From the Laboratory of Molecular Pharmacology and the
Laboratory of Medicinal Chemistry, Division of Basic Sciences,
NCI, National Institutes of Health, Bethesda, Maryland 20892-4255 and
the § Department of Molecular Pharmacology, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105
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ABSTRACT |
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A Ser740 DNA topoisomerases are enzymes that catalyze changes in
the topology of DNA via a mechanism involving the transient breakage and rejoining of phosphodiester bonds in the DNA backbone (1). Studies
in both prokaryotic and eukaryotic cells have demonstrated the
importance of topoisomerases in transcription, DNA replication, and
chromosome segregation. The type II topoisomerases, which make
transient double-strand breaks and change the linking number of DNA in
steps of two, play key roles in chromosome structure. In eukaryotic
cells, these enzymes are essential for chromosome condensation/decondensation and decatenation of chromosome loops during
mitosis (2, 3).
Topoisomerase II (top2)1 has
also been identified as a major target of chemotherapeutic agents that
are specifically active against prokaryotic (4) or cancer cells (5-8).
Fluoroquinolones are antibacterial agents that target DNA gyrase (the
prokaryotic type II topoisomerase) (9), whereas a variety of
DNA-intercalating agents such as anthracyclines, amsacrine, and
mitoxantrone and nonintercalating agents such as the epipodophyllotoxin
etoposide (VP-16) are active against eukaryotic top2 and are clinically important anti-cancer agents (5-8). Azatoxin is also a
nonintercalative top2 poison (10).
Recently, it has been shown that
6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic
acid (CP-115, 953), a fluoroquinolone closely related to
ciprofloxacin, is highly toxic to mammalian cells in culture (11, 12).
Studies in yeast demonstrated that top2 is the primary physiological
target for this quinolone (13). Unlike etoposide, which stabilizes
top2- mediated DNA cleavage primarily by inhibiting the religation
reaction of top2, CP-115,953 stabilizes DNA cleavage by enhancing the
forward rate of cleavage (14).
In Escherichia coli, mutations that lead to quinolone
resistance are most often found in gyrA, the structural gene
for the DNA gyrase A subunit. Ser83 of gyrA is
the amino acid most frequently changed in strains with high levels of
quinolone resistance, although other mutations in either
gyrA or gyrB can lead to quinolone resistance
(15). Mutations that change Ser83 to either leucine or
tryptophan confer high levels of quinolone resistance, whereas changing
Ser83 to alanine results in a low level of quinolone
resistance (16).
A previous study examined the effects of yeast top2 mutations that
change Ser740 (numbering of amino acid residues was
corrected according to Ref. 17; Ser740 was previously
referred to as Ser741 (18)), the amino acid homologous to
Ser83 in gyrA of E. coli (19). A
mutation changing Ser740 Materials, Chemicals, and Enzymes--
Etoposide (VP-16) was
obtained from Bristol-Myers Co., Wallingford, CT. Amsacrine and
mitoxantrone were from the Drug Synthesis and Chemistry Branch (NCI,
National Institutes of Health). Azatoxin was provided by Dr. T. Macdonald, Department of Chemistry of the University of Virginia,
Charlottesville. CP-115,953 was the gift of Drs. P. R. McGuirk and
T. D. Gootz of Pfizer Laboratories. Drug stock solutions were made in
dimethyl sulfoxide at 10 mM. Further dilutions were made in
distilled water immediately before use. Human c-myc inserted
into pBR322, restriction enzymes, T4 polynucleotide kinase,
polyacrylamide/bisacrylamide, and Taq DNA polymerase were
purchased from Lofstrand Labs (Gaithersburg, MD), Life Technologies,
Inc., New England Biolabs (Beverly, MA), or Qiagen Inc. (Valencia, CA).
