From the Department of Biochemistry, Duke University Medical
Center, Durham, North Carolina 27710
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INTRODUCTION |
Type II DNA topoisomerases are ubiquitous enzymes that have
critical functions in many aspects of DNA metabolism, including replication, transcription, recombination, and genome stability (reviewed in Ref. 1). It is dimeric in structure and can pass DNA
through a transient DNA gate generated by reversible
transesterification from a pair of active site tyrosine residues.
In vitro, topoisomerase II (topo
II)1 catalyzes the reaction
of DNA supercoiling and relaxation, catenation/decatenation, and
knotting and unknotting of DNA rings. In vivo, the essential biological functions of topo II are in the segregation of replicated and topologically interlocked chromosomes (Ref. 2; also reviewed in
Ref. 3). Both eukaryotic and prokaryotic topo II share very similar
biochemical mechanisms and are related by strong sequence homologies
(4). The N-terminal and central domains of the eukaryotic topo II
subunit are homologous to the bacterial gyrB and gyrA subunits,
respectively. Recent high-resolution structural data suggest that the
sequence homology between eukaryotic and bacterial topo II can be
translated into structural similarity (5, 6).
The biochemical mechanism of topo II in freely passing DNA helices
through each other is no less complicated than those described for
other macromolecular machineries. The reaction starts with the binding
of a DNA segment to the enzyme surface at the active site tyrosines.
This stretch of DNA will serve as a protein-mediated gate and is termed
the G segment. Upon the binding of ATP, a second segment of DNA is
captured by the closure of the topo II N-terminal protein clamp, known
as the N-gate (7), and transported through the G segment. The binding
of ATP to eukaryotic topo II also induces an allosteric change in the
N-terminal domain in concert with the clamp closure (8, 9). After the
passage of DNA through the reversible break in the G segment, the
transported DNA helix is held and then expelled from the enzyme through
the C-terminal protein clamp, known as the C-gate, formed by the gyrA
homologous domain (10, 11). The hydrolysis of ATP in this reaction
allows the turnover of the catalytic cycle (12-14). Although ATP
binding can trigger the closure of N-gate, its role in other steps of the catalytic cycle, such as the strand passage and exit of DNA from
the C-gate, remains to be delineated. Furthermore, the topoisomer distribution in the topo II-catalyzed reactions including supercoil relaxation, catenation, and knotting is apparently below the
equilibrium position (15). It is likely that the free energy from the
hydrolysis of ATP concomitant with topo II-mediated strand passage
reactions is the driving force to bias the chemical equilibrium. How
this is achieved in biochemical and mechanistic terms is yet to be determined.
We are interested in identifying important residues in the
ATP-dependent reactions of topo II and analyzing the
biochemical consequences of mutations at these residues. Such
information provides insight into the mechanism of topo II reactions
and guides the future experiments to test various hypotheses. We report
here the result from site-specific mutagenesis of a conserved
Lys359 that is situated in close proximity to the
-phosphate of bound ATP. Although the double strand DNA cleavage
reaction is not affected by the mutations tested, Lys359
mutants have greatly reduced catalytic activities and have altered clamp closure response with respect to ATP cofactors. Thus,
Lys359 is implicated in the ATP-triggered reactions of topo
II.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
The expression of
Drosophila topo II was driven by the GAL1
promoter in a YEp24-based yeast vector (16). Site-directed mutagenesis was carried out by the method described by Kunkel (17). Three 21-mer
oligonucleotides, covering the top2 cDNA nucleotides
1325-1345, contain sequence variations of AGG (Arg mutant), GAG (Glu
mutant), and CAG (Gln mutant) in place for the original
Lys359 codon AAG. DNA sequences were determined surrounding
the mutagenic site to confirm that only the desired mutation was
introduced.
Yeast Complementation Assay--
Complementation of yeast
top2ts alleles by Drosophila top2 was
carried out as described (16). The growth of yeast cells at a
nonpermissive temperature of 37 °C in medium containing galactose demonstrated the expression of a functional Drosophila top2.
The color sectoring assay was performed to test the ability of
wild-type and mutant Drosophila top2 to complement a yeast
top2 null mutation (18). The formation of red/white sectored
colony indicated that a Drosophila topo II construct was
functional and could replace the only functional yeast TOP2
on the plasmid (19).
