerase II to DNA was determined in the
absence of a divalent cation. Once again, there was no significant
difference in binding to the two SELEX pools (Fig. 5,
inset). Thus, the increased cleavage complex formation for
the round 20 SELEX pool is not due to an increased binding affinity of
topoisomerase II for the DNA.
Levels of cleavage within a topoisomerase II-DNA complex are dependent
on the relative rates of DNA scission and religation by the enzyme (9,
10, 12, 13). Unfortunately, direct measurement of the rate of cleavage
is technically unfeasible, since it probably does not represent the
rate-determining step of cleavage complex formation. It is possible,
however, to directly measure the apparent first order rate of DNA
religation within cleavage complexes (33, 50). As seen in Fig.
6, rates of DNA religation for the round
20 SELEX pool were ~3-fold faster than those observed for the initial
round 0 substrate. Two conclusions may be inferred from the above
findings. First, the SELEX protocol did not select/evolve DNA sequences
on the basis of decreased religation rates. Second, it is likely that
the average rate of topoisomerase II-mediated scission is considerably
faster for the selected DNA sequences in the round 20 pool than for the
initial random oligonucleotide substrate.

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Fig. 6.
The round 20 SELEX DNA pool is religated
faster than the initial random substrate (round 0 pool).
Topoisomerase II-DNA cleavage complexes were established in the
presence of Ca2+ and trapped by the addition of EDTA.
Religation of the cleaved DNA was initiated by the addition of
Mg2+, and reactions were terminated by the addition of SDS.
Data are a composite of two independent experiments.
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Sequence of Oligonucleotides in the Round 20 SELEX Pool--
The
round 20 SELEX pool was digested with BamHI and
EcoRI restriction endonucleases and ligated into Bluescript
SK phagemid that had been previously digested with these enzymes. This
construct was used to transform E. coli, and the sequences
of 37 of the resultant clones were determined (Fig.
7). All of the clones sequenced were
found to have random regions that were 18 rather than 20 bp in length,
indicating that a 2-bp deletion occurred at some point in the selection
process. As determined by sequence analysis of oligonucleotide pools,
the deletion emerged between SELEX rounds 4 and 8 (data not shown).

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Fig. 7.
The SELEX protocol converges on a predominant
sequence for topoisomerase II-mediated DNA cleavage. The round 20 SELEX DNA pool was ligated into Bluescript vector and used to transform
E. coli. The predominant nucleic acid sequence obtained
(typified by clone 1) is shown along with its frequency (36/37 clones),
as well as the sequence difference found in the remaining clone (clone
17).
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It appears that the SELEX protocol converged on a single preferred
sequence for topoisomerase II-mediated DNA cleavage (Fig. 7). The vast
majority (36/37) of the clones examined contained the same sequence
(typified by clone 1), alternating T and A residues with a centrally
located CA dinucleotide at positions 9 and 10. The remaining clone
(clone 17) was identical to the others, except that it contained a G
(rather than an A) at position 12.
Sites of Topoisomerase II-mediated DNA Cleavage within Preferred
Sequences--
The two sequences selected by Drosophila
topoisomerase II were examined to determine the specific sites of DNA
scission by the enzyme. Oligonucleotides utilized for these studies
consisted of the selected/evolved 18-mer sequences bordered by the
original constant flanking sequences.
Fig. 8 shows a representative cleavage
site map of the predominant sequence (clone 1) that was identified by
SELEX. The enzyme cleaved this oligonucleotide at four principal sites
on each strand, with seven of the eight total sites occurring 3' to a T
nucleotide (Figs. 8 and 9,
top). Additional minor sites of cleavage were also observed.
Sites of cleavage on the two strands occurred immediately 3' to
pyrimidine residues and aligned with the expected four-base stagger
that is characteristic of topoisomerase II-mediated DNA scission (35,
16). In the one clone that differed in sequence (clone 17), the sites
of cleavage observed were the same as for the predominant clone (Fig.
9, top).

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Fig. 8.
Representative map of topoisomerase
II-mediated cleavage of clone 1 DNA. Cleavage reactions for the
top and bottom strands of clone 1 are shown, with the four principal
sites of scission denoted. Reactions shown consisted of a control in
the absence of enzyme (DNA); cleavage in the presence of
topoisomerase II using different divalent cations (Mg2+ or
Ca2+); and cleavage in Mg2+-containing buffer
in the presence of topoisomerase II and drug solvent
(Me2SO; DMSO), or 100 µM etoposide
(Etop) or amsacrine (m-AMSA).
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Fig. 9.