[ Preparation of End-labeled DNA Fragments by PCR--
Three sets
of labeled DNA fragments were prepared from the human c-myc
gene by PCR. A 254-base pair DNA fragment from the first intron was
prepared between positions 3035 and 3288, with numbers referring to
GenBank genomic positions using oligonucleotides 5'-GTAATCCAGAACTGGATCGG-3' for the upper strand and
5'-ATGCGGTCCCTACTCCAAGG-3' for the lower strand (annealing temperature,
56 °C). A 401-base pair DNA fragment from the junction between the
first intron and first exon was prepared between positions 2671 and
3072 using oligonucleotides 5'-TGCCGCATCCACGAAACTTT-3' for the upper
strand and 5'-TTGACAAGTCACTTTACCCC-3' for the lower strand (annealing temperature, 60 °C). A 480-base pair fragment from the first exon containing promoters P1 and P2 was prepared
between positions 2265 and 2745 using the oligonucleotides:
5'-GATCCTCTCTCGCTAATCTCCGCCC-3' for the upper strand and
5'-TCCTTGCTCGGGTGTTGTAAGTTCC-3' for the lower strand (annealing
temperature, 70 °C). Single-end labeling of these DNA fragments was
obtained by 5'-end labeling of the adequate primer oligonucleotide. 10 pmol of DNA was incubated for 60 min at 37 °C with 10 units of T4
polynucleotide kinase and 10 pM [ Overexpression and Purification of Yeast Top2--
Wild-type
yeast top2 and Ser740 Top2-induced DNA Cleavage Reactions--
DNA fragments
(5-10 × 104 dpm/reaction) were equilibrated with
or without drug in 1% dimethyl sulfoxide, 10 mM Tris-HCl,
pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.1 mM Na2EDTA, 1 mM ATP, and 15 µg/ml bovine serum albumin for 5 min
before the addition of 8 units (80 ng) of purified top2 in a 10-µl
final reaction volume.
Calcium-promoted DNA cleavage was performed in the same buffer with 5 mM CaCl2 instead of MgCl2.
Reactions were performed at 37 °C for 30 min and thereafter stopped
by adding 1% SDS and 0.4 mg/ml proteinase K (final concentrations)
followed by an additional incubation at 55 °C for 30 min.
Electrophoresis and Base Preference Analysis--
For DNA
sequence analysis, samples were precipitated with ethanol and
resuspended in 5 µl of loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, and
0.1% bromphenol blue). Samples were heated to 95 °C for 5 min and
thereafter loaded onto DNA sequencing gels (7% polyacrylamide; 19:1
acrylamide/bisacrylamide) containing 7 M urea in 1 ×Tris
borate/EDTA buffer. Electrophoresis was performed at 2,500 volts (60 watts) for 2-3 h. The gels were dried on Whatman No. 3MM paper sheets
and visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA) and ImageQuant software. The determination of preferred bases
around top2 cleavage sites was done as described previously (20,
21).
Mapping and Analysis of the Cleavage Sites Induced by Mutant and
Wild-type Top2 in the Presence of Different Top2 Poisons--
The
cleavage sites induced by the top2S740W protein in the
presence of various top2 poisons were mapped in the upper and lower strands of a fragment of the human c-myc gene. This DNA
fragment includes the junction between the first exon and first intron (28). Fig. 1 presents the cleavage
pattern obtained in the presence of etoposide, azatoxin, the
fluoroquinolone CP-115,953, and the intercalating agents amsacrine and
mitoxantrone. The top2S740W protein was characterized
previously as partially resistant to fluoroquinolones; and when
compared with the wild-type enzyme, several cleavage sites induced in
the presence of CP-115,953 were reduced markedly. For example, cleavage
by top2S740W on the upper strand of the c-myc
DNA fragment at positions 2707, 2718, 2741, 2746, and 2768 was clearly
reduced. By contrast, the mutant protein caused increased cleavage at
specific sites in presence of etoposide, e.g. at positions
2711, 2712, 2781, 3026, and 3019 compared with the wild-type enzyme.