Purification of Wild-type and Mutant Topo
II--
Drosophila topo II was overexpressed in yeast
BCY123 (pep4
, prb1
,
bar1
, lys2::GAL1-GAL4,
ura3
; a generous gift from Dr. Janet Lindsley,
University of Utah). The expression of Drosophila top2
constructs were induced in YEP medium containing 2% galactose. An
8-liter culture of yeast cells overproducing Drosophila topo
II was harvested and lysed as described in Ref. 20. The cleared lysate
was loaded onto a 100-ml of Bio-Rex 70 (Bio-Rad) column. The column was
eluted by a NaCl gradient in Buffer P (15 mM
NaDi, pH 7.0, 0.1 mM EDTA, and 10% Glycerol), with topo II coming off at 0.5 M NaCl. The active fractions
were then pooled, diluted 1:1 with 0.1 M Tris-HCl (pH 8.0),
and loaded onto a 7.8-ml Poros HQ column (PerSeptive Biosystems,
Framingham, MA). The column was eluted by a NaCl gradient in 20 mM Tris-HCl (pH 8.0), and topo II was eluted at 0.8 M NaCl. The peak fractions were combined, diluted 4-fold
with 50 mM NaPi (pH 8.0), and loaded onto a
1.7-ml heparin column (Poros HE, PerSeptive Biosystems). The Poros HE
column was eluted by a NaCl gradient in 10 mM
NaPi (pH 8.0). The peak fractions eluted at 0.3 M NaCl were then pooled and dialyzed against the storage
buffer containing 15 mM NaPi, pH 7.0, 0.1 mM EDTA, 0.1 M NaCl, and 50% glycerol for
16 h. 0.1 mM of dithiotreitol and a mixture of
protease inhibitors including 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A were included
in all solutions used in the purification procedures. Protein
concentrations were determined by Coomassie dye-binding assay (21), and
purification at each step was monitored by SDS-PAGE (22).
CsCl Density Gradient Ultracentrifugation--
A 40-µl
reaction mixture containing 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 100 mM NaCl, 10 mM
MgCl2, 0.1 mM EDTA, 50 µg/ml bovine serum
albumin, 25 nM of pCaSpeRhs83 DNA (circular or linearized), and 75 nM of topo II proteins was incubated at 30 °C for
15 min. Some reactions also included 0.5 mM of a nucleoside
triphosphate cofactor (ATP or AMPPNP). The reaction was terminated by
adding a chilled mixture of 330 µl of saturated CsCl solution and 107 µl of 10 mM Tris-HCl, pH 8.0, to make the final density
of the solution 1.65 g/ml. The mixture was spun at 40,000 rpm in an
analytical ultracentrifuge (XL-A ultracentrifuge, Beckman Instruments)
at 20 °C for 36 h before scanning the DNA concentrations across
the gradient at wavelengths of 260 and 280 nm. The density difference between protein/DNA complex and DNA was used to calculate the protein
molecular weight in the salt-stable complex (23). The plot of
(1/
c-n
1/
d)/(1/
p
1/
c-n)·MWd versus n produced a straight
line with slope MWp, the molecular weight of protein moiety in
the complex. In this equation, n is the number of protein
molecules bound in the complexes, MWd is the DNA molecular
weight, and
p,
d, and
c-n are the
buoyant densities of protein, DNA, and a complex of DNA bound with
n protein molecules, respectively.
Other Methods--
Topo II strand passage activities were tested
by the unknotting of P4 knotted DNA, supercoiled relaxation,
catenation, and decatenation assays as described in (24). ATPase assay
was done as described in Ref. 25 except that a 8.6-kilobase plasmid
DNA, pCaSpeRhs83 (26), was used, and the radioactivities were
quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Topo
II-mediated double-stranded DNA cleavage was carried out using plasmid
DNA substrate as described in (27). The uniquely end-labeled DNA containing the intergenic region of heat shock gene HSP70
was used to map the topo II cleavage sites as described earlier (28). Protein-mediated binding of DNA to GF/C glass fiber filters (29) was
done as described by Roca and Wang (7). The radiolabeled DNA substrate
used in the filter binding assays was generated by nick translation of
nicked pHC 624 (30) carried out in the presence of
[
-32P]TTP (0.5 Ci/mmol) for 1 h followed by
religation with T4 DNA ligase in the presence of 5 µg/ml ethidium
bromide for 3 h. The mixture was then phenol extracted and run
through a Sephadex G-50 spin column (Amersham Pharmacia Biotech) to
remove the unincorporated nucleotides. Half of the labeled DNA sample
was linearized by the restriction enzyme EcoRI. An equal
mixture of linear and circular DNA samples was used in each assay.