DNA sequences generated by the SELEX protocol
are cleaved at multiple sites by topoisomerase II. The preferred
sequence (exemplified by clone 1) as well as the single variant (clone
17) generated by SELEX are shown. The nucleotide positions of the four
principal sites of cleavage on the top and bottom strands are indicated
by the arrows. Relative levels of DNA cleavage complex
formation in the presence of either Mg2+ or
Ca2+ at each site of clone 1 (as determined by
PhosphorImager analysis of gels represented in Fig. 8) are also shown.
Data are normalized to cleavage at the top strand of site 1 in the
presence of Mg2+ and are the average of two independent
experiments. The S.E. is denoted by the error
bars.
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It should be noted that the multiple sites of topoisomerase II-mediated
DNA scission within the selected/evolved sequences do not result from
the formation of multiple cleavage complexes on individual
oligonucleotide molecules. Since the enzyme protects at least 28 bp of
DNA, as determined by footprint analysis (41, 42), only a single
topoisomerase II homodimer is capable of forming a cleavage complex
within the preferred sequence of any of these oligonucleotide molecules
at any given time.
Levels of cleavage at specific sites in the predominant sequence (clone
1) were generally 2-4-fold higher in the presence of Ca2+
than in Mg2+, site 3 being the notable exception (Fig. 9,
bottom). Similar levels of scission were observed for clone
17, except that cleavage at site 3 (which encompasses the A:T
G:C
substitution) was decreased by ~50% relative to site 3 in the
predominant sequence (data not shown).
Drug Stimulation of Topoisomerase II-mediated DNA Cleavage at
Specific Sites--
The stimulation of DNA cleavage observed for SELEX
products in the presence of drugs (see Fig. 3) suggests that at least
some of the sites within the selected/evolved sequences are
drug-inducible. Fig. 10 shows the
effects of Me2SO (solvent), etoposide, and amsacrine on the
relative levels of cleavage at the four principal sites of complex
formation observed in the absence of drugs. While Me2SO had
no significant effect on the levels of complex formed, both etoposide
and amsacrine stimulated topoisomerase II-mediated DNA cleavage within
the clone 1 sequence.

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Fig. 10.
Anticancer drugs stimulate cleavage of clone
1 in a site-dependent manner. Relative levels of
cleavage complex formation are shown for each principal site in the
presence of solvent (Me2SO; DMSO), etoposide, or
amsacrine (m-AMSA). Data were obtained by PhosphorImager
analysis of gels such as that shown in Fig. 8. Results were normalized
to cleavage at the top strand of site 1 in the presence of
Me2SO and are the average of two independent experiments.
The S.E. is denoted by the error bars.
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In the presence of etoposide, site 3 exhibited the highest level of
cleavage. This is consistent with the reported preference for a C
residue immediately 5' to the site of etoposide-stimulated DNA cleavage
(see Fig. 9, top) (51). Cleavage at site 2 in clone 17 increased 3-fold relative to that in clone 1 (data not shown), consistent with the T
C substitution 5' to the site of scission on
the bottom strand. Site 3 in clone 17 was cleaved at ~50% of the
level observed in the predominant clone (data not shown).
In the presence of amsacrine, site 2 exhibited the highest level of
cleavage. Both halves of this site match the reported preference for an
A residue immediately 3' to the site of amsacrine-stimulated DNA
cleavage (51). Although site 4 also contains A residues 3' to both
sites of cleavage, levels of amsacrine-induced scission were
considerably lower than observed for site 2. The reason for this
difference is not apparent, but it is notable that cleavage at site 4 was poor (relative to the other sites) under every condition tested.
Topoisomerase II-mediated Cleavage of the Preferred DNA Sequence Is
Double-stranded--
The SELEX protocol took advantage of the fact
that topoisomerase II generates significant levels of single-stranded
DNA breaks in the presence of Ca2+ (33). This raises the
question as to whether the enzyme cleaves the preferred sequence in a
double-stranded fashion in the presence of its physiological divalent
cation, Mg2+ (45). To address this issue, cleavage
complexes trapped with clone 1 in the presence of Mg2+ were
subjected to electrophoresis in a 14% nondenaturing polyacrylamide gel. As seen in Fig. 11, multiple DNA
cleavage products (consistent with the multiple sites of cleavage
within the oligonucleotide) were observed under nondenaturing
conditions. Thus, topoisomerase II-mediated scission of the preferred
sequence is (to at least some extent) double-stranded in nature.

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Fig. 11.