Azatoxin, another nonintercalating top2 poison (10), also showed
enhanced DNA cleavage at several sites. It should be noted that reduced
cleavage in the presence of etoposide occurs at other sites,
e.g. 2839 and 2776. The intercalating agent mitoxantrone
resulted in less cleavage with the top2S740W protein at
sites such as 2707, 2735, 2785, 2807, 2953, and 2974, whereas at other
sites, less cleavage was seen with the wild-type protein
(e.g. positions 2813, 2898, 2901, and 3011). Similarly, multiple changes in the cleavage sites induced in the presence of
amsacrine were seen. The result with amsacrine is particularly interesting because the wild-type and top2S740W protein
have similar sensitivities to amsacrine in vivo (25). Taken
together, these results show that the Ser740 Calcium-promoted DNA Cleavage Sites Differ between the
Top2S740W and the Wild-type Enzyme--
To investigate
whether the altered DNA cleavage activity of the top2S740W
was drug-dependent, we compared the calcium-promoted DNA
cleavage (29) for the Ser740 Altered Base Preference of the Etoposide-stabilized Cleavage
Complexes for the Mutant Top2S740W--
As described
above, the top2S740W protein is hypersensitive to
etoposide. It was therefore of considerable interest to examine the
effect of this mutation on the DNA base preference of top2 in the
presence of this drug (Fig. 3). Cleavage
sites for three c-myc DNA fragments (see "Experimental
Procedures") were analyzed for both DNA strands. For the wild-type
top2 protein, etoposide preferentially stabilized sites with C Enhanced Salt and Heat Stability of the Cleavage Complexes Mediated
by Top2S740W in the Presence of Etoposide--
The effect
of the Ser740
To focus on the role of etoposide-protein-DNA interactions on the
stability of the DNA-enzyme interaction, the salt reversibility (0.5 M NaCl final concentration) of the calcium-promoted or
etoposide-stabilized cleavage complexes was examined (Fig.
5). The calcium-promoted cleavage
complexes were readily salt reversible for both top2S740W
and the wild-type enzyme (Fig. 5A). By contrast, all
etoposide-stabilized cleavages induced by top2S740W were
completely salt-stable even after 30 min. Most of the cleavage complexes reversed at least partially for the wild-type top2 (Fig. 5B). These results support previous results suggesting that
etoposide more strongly stabilizes the top2·DNA complexes formed with
the top2S740W enzyme.
Lee and Hsieh (31) showed previously that heat or salt incompletely
reversed teniposide-stabilized covalent complexes induced with
Drosophila top2. They did not observe stable DNA
double-strand cleavage following heat or salt reversal. To determine
whether the etoposide-stabilized, heat-stable top2S740W
cleavages were predominantly DNA single- or double-strand breaks, we
performed nondenaturing gel electrophoresis (Fig.
6). Several strong cleavage sites were
observed on nondenaturing gels, indicating that the
top2S740W protein generates stable double-as well as
single-strand breaks. Slight heat stability was also seen with the
wild-type top2 mediated at sites 3171, 3175, and 3238, but the
stability was consider-ably less than was seen with the
top2S740W protein.
Reversibility of the Etoposide-induced Cleavage Complexes Is
Base Sequence-dependent--
Because the
top2S740W exhibited a large number of cleavage sites with
different reversal kinetics after heat treatment, we sequenced these
sites to study the influence of local base preference on top2
religation kinetics for the top2S740W (Fig.
7). Etoposide-stabilized cleavage sites
were divided into rapidly reversible (complete reversal within 2 min at
65 °C) or slowly reversible (incomplete reversal after 2 min).
Cleavage sites with slow reversibility exhibited highly significant
preference for C A number of factors contribute to the sensitivity of cells toward
agents targeted to the type II topoisomerases (32-35). Top2 mutations
that alter drug-induced DNA cleavage result in marked alterations,
ranging from high resistance (26, 36, 37) to severalfold
hypersensitivity (19, 26). However, the mechanisms by which mutations
within the enzyme alter drug sensitivity have not been defined.