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RESULTS |
Lys359 Mutants of Drosophila Topoisomerase II--
The
x-ray crystal structure of the 43-kDa N-terminal fragment of gyrB
protein reveals a two-domain structure: the N-terminal half (residues
2-220) is the ATP-binding moiety and the C-terminal half (residues
221-392) forms a 20-Å cavity (31). Examining the cavity-forming
domain shows that a loop with a sequence of QTK (residues 335-337)
extends away from the rest of the domain and is in close contact with
-phosphate of the bound ATP analog (Ref. 31; see Fig.
1A). The N---O distance
between the
-amino group in Lys337 and
-phosphate in
ATP is 2.6 Å, suggesting that either hydrogen bonding or ionic
interaction could exist between them. It is also interesting to note
that QTK and the sequence surrounding it are highly conserved among
topo II (Fig. 1B).

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Fig. 1.
A, AMPPNP and some of its interacting
residues in gyrB. The diagram was drawn with Kinemage software (51)
using the coordinates from the crystal structure of a 43-kDa E. coli gyrB fragment complexed with AMPPNP (31). AMPPNP and the
Lys337 side chain are shown in black. The side
chains from other residues (Glu42, Lys103,
Gln335, and Thr336) and backbone C- s are in
dark gray and light gray, respectively. The
dotted line marks the 2.6-Å interatomic distance from
-amino nitrogen to the -phosphate oxygen in AMPPNP. Earlier works
demonstrated that mutations in Glu42 (45) or
Lys103 (50) inactivate DNA gyrase. B, amino acid
sequence alignment surrounding Lys337 of gyrB. The highly
conserved residues, either identities or homologous substitutions, are
boxed for sequences of gyrB from E. coli
(EcGyrB) and Bacillus subtilis
(BsGyrB), gene 39 protein from phage T4 topo II, human topo
II (HumA), and topo II from Saccharomyces
cerevisiae (Sc), Schizosaccharomyces pombe
(Sp), and Drosophila melanogaster
(Dm). The Lys359 substitution mutants reported
in this work are given under the sequence for Dm.
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To test the importance of the lysine residue in the QTK loop in topo II
reactions, we generated site-specific mutations converting lysine to
arginine, glutamine, and glutamate (see "Experimental Procedures").
The homologous residue of Lys337 of gyrB is
Lys359 in Drosophila enzyme (Fig.
1B). The activity of the site-specific mutants was first
tested by genetic complementation assays that were based on our
previous observation that a functional Drosophila top2 gene
could rescue either a conditional yeast top2 mutation (16)
or a null mutation (18). In these in vivo functional assays,
the K359R mutant is active but K359Q and K359E are not (data not
shown). These results are also confirmed by the topoisomerase assays in
which the crude extracts yeast cells expressing K359R are active,
whereas those expressing K359Q and K359E are inactive (data not shown).
Therefore, both in vivo and in vitro assays suggest that lysine is critical for topoisomerase functions; a homologous substitution with arginine does not significantly alter topoisomerase activities, and mutations converting it into glutamine and glutamate result in a loss of its activities.
Purification of K359Q and K359E Mutant Proteins and Their
Biochemical Characterizations--
To analyze the effects of
substitutions of Lys359, we have purified wild-type topo II
and K359Q and K359E mutant proteins from overproducing yeast cells. The
cleared extracts were fractionated on a cation-exchange column
(BioRex-70), followed by anion exchanger chromatography (Poros HQ), and
affinity chromatography (Poros HE). Wild-type and mutant proteins all
had identical elution profiles in these chromatographic steps (see
"Experimental Procedures") and were purified to over 90%
homogeneity (Fig. 2).

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Fig. 2.
Purification of wild-type and mutant
Drosophila topo II. 2 µg each of molecular weight
size markers, wild-type, and two Lys359 mutant proteins
were run in a 7% Laemmli gel (22) and stained with Coomassie
Blue.
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The DNA strand passage activity of these proteins were determined by
the P4 DNA unknotting assays (Fig. 3).
Two-fold serial dilutions in the linear range of activity were assayed
for each protein. With 3.2 fmol of wild-type protein, P4 DNA could be
completely unknotted (Fig. 3, lane 2). This corresponds to a
specific activity of about 106 units/mg, which is the same
as topo II purified from Drosophila cells (25, 27). Even
with the highest amount of mutant proteins tested here, about 500-fold
more than the wild-type enzyme, the unknotting reaction was only about
50 and 10%, respectively, in completion (Fig. 3, lanes 7 and 11). Other strand passage assays including supercoil
relaxation, catenation, and decatenation of circular DNA produced
similar results (data not shown). Furthermore, the unknotting activity
is ATP-dependent; no unknotting was observed for wild-type
and mutant proteins at the highest concentrations used in these assays
in the absence of ATP (Fig. 3, lanes 1, 6, and
10). Other strand passage assays are also
ATP-dependent (data not shown). Therefore, substitution of
Lys359 with a neutral residue (K359Q) has resulted in
103-fold reduction in topoisomerase activity, whereas its
substitution with a negatively charged residue (K359E) further lowers
the enzymatic activity.