Cleavage of the selected/evolved sequence is
double-stranded in the presence of Mg2+. Topoisomerase
II-DNA cleavage complexes were established with clone 1 oligonucleotide
in Mg2+-containing buffer, trapped by the addition of SDS,
digested with proteinase K, and subjected to electrophoresis in a 14%
nondenaturing polyacrylamide gel at 10 °C. Samples shown are a
control in the absence of enzyme (DNA), cleavage with
topoisomerase II (Topo), cleavage with enzyme but omitting
the proteinase K treatment ( Pro K), and cleavage with
enzyme that was reversed by the addition of EDTA prior to SDS
(+EDTA). Locations of the origin, intact substrate DNA, and
heterogenous double-stranded cleavage products are indicated.
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Fig. 11 also shows that cleavage complexes formed with clone 1 do not
enter the gel if treatment with proteinase K is omitted (lane 3), demonstrating that the DNA in these
complexes is protein-associated. Furthermore, the formation of cleavage
complexes is reversed by the addition of EDTA prior to SDS
(lane 4). Both of these characteristics are
hallmarks of topoisomerase II-mediated DNA scission (9, 10, 12).
The Preferred Topoisomerase II DNA Cleavage Sequence Is
Dramatically Overrepresented in the Euchromatic Genome of D. melanogaster--
Statistically, an exact match for an 18-mer sequence
should occur once in every 70 billion bp of random DNA (i.e.
once in 418 bp). Consequently, it is doubtful that this
sequence should appear by random chance even a single time in the
250-megabase pair genome of D. melanogaster. The odds of
finding a match for such a sequence are reduced even further at the
present time in light of the fact that <15 megabase pairs of the
Drosophila euchromatic genome have been sequenced. To test
this assumption, 10 individual 18-mer sequences were generated in a
random fashion and used to search the European Drosophila
Genome Project data base. No exact matches in the
Drosophila genome were found for any of the 10 random
sequences examined. Of the 10, eight did not even yield partial matches (using the default settings for the blastn search routine with the
threshold set at 1000). The other two each yielded a single partial
match at 15 of 18 or 17 of 18 positions (the latter with an insertion).
In marked contrast, a search of this same data base revealed >60 exact
matches for the predominant Drosophila topoisomerase II DNA
cleavage sequence in the euchromatic genome of this organism. Of these
exact matches, 20 were found in known gene sequences, with 16 of them
occurring near the 5'- or 3'-end of the genes (Fig.
12). Thus, it appears that the
preferred topoisomerase II DNA cleavage sequence selected by SELEX is
dramatically overrepresented in the Drosophila genome.
Statistically (based on the total number of matches), it occurs
approximately once every 250,000 bases. (A similar prevalence of this
sequence was found in the 14-megabase pair S. cerevisiae
genome, consistent with the finding that it also is a preferred
substrate for yeast topoisomerase II.) Even accounting for the
increased A-T content (~57%) of the Drosophila euchromatic genome, this sequence should appear less than once every 8 billion base pairs. Therefore, the sequence TATATATACATATATATA is
present at a level that is at least 10,000 times higher than predicted
by random chance. In addition, an exact match for this sequence was
found within the mitochondrial genome of Drosophila, which
is <20 kilobase pairs in length. Taken together, these findings suggest that the DNA cleavage sequence selected by topoisomerase II is
of physiological importance.

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Fig. 12.
Occurrence of the preferred DNA cleavage
site in Drosophila gene sequences. Shown are the
preferred 18-mer sequence and 15 bp immediately 5' and 3' to the
preferred sequence. Position indicates the sequence location
of the preferred 18-mer in the gene, followed by the complete length of
the gene entry in the data base. Gene indicates the name of
the entry in the data base.
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It is notable that clone 17 (TATATATACATGTATATA), which differed from
the predominant cleavage sequence by only an A
G substitution, yielded one exact match in the Drosophila euchromatic
genome, at the 3'-end of the gene encoding the kinase suppressor of
ras. The frequency with which this cleavage sequence occurs
in the genome relative to that of clone 1 (1 out of 60) is comparable with the frequency with which it was found in the round 20 SELEX pool
(1 out of 37). No exact matches for this sequence were found in the
yeast genome data base.
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DISCUSSION |
Topoisomerase II is an essential enzyme that is involved in
virtually every aspect of DNA metabolism (1, 2). Fundamental to all
aspects of its catalytic function, topoisomerase II must create
transient double-stranded breaks in the backbone of the genetic
material (9-12). Although the enzyme displays a reproducible pattern
of cleavage on any given DNA substrate, the factors that underlie its
nucleotide specificity remain an enigma. In an attempt to define the
DNA site specificity of topoisomerase II, previous studies have
determined consensus sequences for enzyme action based on the
nucleotide analysis of multiple (ranging from 16 to 93) cleavage sites
(19-22). In general, the consensus sequences reported from this
approach have been weak and bear little relationship to one another.