Decreased drug binding to the top2·DNA complex has been reported for
quinolone-resistant DNA gyrase with a Ser83 The results presented in this report show that the Ser740
Trp
mutation in yeast topoisomerase II (top2) and of the equivalent
Ser83 in gyrase results in resistance to quinolones and
confers hypersensitivity to etoposide (VP-16). We characterized the
cleavage complexes induced by the top2S740W in the human
c-myc gene. In addition to resistance to the
fluoroquinolone CP-115,953, top2S740W induced novel DNA
cleavage sites in the presence of VP-16, azatoxin, amsacrine, and
mitoxantrone. Analysis of the VP-16 sites indicated that the changes in
the cleavage pattern were reflected by alterations in base preference.
C at position
2 and G at position +6 were observed for the
top2S740W in addition to the previously reported C
1 and
G+5 for the wild-type top2. The VP-16-induced top2S740W
cleavage complexes were also more stable. The most stable sites had
strong preference for C
1, whereas the most reversible sites showed no
base preference at positions
1 or
2. Different patterns of DNA
cleavage were also observed in the absence of drug and in the presence
of calcium. These results indicate that the Ser740
Trp mutation alters the DNA recognition of top2,
enhances its DNA binding, and markedly affects its interactions with
inhibitors. Thus, residue 740 of top2 appears critical for both DNA and
drug interactions.
INTRODUCTION
Top
Abstract
Introduction
References
Trp resulted in resistance to
CP-115,953 and hypersensitivity to etoposide (19). To investigate the
basis for the differential responses of the top2S740W, we
analyzed the base sequence preference (20-23) and stability of the
top2 cleavage complexes in the absence and presence of top2-targeting drugs.
EXPERIMENTAL PROCEDURES
-32P]ATP was purchased from NEN Life Science
Products. PCR oligonucleotide-primer were obtained from Life
Technologies, Inc. (Gaithersburg, MD).
-32P]ATP
(100 µCi) in kinase buffer (70 mM Tris-HCl, pH 7.6, 0.1 M KCl, 10 mM MgCl2, 5 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin).
Reactions were stopped by heat denaturation at 70 °C for 15 min.
After purification using Sephadex G-25 columns (Boehringer Mannheim),
the labeled oligonucleotides were used for PCR. Approximately 0.1 µg
of the c-myc DNA that had been restricted by
SmaI and PvuII (fragment 2265-2745),
XhoI and XbaI (fragment 2671-3072 and fragment
3035-3288) was used as template for the PCR. 10 pmol of each
oligonucleotide primer, one of them being 5'-labeled, was used in 22 temperature cycle reactions (each cycle with 94 °C for 1 min,
annealing for 1 min, and 72 °C for 2 min). The last extension was
for 10 min. DNA was purified using PCR Select-II columns
(5Prime-3Prime, Inc. Boulder, CO).
Trp proteins were overexpressed
using YEpTOP2-PGAL1 or YEptop2-S*W-PGAL1 using yeast strain JEL1t1
(24) and purified to homogeneity as described
previously (25). The detailed procedure has been described elsewhere
(26). Top2 reactions were carried out as reported (26, 27) using either supercoiled pBR322 to monitor ATP-dependent relaxation or
kinetoplast DNA isolated from Crithidia fasciculata to
monitor decatenation.
RESULTS
Trp
mutation affects the DNA cleavage patterns induced by both intercalating and nonintercalating drugs.
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Fig. 1.