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Fig. 3.
Unknotting assays of wild-type and mutant
topo II. P4 knotted DNA rings were used as the substrate, and the
reactions were carried out either without ATP (lanes 1, 6,
and 10) or in the presence of 1 mM ATP
(all other lanes). The amounts of topo II added to reaction
mixtures were 3.2, 3.2, 1.6, and 0.8 fmol for wild-type (WT,
lanes 1-5), 1.8, 1.8, 0.9, and 0.4 pmol for the K359Q mutant
(lanes 6-9), and 2.2, 2.2, 1.1, and 0.5 pmol for the K359E
mutant (lanes 10-14). Lane 14 shows the control,
with no enzyme. The positions of nicked circle and knotted nicked
circle are marked by NC and KNC,
respectively.
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In parallel with the greatly reduced topoisomerase activity, K359Q and
K359E also lost most of their ATPase activity (Fig. 4). The DNA-dependent ATPase
activity of glutamine and glutamate mutants dropped to 5.9 and 4.0%,
respectively, of the wild-type enzyme activity (Fig. 4, solid
columns). Most of these residual ATPase activities in the mutant
proteins can be attributed to the DNA-independent activities, which are
not significantly altered among the topo II proteins tested here (Fig.
4, shaded columns). This DNA-independent ATPase activity is
likely intrinsic to topoisomerase proteins rather than from a
contaminating ATPase. We tested the sensitivity of ATPase activity
toward two topo II-targeting, antitumor drugs: ICRF159 and teniposide
(VM26). ICRF159 and other drugs in the family of bisdioxopiperazine
inhibit the catalytic activities of topo II (32) and can promote the
clamp formation of yeast topo II (33). Bisdioxopierazines can also
inhibit the ATPase activity of yeast topo II (33). In the presence of
ICRF159, the DNA-independent ATPase activity was reduced by 50% (Fig.
4, hatched versus shaded columns), a level of inhibition similar to
that observed for yeast topo II. The same level of inhibition by
ICRF159 was also demonstrated for K359Q and K359E mutant proteins (Fig.
4, hatched versus shaded columns). A second topo II
inhibitor, teniposide, can promote the formation of the DNA cleavage
complex of eukaryotic topo II (reviewed in Refs. 34 and 35; see also Ref. 36), and it can inhibit the ATPase activities in the mutant protein to a similar extent as ICRF159 (data not shown).

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Fig. 4.
ATPase activities of wild-type and mutant
topo II. ATPase assays contained 0.9 pmol of topo II proteins
either with 2.6 pmol of a 8.6-kilobase pair plasmid DNA pCaSeRhs83
(solid) or without (shaded). ATPase assays were
also carried out in the absence of DNA but with the addition of 1 mM of ICRF159 (hatched). The rates were
determined from the linear regression analysis of the linear portion of
the rate curve for ATP hydrolysis and were the averages of at least
five independent measurements. The bar above each
column shows the S.D. of these measurements. The ATPase
rates were normalized against the rate of DNA-dependent ATP
hydrolysis for wild-type topo II, which was determined to be 5.8 µM min 1.
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DNA Cleavage Reactions for Glutamine and Glutamate Mutants--
In
addition to DNA strand passage and hydrolysis of ATP, another hallmark
reaction of topo II is the double strand DNA cleavage. In this
reaction, the addition of a strong denaturant traps the formation of a
DNA cleavage complex in which the active site tyrosine of topo II is
covalently linked to the phosphoryl end at DNA cleavage site. We used a
8.6-kilobase pair plasmid DNA, pCaSpeRhs83, as the substrate, and the
cleavage reaction was monitored by the appearance of linear DNA
fragments (Fig. 5). In contrast to
topoisomerase and ATPase activities, the double strand DNA cleavage in
the absence of any cofactors is comparable for all three proteins (Fig.
5, lanes 1-3). Although ATP stimulates the DNA cleavage by
wild-type and K359Q proteins, it only has a marginal effect on K359E
protein (Fig. 5, lanes 10-12). The ATP-stimulatory effect
appears to correlate with the ATP-dependent strand passage
activity in these mutant proteins.

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Fig. 5.
