From the Salk Institute for Biological Studies, La Jolla,
California 92037 and the Biology Department, San
Diego State University, San Diego, California 92182-4614
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ABSTRACT |
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Eukaryotic type 1B topoisomerases act by forming
covalent enzyme-DNA intermediates that transiently nick DNA and thereby
release DNA supercoils. Here we present a study of the topoisomerase
encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site
than the 5'-(T/C)CCTT-3' sequence previously identified for poxvirus
topoisomerases. Separate assays of initial DNA binding and covalent
complex formation revealed that different DNA sequences were important
for each reaction step. The location of the protein-DNA contacts was
mapped by analyzing mutant sites and inosine-substituted DNAs. Some of
the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively
important for covalent complex formation but not initial DNA binding.
Interactions important for catalysis were probed with 5'-bridging
phosphorothiolates at the site of strand cleavage, which permitted
covalent complex formation but prevented subsequent religation. Kinetic
studies revealed that the flanking sequences that promoted recovery of
covalent complexes increased initial cleavage instead of inhibiting
resealing of the nicked intermediate. These data 1) indicate that
previously unidentified DNA contacts can accelerate a step between
initial binding and covalent complex formation and 2) help specify
models for conformational changes promoting catalysis.
Topoisomerases play important roles in DNA replication,
recombination, and transcription (1). Type 1B topoisomerases act (Fig.
1) by first binding double-stranded DNA
and then cleaving substrate DNA using a conserved enzyme tyrosine
residue, forming a 3'-O-phosphotyrosine linkage. The DNA
strands then isomerize, either by passing one strand through the break
in the other or by rotation of the covalent complex, relaxing the DNA.
The broken strand is then resealed by nucleophillic attack of the
5'-hydroxyl on the phosphotyrosine bond. Last, the topoisomerase
dissociates from substrate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Reaction pathway for type 1 topoisomerases. Topoisomerase is indicated by the
shaded circle.
All the poxviruses studied to date encode a type 1B topoisomerase (2-7) that is inferred to be required for viral replication (8). Poxvirus topoisomerases bind to the sequence 5'-(T/C)CCTT-3' and cleave just 3' of the last T (designated position +1), to form the covalent intermediate (7, 9, 10). Although many topoisomerases have been found to show some DNA sequence preferences (11-14), the high degree of specificity seen with the poxvirus topoisomerases is unique.
In the case of vaccinia virus, the most thoroughly studied model, the
topoisomerase enzyme consists of two domains linked through a
trypsin-sensitive bridge (15, 16). The N-terminal domain (amino acids
1-80) contacts the 5'-(T/C)CCTT-3' element on the major groove side of
the DNA (15-17). Chemical footprinting and interference studies
indicate that the enzyme contacts several phosphates, including the
scissile phosphate, on both strands and the minor groove side as well.
Recently, the crystal structure of the C-terminal catalytic domain of
vaccinia topoisomerase (amino acids 81-314) was determined and found
to be a member of the -integrase family (18). The topoisomerase of
MCV1 is 54% identical to
that of vaccinia and presumably resembles the vaccinia enzyme closely
in structure and mechanism.
Here we present a study of the role of DNA sequence in the function of
MCV topoisomerase. Poxvirus topoisomerases are experimentally tractable
due to their small size (33 kDa) and function as monomers (19). The
high degree of sequence specificity allows the enzyme to be placed
precisely on test DNA substrates for analysis. In addition, MCV causes
medically important opportunistic infections in immunocompromised
patients (20-22), adding interest to understanding the mechanism in
the hope of aiding the development of antiviral agents. We investigated
DNA contacts important in different parts of the topoisomerase
catalytic cycle. We found that previously unidentified DNA contacts
increase the rate of a limiting step between initial binding and
covalent bond formation.
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EXPERIMENTAL PROCEDURES |
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Enzyme Purification-- MCV topoisomerase was overexpressed in Escherichia coli and purified as described by Hwang et al. (7). The active fraction of MCV topoisomerase monomers was measured by titrating the enzyme with a suicide substrate derived from substrate a DNA (7). In this substrate (sequence 5'-TCCGTGTCGCCCTTATTCC-3' and 5'-GAGGCAAAATGCCGCAAAAAGGGAATAAGGGCGACACGG-3') the short duplex extension 3' of the 5'-CCCTT-3' sequence can dissociate upon covalent complex formation, trapping the covalent intermediate and allowing the active fraction to be determined.
Noncovalent Complex Formation--
Oligonucleotide substrates
were end-labeled at the 5'-end by treatment of one strand with
[-32P]ATP and T4 polynucleotide kinase. After
labeling, the oligonucleotide was purified in a Sephadex G-25 spin
column and then hybridized to the complementary oligonucleotide by
heating and slow cooling. Reaction mixture contained 20 mM
Tris-Cl (pH 8.0), 200 mM potassium glutamate, 1 mM dithiothreitol, 0.1% Triton X-100, 1 mM
EDTA, and 50 nM of 5'-32P-end-labeled DNA
substrate. Reaction mixtures were prewarmed at 37 °C and then
initiated by the addition of topoisomerase. Reaction mixtures were
incubated for 5 min and cooled in ice water bath for 5 min. Reaction
products were separated on 5% polyacrylamide gels, visualized by
autoradiography, and the radioactivity was quantitated by
PhosphorImager. All reactions were repeated and quantitated 2-4 times.