Ser740 Trp mutation in yeast top2 alters DNA cleavage patterns induced
by various top2 inhibitors in the human c-myc
gene. A DNA fragment from the junction between the
c-myc first intron and first exon between positions 2671 and
3072 was prepared by PCR using one primer labeled with 32P
at the 5'-terminus. Panel A, labeling of the upper DNA
strand at position 2671. Panel B, labeling of the lower DNA
strand at position 3072. Top2 reactions were performed at 37 °C for
30 min and stopped by adding SDS and proteinase K (1% and 0.4 mg/ml
final concentrations, respectively). DNA electrophoresis was in 7%
denaturing acrylamide gels (7 M urea) in TBE buffer. Drugs
are indicated above each lane (100 µM
etoposide, 100 µM CP-115,953, 200 µM
azatoxin, 100 µM amsacrine, 1 µM
mitoxantrone). The purine ladder was obtained after formic acid
reaction. Top2, no drug treatment; Control, no
top2, no drug treatment. Numbers correspond to genomic
positions of the nucleotide covalently linked to top2. YWT,
yeast wild-type enzyme; yS740W, top2S740W.
Double-headed arrows correspond to DNA cleavage sites with a
4-base pair stagger and represent potential DNA double-strand
breaks.
Trp protein
and the wild-type enzyme in the two strands of the c-myc
first intron fragment (Fig. 2). Even in
the presence of magnesium, differences in the cleavage patterns could
be observed. When magnesium was replaced by calcium, higher levels of
DNA cleavage were seen with both proteins. DNA cleavage sites common to
both proteins were seen in the presence of Ca2+; however,
there were also major differences in the intensity of cleavage at other
sites. Most of the sites of DNA cleavage in the upper and lower strands
were staggered by 4 base pairs with a 5'-overhang, as expected for
concerted top2-induced double-strand cleavage (2, 5, 7). These results
suggest that the top2S740W mutation also alters DNA
cleavage in the absence of a topoisomerase II poison.
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Fig. 2.
Ser740 Trp mutation in yeast top2 alters the enzyme-mediated DNA
cleavage sites in the absence of inhibitors. A 254-base pair DNA
fragment from the first c-myc intron was prepared between
positions 3035 and 3288 by PCR using one primer labeled with
32P at the 5'-terminus. Panel A, labeling of the
upper DNA strand at position 3035. Panel B, labeling of the
lower DNA strand at position 3288. Top2 reactions were performed at
37 °C for 30 min in the presence of 5 mM
MgCl2 or 5 mM CaCl2 as indicated
and stopped by adding EDTA and SDS (25 mM and 1% final
concentrations, respectively). The purine ladder was obtained after
formic acid reaction. yWT, yeast wild-type top2;
yS740W, top2S740W. Double-headed
arrows correspond to DNA cleavage sites with a 4-base pair stagger
which represent potential DNA double-strand breaks.
1 (103 out of 167 sites) (Fig. 3C). This result agrees well with
previous analyses of cleavage of the same DNA fragments by human top2
in the presence of etoposide (5, 30). Preference on the opposite strand
showed a complementary (although slightly weaker) preference for G+5.
Top2S740W also demonstrated a strong preference for C
1.
In addition, a novel preference for C at position
2 (94 out of 176 sites) in combination with the complementary G at position +6 (84 out
of 176 sites) was also seen. To focus on the impact of the
Ser740
Trp mutation on base preference, we analyzed
separately those DNA cleavage sites exclusively detectable for
top2S740W or for the wild-type enzyme (Fig. 3B).
These unique DNA cleavage sites did not show any clear C
1 or G+5
preference for either protein, which is expected because both proteins
have a C
1/G+5 preference. However, top2S740W still showed
a significant preference of C at position
2 (45 out of 63 sites) in
combination with a preference for the complementary G at position +6
(39 out of 63 sites). In addition, a chi-square test indicated that the
combination of the C
1 and C
2 preference in the
top2S740W was not significantly more frequent than having
C
1 or C
2 alone. Thus, the novel C
2 base preference in the
top2S740W is independent of the C
1 preference. In
contrast, the cleavage sites unique for the wild-type protein tended to
exclude sites with C+2 (7 out of 54 sites) and with G+6 (9 out of 54 sites). These data show a change in the protein-DNA interaction
resulting from the Ser740
Trp mutation, leading to
an extension of the base preference for the C
2 position in the
presence of etoposide.