Wild-type and mutant topo II have comparable
double strand DNA cleavage activities. In the DNA cleavage assays,
1.5 pmol of wild-type topo II (K, lanes 1, 4, 7, 10, 13, and
16), K359Q (Q, lanes 2, 5, 8, 11, 14, and
17), or K359E (E, lanes 3, 6, 9, 12, 15, and
18) were incubated at 30 °C for 30 min with 0.05 pmol of
supercoiled pCaSpeRhs83 DNA in either the presence (lanes
10-18) or absence (lanes 1-9) of 1 mM
ATP. The effect of topo II drugs was tested by including 1 µM of VM26 (lanes 4-6 and 13-15)
or mAMSA (lanes 7-9 and 16-18). Lane
19 shows the control, with no enzyme, and the supercoiled plasmid
is marked SC. Lane 20 shows the linearized
plasmid DNA (L). Note that both wild-type and K359Q topo II
have higher strand passage activity, and under the low-salt DNA
cleavage reaction conditions, they could promote the formation of
catenated networks (CAT; see lanes 10 and
11).
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A unique feature in the DNA cleavage reaction mediated by topoisomerase
is that it can be stimulated by a number of antitumor agents (reviewed
in Refs. 34 and 35). To examine the effects of antitumor drugs on the
cleavage by mutant proteins, we have used representatives from two
distinct classes of topo II-targeting agents: amsacrine (mAMSA) and
teniposide of epipodophyllotoxin (VM26) (36, 37). They differ by their
ability to intercalate into DNA. Although mAMSA can inhibit the strand
passage step of topo II, another member of epipodophyllotoxin drugs,
etoposide, does not affect this step in the topo II reaction (38). In
the absence of ATP, VM26 at a concentration of 1 µM can
stimulate the DNA cleavage by all three proteins to identical levels
(Fig. 5, lanes 7-9). mAMSA at the same concentration also
affects the DNA cleavage to a similar degree (Fig. 5, lanes
4-6). The presence of ATP greatly stimulates the drug-promoted
DNA cleavage, as evidenced by the nearly quantitative conversion of
plasmid DNA into full-length linear molecules and smaller fragments, by
wild-type and glutamine mutant proteins (Fig. 5, lanes 13 and 16 and lanes 14 and 17, respectively). For the glutamate mutant, the ATP stimulatory effect is
negligible (Fig. 5, lanes 15 and 18). Despite the
greatly reduced catalytic activities for Lys359 mutants,
these mutations do not affect the formation of DNA cleavable complexes
and also do not interfere with the interaction of topo II-targeting
drugs. However, depending on the lysine substitutions, the effects of
ATP cofactor in these reactions are variable.
Sequence Specificity in the Topo II-mediated DNA Cleavage
Reaction--
The DNA cleavage reaction has been used to assess the
sequence preference in the topo II/DNA interactions (39-41).
Previously, we have used DNA cleavage and nuclease footprinting to
demonstrate the coincidence of topo II cleavage and binding sites in
the intergenic region of a heat shock gene HSP70 (28). For
this study, we prepared a similar end-labeled DNA substrate to map the
DNA cleavage sites for wild-type and mutant proteins (Fig.
6). Without any added cofactors,
identical DNA cleavage patterns were observed for all three proteins,
and the relative intensities at different cleavage sites are consistent
with earlier data (Fig. 6, lanes 1-3). The strongest
cleavages occur in sites marked as T4-T6, which contain tracks of alternating purine/pyrimidine sequences. Addition of teniposide, VM26, greatly stimulates the cleavage by topo II and mutant
proteins to a similar extent, except for site T3, where cleavage is
enhanced to a lesser degree with mutant proteins (Fig. 6, lanes
4-6). We have tested the stimulation of topo II-mediated, double-strand DNA cleavage by VM26 at concentrations ranging from 0.2 to 25 µM. At higher concentrations, the drug stimulated
DNA cleavage to a higher extent, and the degree of such stimulation was
similar for both the wild-type and mutant proteins (data not shown).
Under these conditions of DNA cleavage, most of the DNA substrate
remained uncleaved, and the cleavage was correlated with the amount of
topo II protein present in the reaction (data not shown). The data from
DNA cleavage experiments using either the plasmid or the end-labeled
DNA substrate demonstrate that Lys359 mutations do not
affect DNA cleavage reactions of topo II and do not alter the sequence
specificity in their interactions with DNA substrate.

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Fig. 6.