Consequently, they have not proven to be as useful a tool for
elucidating the DNA site specificity of topoisomerase II as originally hoped.
It is not entirely surprising that consensus sequences reported for
enzyme action have been weak in nature. First, since topoisomerase II
displays at least some level of specificity for an average of eight
nucleotide positions (based on consensus sequences), cleavage over a
large number of base pairs (approaching 48) would have to
be analyzed in order to define a consensus with a high degree of
confidence. Clearly, this is a difficult criterion to meet and has not
been approached in previous studies. Second, topoisomerase II probably
carries out general functions (such as the control of superhelical
density or DNA untangling) in a global, rather than a highly specific,
manner (1, 2). Therefore, to fulfill this aspect of its physiological
role, the enzyme must be able to act at a wide variety of DNA sequences.
Beyond its global functions, however, topoisomerase II plays specific
roles in chromosome organization, condensation/decondensation, and
segregation (1, 2, 9, 10, 12, 52). To fulfill these latter
responsibilities, the enzyme appears to act at specific regions in the
genetic material (matrix/scaffold attachment regions (i.e.
MAR and SAR sequences) and centromeric sequences, for example) (27, 48,
53, 54). Thus, against a background of low stringency sites,
topoisomerase II may have highly preferred sites of action within the
genome. The present study used a SELEX protocol to identify a candidate
for such a site from Drosophila.
The cleavage sequence that was selected/evolved
(TATATATACATATATATA) is rich in A:T base pairs and is made
up of alternating purine/pyrimidine residues. Earlier studies that
derived a consensus DNA cleavage sequence for Drosophila
topoisomerase II (19) or characterized interactions between
topoisomerase II and MAR/SAR sequences (25, 48, 53) suggested that A/T
richness contributes to topoisomerase II-mediated DNA scission.
Furthermore, a study that overlaid several consensus sequences for the
enzyme demonstrated that topoisomerase II cleaves runs of alternating
purines and pyrimidines (55). Thus, the preferred sequence identified
in the present work supports these previous observations.
Several aspects of the sequence identified by the SELEX protocol merit
special note. First, the DNA site that was selected/evolved for
cleavage by Drosophila topoisomerase II in the absence of DNA cleavage-enhancing drugs was also a preferred sequence for the
action of anticancer agents targeted to the enzyme. This agrees with
the previous hypothesis (12, 46) that drugs stimulate DNA cleavage at
sites for which topoisomerase II has some level of intrinsic specificity.
Second, the selected sequence contains multiple sites for topoisomerase
II-mediated DNA scission. Clustering of strong DNA cleavage sites for
the enzyme has been observed previously (46). The relationship between
site proximity and strength is not known. Due to the small distance
between the sites in this sequence, it is clear that only a single
molecule of the enzyme can be accommodated on this sequence at a given
time. Therefore, increased DNA scission cannot be due to multiple
simultaneous cleavage events. More likely, the presence of multiple
cleavage sites in close proximity may impede linear diffusion of
topoisomerase II to distal sites on the DNA, thereby increasing the
local concentration of the enzyme.
Third, this DNA sequence was a poor cleavage substrate for human type
II enzymes. This latter result implies that the assumed "universal"
nature of topoisomerase II site specificity (20, 22, 23, 47, 48) may
not hold true for highly preferred sites of scission.
Finally, the selected/evolved topoisomerase II DNA cleavage sequence is
dramatically overrepresented in the Drosophila genome and is
often found at the 5' or 3' extremes of expressed genes. A previous
study that mapped (at low resolution) topoisomerase II cleavage sites
within 830 kilobase pairs of cloned Drosophila DNA found a
correlation between fragments that contained SAR sequences and those
rich in enzyme-mediated scission (27). Since MAR/SAR sequences
generally display a high A/T content (56), and the frequency of the
preferred topoisomerase II cleavage sequence in the
Drosophila genome (every ~250 kilobase pairs) is on the order of the size of chromosomal loop domains (57), it is tempting to
speculate that the sequence obtained in the present study represents an
attachment region. However, further in vivo experimentation will be required to determine the functional significance of the sequence and its potential physiological interactions with
topoisomerase II.
In summary, the sites of action of topoisomerase II on DNA profoundly
influence its catalytic activity. To further our understanding of how
the enzyme recognizes its DNA substrate, a SELEX protocol was used to
identify a highly preferred sequence for DNA cleavage mediated by
Drosophila topoisomerase II. Results of the present study
afford a unique perspective toward defining the intrinsic DNA cleavage
specificity of the type II enzyme and may ultimately reveal
relationships that link the site specificity of topoisomerase II to its
physiological functions.