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Fig. 3.
Probability of the observed base frequency
deviations at top2 cleavage sites for the wild-type enzyme or for the
Ser740 Trp mutant top2 in the
presence of 100 µM etoposide.
Position 0 corresponds to the cleavage site. Panels A and
B, probability of the observed base frequency deviations
from expectation. In the y axis, p is the probability of
observing that deviation or more, either as excess (above base line) or
deficiency (below base line) relative to the expected frequency of each
individual base (20, 21). Panel A, all cleavage sites were
analyzed. Panel B, only specific cleavage sites were
analyzed. Panel C, base distribution at each position.
Underlined numbers represent base frequencies significantly
(p < 0.001) greater or lower than expected.
Trp mutation on the stability of specific
top2·DNA cleavage complexes was determined by examining the salt and
heat reversibility of the ternary complex formed with drug, protein,
and DNA (Fig. 4). Cleavage reactions were
carried out with the wild-type or the Ser740
Trp top2 for 30 min at 37 °C. The reactions were
heated to 65 °C for various times before the addition of SDS. Fig. 4
shows the result for the upper (panel A) and lower strand
(panel B) of the c-myc fragment corresponding to
the first intron. Most of the etoposide-stabilized cleavage sites were
readily reversible for the wild-type protein. In contrast, a number of
cleavage sites induced by top2S740W showed slow reversal
(e.g. positions 3073, 3091, 3163, 3223, and 3183) or no
detectable reversal after a 30-min incubation at 65 °C
(e.g. positions 3167, 3171, 3241, 3174, and 3170). Enhanced heat stability of the DNA cleavage sites induced by
top2S740W was also observed in the other c-myc
DNA fragments (data not shown).
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Fig. 4.
Cleavage complexes stabilized by
100 µM etoposide with
top2S740W exhibit enhanced heat stability. The DNA
fragments were the same as described in Fig. 2. Panel A,
labeling of the upper DNA strand at position 3035. Panel B,
labeling of the lower DNA strand at position 3288. Top2 reactions were
performed at 37 °C for 30 min in the presence of 100 µM etoposide. The reactions were then incubated at
65 °C for the indicated times before the addition of SDS and
proteinase K. Top2, no drug treatment; Control,
no top2, no drug treatment. Numbers correspond to genomic
positions of the nucleotide covalently linked to top2. yWT,
yeast wild-type top2; yS740W, top2S740W.
Double-headed arrows correspond to DNA cleavage sites with a
4-base pair stagger and represent potential DNA double-strand
breaks.
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Fig. 5.
Cleavage complexes stabilized by 100 µM etoposide with top2S740W
exhibit enhanced salt stability. The DNA fragment was the same as
in Fig. 2A. Panel A, salt stability of
calcium-promoted (5 mM) cleavage sites. Panel B,
salt stability of 100 µM etoposide-stabilized cleavage
complexes.Top2 reactions were performed at 37 °C for 30 min. After
the addition of NaCl (0.5 M NaCl final concentration), the
reactions were incubated at 37 °C for the indicated times before the
addition of SDS and proteinase K. Top2, no drug treatment.
Numbers correspond to genomic positions of the nucleotide
covalently linked to top2. yWT, yeast wild-type top2;
yS740W, top2S740W.
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Fig. 6.
Cleavage complexes induced by
top2S740W consist predominantly of DNA double-strand
breaks. The DNA fragment was the same as in Fig. 2A.