Mutant and wild-type topo II have similar DNA
sequence specificity in the cleavage reaction. Plasmid DNA 6/122,
a derivative of pBR322 with an insertion of 1.7-kilobase pair
SalI fragment from the intergenic region of
Drosophila heat shock gene HSP70, was used to
prepare the uniquely end-labeled linear DNA. 6/122 was linearized by
HindIII, radiolabeled at its 5'-ends, and treated with
EcoRI to remove one of the end labels. 75 nM
wild-type topo II (K, lanes 1 and 4), K359Q
(Q, lanes 2 and 5), or K359E (E, lanes
3 and 6) were incubated at 30 °C for 30 min with 0.5 nM of 5'-end labeled 6/122 DNA (2.5 × 106
cpm/pmol) in either the presence (lanes 4-6) or absence
(lanes 1-3) of 1 µM VM26. Lane 7 shows the control, with no enzyme. The strong topo II binding and
cleavage sites, marked as T1-T6 in order of increasing
strengths (28), and size markers (in base pairs) are shown on the
left and right sides, respectively, of the
autoradiogram.
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Closure of N-Gate in the Protein Clamps of Topo II and
Lys359 Mutants--
Recent structural and biochemical data
suggest a two-gate model for DNA transport by topo II (7, 10, 11). A
key element in this proposed mechanism is that the jaws of the N-gate
are closed upon the binding of ATP and are open when ATP is hydrolyzed and dissociated. We have used a glass fiber filter binding assay (7,
29) to test the retention of circular versus linear DNA by
topo II proteins. An equal-weight mixture of negatively supercoiled and
linear DNA was used in these binding assays. The results obtained with
the wild-type protein have demonstrated that the formation of a
salt-stable complex with circular DNA is only observed in the presence
of a nonhydrolyzable ATP analog, AMPPNP, and the linear molecules were
found almost exclusively in the high-salt eluate (Fig.
7, lanes 7 and 8).
In the absence of any cofactors, there was no filter-retainable,
salt-stable complex (Fig. 7, lane 4). The total absence of
filter-retainable complex in the presence of ATP (Fig. 7, lane
6) is presumably due to the continuing hydrolysis of ATP and
opening of the protein clamp. In an interesting contrast, for K359Q and
K359E mutants, clamp complex formation is detectable in the conditions
of no cofactors or with ATP (Fig. 7, lanes 10 and
12 and lanes 16 and 18, respectively).
The inclusion of AMPPNP stimulates the clamp complex formation for
K359Q (Fig. 7, lane 14), but it has no effect for K359E
(Fig. 7, lane 20). The efficiency of the clamp complex
formation with the mutant proteins under the conditions tested here is
always lower than that for the wild-type protein in the presence of ATP
analog, suggesting that Lys359 is critical for the clamp
closure reaction. The mutant protein with glutamate substitution is
less efficient in the clamp complex formation than that of the
glutamine mutant. Furthermore, the formation of clamp complex either
without any cofactor or with ATP suggests that Lys359 may
be involved in the pathway for ATP-induced closure of N-gate, and its
substitutions with other residues result in an anomaly in this
signaling process.

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Fig. 7.
Lys359 mutant topo II proteins
can form salt-stable, filter-retainable complex with circular DNA.
Wild-type topo II (lanes 3-8), K359Q (lanes
9-14), K359E (lanes 15-20), or control, with no
enzyme (lanes 21 and 22), were incubated with a
mixture of labeled linear and circular DNA either in the presence of 1 mM ATP (lanes 5, 6, 11, 12, 17, and
18), in the presence of 1 mM AMPPNP (lanes
7, 8, 13, 14, 19, and 20), or without any cofactors
(lanes 3, 4, 9, 10, 15, and 16). The binding
reactions were quenched by the addition of NaCl to 1 M and
filtered through GF/C filters. The filters were further washed with 1 M NaCl before final elution with 1% SDS. The combined
filtrates and 1 M NaCl eluates (odd-numbered lanes
3-21) and SDS eluates (even-numbered lanes 4-22) were
analyzed by 1.6% agarose gel electrophoresis in the presence of 1 µg/ml ethidium. The gel was dried and autoradiographed. Lane
1 shows the linear DNA marker, and lane 2 shows the DNA
substrates used in the filter-binding assays. The positions for
supercoiled DNA (SC), relaxed circle (RC), nicked
circle (NC), linear (L), and supercoiled dimer
(SCD) are marked on the left. Circular DNA can
form stable complex with mutant topo II in high salt even without
cofactors (lanes 10 and 16) or with ATP
(lanes 12 and 18).
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A unique feature for the clamp complex is that the topological link
between a protein ring and circular DNA is stable in high salt; the
filter-retainable complex is resistant to 1 M NaCl wash (Ref. 7; also Fig. 7). Earlier data also demonstrated by gel mobility
shift assays that the complex formed by topo II and circular DNA in the
presence of AMPPNP is resistant to dissociation in 0.5 M
NaCl (14). We used the analytical ultracentrifugation to monitor the
formation of these salt-stable complex in CsCl density gradient. In the
buoyant density gradient experiments, circular DNA with increasing
numbers of complexed topo II molecules band at distinct positions with
progressively lighter buoyant densities (Fig.