The upper DNA strand was labeled at position 3035. Top2 reactions were
performed at 37 °C for 30 min. The reactions were then incubated at
65 °C for the indicated times before the addition of SDS and
proteinase K. For DNA sequence analysis, samples were precipitated with
ethanol and resuspended in 5 µl of loading buffer (30% glycerol, 1 mM EDTA, pH 8, 10 mM Tris, pH 7.4, and 0.1%
bromphenol blue). Samples were loaded onto nondenaturing DNA sequencing
gels (7% polyacrylamide; 19:1 acrylamide/bisacrylamide in 1× Tris
borate/EDTA buffer). Electrophoresis was performed at 45 watts for 2-3
h. Top2, no drug treatment; Control, no top2, no
drug treatment; yWT, yeast wild-type top2;
yS740W, top2S740W.
1 (78 out of 89 sites) in combination with a less
strong C
2 preference (54 out of 89 sites) (Fig. 7B).
Complementary preference was observed on the opposite strand with
preference (although weaker) for G+5 (53 out of 89 sites) and G+6 (36 out of 89 sites) as well. In contrast, rapidly reversible cleavage
sites did not show any base preference at positions
1 and
2.
However, they displayed a significant G+5 and G+6 base preference,
indicating DNA cleavages in the complementary strand with a C
1 and
C
2 base preference. These data are consistent with the formation of
stable cleavage complexes when the preferred base(s) occur(s) on the DNA strand that is cleaved by the enzyme. Religation is influenced less by the base sequence on the opposite strand.
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Fig. 7.
Probability of the observed base frequency
deviations at top2S740W cleavage sites showing fast or slow
reversibility. Reactions were performed in the presence of 100 µM etoposide. Position 0 corresponds to the cleavage
site. Panel A, probability of the observed base frequency
deviations from expectation. In the y axis, p is the
probability of observing that deviation or more, either as excess
(above base line) or deficiency (below base line) relative to the
expected frequency of each individual base. Panel B reflects
the base distribution at each position. Underlined numbers
represent base frequencies significantly (p < 0.001)
greater or lower than expected.
DISCUSSION
Trp
mutation in the A subunit (7, 38). A homologous mutation in the yeast
top2 gene, which changes Ser740 into Trp, also results in
quinolone resistance (19). The same region of the protein is clearly
important for determining sensitivity to eukaryotic topoisomerase II
poisons because the mutation also causes hypersensitivity to etoposide
(19).
Trp mutation in yeast top2 affects the DNA-protein interactions. In
the absence of any drug, the calcium-promoted DNA cleavage sites of
top2S740W were clearly different from those induced by the
wild-type enzyme, indicating a change in DNA recognition. Amino acid
residue 740 is part of the
4 DNA-recognition helix within the
helix-turn-helix (HTH) motif of top2 (39). This HTH motif and its
counterpart in E. coli gyrase are mutational hotspots for
resistance to drugs that stabilize the cleaved state of DNA (15, 39).
DNA footprinting has shown that for top2 and DNA gyrase approximately
15-35 base pairs of DNA are protected by the enzyme (40, 41). In
addition, a 29-kDa fragment containing the active-site tyrosine and the HTH motif can be cross-linked to DNA (42), and protein footprinting has
demonstrated that the presence of DNA protects the HTH motif from
chemical modification (43). The data of this study are consistent with
a direct interaction of the HTH motif with DNA (44). Peptides
containing aromatic amino acids are known to be capable of partial
intercalation with DNA (45). NMR titrations with complexes between
double-strand DNA and tryptophan-containing peptides confirmed the
possibility of intercalation (46). Consistent with this possibility,
Fig. 8 presents the position of
Ser740 on the protein surface and its close proximity to
the DNA. In addition, Ser740 is approximately 3.0 Å from
Tyr734, and these residues could form a hydrogen bond.
Thus, the Ser740
Trp mutation could alter the DNA-top2
interaction directly by intercalation as well as indirectly by changing
the enzyme conformation and modifying DNA recognition.
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Fig. 8.