8). The wild-type topo II did not form
any stable complex with circular DNA either without any cofactors or
with ATP, because only a single free DNA peak appeared in the bottom of
a density gradient (Fig. 8, A and B). In
contrast, the presence of AMPPNP resulted in the generation of DNA
species with multiply interlocked protein rings (Fig. 8C).
For the K359E mutant, the formation of these complexes can be observed
regardless of the cofactor present (Fig. 8, G, H, and
I). The complex with K359Q can also form without any
cofactors (Fig. 8D) but it can be stimulated by ATP and,
more efficiently, by AMPPNP (Fig. 8, E and F).
The control experiments with linear DNA suggest that the complex formed is a result of the capture of DNA by the closure of a protein clamp
(Fig. 9). The protein clamps can slide
off a linear DNA substrate under high salt conditions. No complex with
linear DNA was observed in the absence of cofactors or in the presence
of ATP for K359Q and K359E mutant proteins (Fig. 9, D, G, E,
and H), and only a minor DNA/protein complex peak was
observed for wild-type topo II (Fig. 9, A and B).
In the presence of AMPPNP, a peak was observed for linear DNA complexed
with wild-type topo II (Fig. 9C) or K359Q (Fig.
9F), but none with K359E (Fig. 9I). The
salt-stable complex formed between topo II and linear DNA is most
likely due to a DNA cleavage complex with one of topo II subunits
covalently linked to a single strand DNA break (42-44). This was also
confirmed by the following experiment. When wild-type topo II was
incubated with labeled linear DNA in the presence of AMPPNP, a trace
amount of DNA was retained on the glass fiber filter, which was
analyzed by two-dimensional agarose gel electrophoresis and was found
to comigrate with the full-length linear DNA (data not shown). This
result supports the notion that the complex formed under these
conditions is due to single-strand DNA cleavage, because DNA in such a
complex should remain as a full-length linear molecule. In all
instances, the complex formed between linear DNA and topo II is always
much less than those with circular DNA under identical conditions. The
results obtained from CsCl density gradient experiments are in complete
accord with the data from filter binding assays. Furthermore, it shows
the protein clamp due to the closure of N-gate is stable in very high
concentration of salt solutions (~5 M CsCl) and for a
period at least as long as ultracentrifugation time (~30 h). The
buoyancy shifts between the DNA/protein complex and DNA can be used to
determine the molecular weight of protein in the complexes (23) (see
"Experimental Procedures"). For all three proteins, it can be shown
that the multiple species of complexes correspond to a single DNA
circle interlocked with multiple molecules, up to 4, of a protein with
a molecular mass of 350 kDa, consistent with it being a dimer of the
170 kDa topo II subunit.

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Fig. 8.
Formation of salt-stable topo II/DNA complex
in CsCl density gradient ultracentrifugation. The major peak at
the bottom of each gradient corresponds to DNA without any bound
protein, whereas the lighter species are complex of DNA with different
numbers of protein molecules. The binding reactions were carried out
with circular pCaSpeRhs83 DNA and wild-type topo II (A-C),
K359Q (D-F), or K359E (G-I) in the presence of
ATP (B, E, and H), AMPPNP (C, F, and
I), or no cofactors (A, D, and G). The
minor species observed between major bands (see C and
F) correspond to topo II complexed with dimer DNA, which is
present in the plasmid DNA preparations.
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Fig. 9.
No topo II clamp complex is formed with
linear DNA in CsCl density gradient ultracentrifugation. In
contrast to the multiple peaks corresponding to multiple molecules of
topo II complexed with circular DNA, very little complex was observed
with linear DNA under identical conditions. The reaction conditions
were similar to those described for Fig. 8, except that
XhoI-digested pCaSpeRhs83 DNA was used in all experiments.
Binding reactions contained either wild-type topo II (A-C),
K359Q (D-F), or K359E (G-I) in the presence of
cofactor ATP (B, E, and H) or AMPPNP (C,
F, and I) or no cofactor (A, D, and
G). The minor peak observed in A-C and
F is likely from the single strand DNA cleavage complex in
which one of topo II subunits is covalently linked to the single strand
break in a linear duplex molecule.
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DISCUSSION |
A key question in understanding the mechanism of topo II is to
elucidate the role of ATP binding and hydrolysis in the overall process
of DNA strand passage reaction. One approach is to identify the crucial
residues in topo II that interact with ATP and analyze their functions.