Position of the Ser740
Trp mutation in the yeast top2 and hypothetical
DNA interactions. Drawings were generated using the program Quanta
(Version 97) based on coordinates reported in Ref. 17. Panel
A, ribbon representation of the crystal structure of the 92-kDa
fragment of the yeast enzyme (17) with a DNA fragment modeled into each
of the putative DNA binding sites (18). The positions of the
Ser740 in each top2 subunit are shown as van der Waals
spheres and are highlighted by green arrows.
Panel B is a close view of the DNA binding region,
presenting the proximity of the Ser740 mutation to the DNA.
Ser740 is shown in a ball-and-stick model. The
numbering of amino acid residues was corrected according to Ref.
17.
The Ser740 Trp mutation in yeast top2 also markedly
affected the protein-drug interaction. We presented two lines of
evidence that, compared with the wild-type protein, the increased heat and salt stability of top2S740W-induced DNA cleavages are
dependent on etoposide interaction. First, calcium-promoted cleavages,
although presenting an altered DNA-protein interaction, were readily
reversible. Second, cleavages on a DNA strand without the
etoposide-preferred bases by cooperative effects with the other subunit
are also readily reversible (30, 47). The fact that a single mutation
at amino acid residue 740 changed the quinolone as well as the
etoposide sensitivity is consistent with previous studies suggesting
that quinolones share a common interaction domain on eukaryotic top2
with other DNA cleavage-enhancing drugs (48). Based on drug-associated
preferences for the bases immediately flanking the top2-linked DNA
cleavage site, we also proposed a drug-stacking model in which the
drugs generally occupy a common site at the interface of the enzyme and
the ends of the cleaved DNA (5, 20-22).
Our data provide evidence that the Ser740 Trp mutation
might directly or indirectly change at least overlapping
quinolone-protein and etoposide-protein interaction domains.
Furthermore, we found a novel C
2 base preference in the
top2S740W in the presence of etoposide. This new C
2
preference proved to be statistically independent of the common C
1
preference. These findings suggest a model of a more relaxed drug
binding site for the Ser740
Trp enzyme allowing
etoposide to interact with C
2 in addition to C
1. This model does
not conflict with the hypothesis of etoposide acting at the DNA-protein
interface (5, 21, 30) because the altered interface between the
top2S740W and its DNA substrate is likely to increase the
etoposide binding affinity and hence to increase the stability of the
ternary complex.
The results of this study suggest a DNA-top2 binding site on the protein surface, which is directly or indirectly affected by amino acid residue 740 and hence controls the binding of drugs including quinolones and etoposide to the top2·DNA complex.
Further structural studies with wild-type and Ser740
Trp mutant top2 in the presence of DNA and inhibitors are awaited.
A DNA fragment of the c-myc first intron between positions
3185 and 3168, containing several highly salt- and heat-stable cleavage sites, could be a suitable DNA substrate.
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ACKNOWLEDGEMENTS |
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We thank Drs. Q. Liu and J. C. Wang for providing data to generate Fig. 8. We also thank Dr. T. Macdonald, Department of Chemistry, University of Virginia, Charlottesville, and Drs. P. R. McGuirk and T. D. Gootz, Pfizer, for providing topoisomerase II inhibitors.
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FOOTNOTES |
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* 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.
Supported by Deutsche Forschungsgemeinschaft Grant Str 527/1-1,
Bonn, Germany.
¶ Supported in part by Grant CA52814 from the NCI, National Institutes of Health, and the American Lebanese Syrian Associated Charities.
** To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, Bldg. 37, Rm. 5D02, NIH, Bethesda, MD 20892-4255. Tel.: 301-496-5944; Fax: 301-402-0752; E-mail: pommier{at}nih.gov.
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ABBREVIATIONS |
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The abbreviations used are: top2, topoisomerase II; PCR, polymerase chain reaction; HTH, helix-turn-helix.
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REFERENCES |
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