Examining the structure of the 43-kDa fragment of gyrB shows three
regions of the enzyme that directly interact with the
and
phosphates of the bound ATP: His38-Asn46,
His99-Gly119, and
Pro330-Asp338. The first region is part of a
helix in which a conserved Glu42 was identified to be
critical to the ATPase and DNA supercoiling activities of
Escherichia coli gyrase (45). The second region, which forms
a loop and part of a short helix, contains a
GXXGXG motif found in a variety of ATP binding
proteins (46, 47). Located here are two lysine residues,
Lys103 and Lys110, which were specifically
modified with an ATP analog in affinity labeling studies (48). Changing
the Gly144 of the GXXGXG
motif in the yeast enzyme to Ala and the mutations of
Lys103 in gyrase result in the complete loss of
ATP-dependent topo II activities (49, 50). The third region
is a loop of Pro330-Asp338 that is a part of
the domain that forms the 20 Å hole. Within this loop, both
Gln335 and Lys337 are in contact with oxygens
in the
-phosphate of the bound AMPPNP. The unique molecular
architecture of this loop suggests a potential mechanism to relay the
signaling in the binding and hydrolysis of ATP to induce the
conformational changes in the rest of the topo II molecule (31).
Our data presented here demonstrate that Lys359 in
Drosophila topo II, the homologous residue of
Lys337 in gyrB, is critical for the catalytic activities of
topo II. Although a conservative substitution with arginine does not
inactivate the enzyme, changing it to either glutamine or glutamate
greatly reduces both the strand passage and DNA-dependent
ATPase activity. Because these mutant proteins have chromatographic
behavior identical to the wild-type enzyme during the purification
steps and form dimers based on the CsCl density gradient experiments,
we believe that there is no gross misfolding in the mutant proteins.
Furthermore, the apparent similarity in DNA cleavage activity, cleavage
site specificity, and sensitivity toward topo II-targeting drugs
indicates that these mutants still retain part of the wild-type topo II functions. Therefore, the reduced strand passage activity seems to be
due to a defect in specific steps involving ATP-dependent functions rather than in the formation of a protein-mediated DNA gate.
There is a correlation among the ATP-dependent activities in the mutant proteins. For instance, although the K359E mutant is less
active in the unknotting assays than the K359Q mutant, it has also lost
most of the ATP-stimulation in both DNA cleavage and protein-clamp
assays. This defect in ATP-induced response could be either because the
mutation has abolished the ability of topo II to bind ATP or because
the defect is in a step subsequent to the binding of ATP. Our data
favor the latter possibility because the residual strand passage
activity in the mutant proteins is still ATP-dependent
(Fig. 3), and the Km of ATP in this reaction for
both mutant proteins is comparable with the wild-type enzyme (data not
shown). Furthermore, at least for K359Q protein, ATP can stimulate both
the DNA cleavage reaction and protein clamp formation.
In an interesting contrast with the wild-type topo II,
Lys359 mutants can form a salt-stable protein clamp complex
with circular DNA even in the presence of ATP or in the absence of any
nucleotide cofactors. The clamp closure for mutant proteins with ATP
could be due to their lower ATPase activity, thereby prolonging the ATP-bound state. However, both mutant proteins have their ATPase activities reduced to a similar extent, whereas the formation of clamp
complex with K359Q mutant is more efficient than K359E, suggesting the
possible involvement of other factors, such as the efficiency of ATP
signaling in clamp closure. It is possible that there is a dynamic
equilibrium between the open-form and closed-form of the protein clamp.
In the presence of DNA, this equilibrium can be affected upon the
binding of nucleotide cofactor. For wild-type enzyme, binding of ATP
can shift this equilibrium toward the closed clamp conformation and
initiate the subsequent conformational changes that are necessary for
the strand passage reaction (5). Replacing Lys359 with
either Gln or Glu interferes with the ATP-signaling mechanism, and the
equilibrium between open/closed clamp complex. The fact that K359Q and
K359E could form protein clamps in the absence of nucleotide cofactor
suggests that the basal equilibrium is shifted toward closed clamp
conformation in these mutant proteins. Although the site-specific
mutants presented in this paper are unique in that they showed altered
response of clamp closure to triphosphate cofactors in addition to the
loss of strand passage activity, the complexity in the clamp closure
process suggests that future site-specific mutagenesis experiments can
identify other critical residues involved in the movement of this
intricate macromolecular machinery.
We thank Dr. Harvey Sage for assistance in
the analytical ultracentrifuge runs and Dr. Jane Richardson for
discussion of the use of kinemage.