Note that the difference between the amount of covalent complexes
visualized on SDS-PAGE and complexes visualized on native gels allows
the amount of noncovalent complexes to be determined by subtraction.
Covalent Complex Formation-- Reaction mixtures contained 20 mM Tris-Cl (pH 8.0), 200 mM potassium-glutamate, 1 mM dithiothreitol, 0.1% Triton X-100, 1 mM EDTA, 50 nM of 5'-32P-end-labeled DNA substrate and 20 nM topoisomerase. Reactions were prewarmed at 37 °C and then initiated by the addition of topoisomerase. Reactions were incubated for 5 min and stopped by the addition of SDS to a 1% final concentration. Reaction products were heated at 95 °C for 5 min and then analyzed on 12% polyacrylamide gels containing 0.1% SDS. The reaction products were visualized by autoradiography and quantitated by a PhosphorImager. All reactions were repeated and quantitated 2-4 times.
DNase I Footprinting Experiments-- A synthetic duplex 62-mer oligonucleotide matching substrate a was used as a footprinting substrate. Reaction mixtures (10 µl) contained 20 mM Tris-Cl (pH 8.0), 200 mM potassium glutamate, 1 mM dithiothreitol, 0.1% Triton X-100, 1 mM EDTA, 50 nM of 5'-32P-end-labeled DNA substrate, and topoisomerase. Reaction mixtures were prewarmed at 37 °C, initiated by the addition of topoisomerase, and incubated for 5 min at 37 °C. Samples were adjusted to 2.5 mM MgCl2 and incubated for an additional 5 min at room temperature in the presence or absence of 0.001 units/µl DNase I. Reaction products were stopped by the addition of 1% SDS. The reaction mixtures were then digested with 10 µg of proteinase K for 30 min at 45 °C. The samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1). An aliquot of the aqueous extract was analyzed on 12% polyacrylamide gels containing 7.5 M urea. The reaction products were visualized by autoradiography.
Synthesis of the 5'-Bridging Phosphorothiolate
DNA--
5'-bridging phosphorothiolates (23) were synthesized using
5'-(S-dimethoxytrityl)-adenosine (synthesis to be reported
elsewhere).2 For cleavage
with heavy metals, 5'-end-labeled oligonucleotides were precipitated
with ethanol and carrier salmon sperm DNA (1 µg), dried, and
dissolved in 30 µl of distilled water. Subsequent steps were carried
out in a fume hood using chemical gloves due to the extreme hazard of
phenyl mercuric acetate. 5 µl of each oligonucleotide was incubated
with 5 µl of water saturated phenyl mercuric acetate (Sigma) or water
control for 5 min at room temperature. Water (40 µl) was then added
to all tubes, and samples were extracted three times with 100 µl of
ethyl acetate (Aldrich) to remove phenyl mercuric acetate.
Oligonucleotides were then precipitated with ethanol, dried, and
resuspended in 10 mM thiourea (included to chelate heavy
metals). One-µl samples were analyzed on 10% DNA sequencing-type
polyacrylamide gels and analyzed by autoradiography.
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RESULTS |
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Specificity of DNA Binding and Covalent Complex Formation by MCV Topoisomerase-- In previous studies, DNA sequences favored for initial binding and covalent complex formation with purified MCV topoisomerase were investigated. Enzyme was incubated with labeled pUC19 DNA, and seven preferred cleavage sites were identified (7). Each contained the 5'-(T/C)CCTT-3' sequence favored by poxvirus topoisomerases (2, 10). One site was cleaved with much higher efficiency than the others (Table I, substrate a). Interestingly, there are eight 5'-CCCTT-3' sequences in pUC19 (Table I, substrates a-c and g-k), but only four were cleaved detectably (Table I; substrates a, b, c, and g). This indicated that the conserved pentamer was not sufficient for high levels of covalent complex formation. A similar conclusion was also reached in studies of the vaccinia topoisomerase (9). Three additional favored sites had the sequence 5'-TCCTT-3' (Table I; substrates d, e, and f).
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To study the function of MCV topoisomerase in more detail, sequences
from pUC19 were synthesized as oligonucleotides and annealed to form
duplexes. Their binding activities were then studied in gel retardation
assays (summarized in Table I). The binding measured by the band shift
assay represented contributions from both covalent and noncovalent
complexes. The fraction of covalent complexes was determined as
described below, allowing the fraction of noncovalent complexes to be
deduced by subtraction. For example, for substrate a, 57% of DNA was
bound in Fig. 2, lane
4, and of this 33% was in noncovalent and 24% in covalent
complexes. The fraction bound in noncovalent complexes ranged from 33%
(defined as 100% in Table I) for substrate a, to 3% for substrate
substrate k. Evidently, the sequence determinants for binding extended
beyond the 5'-(T/C)CCTT-3' pentanucleotide found in all substrates.
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DNA binding by the various substrates was tested under several solution conditions. Binding was assayed in the presence of 200 mM potassium glutamate, since this promotes accumulation of the covalent complex intermediate in the assays described below. Binding was also assayed in the presence of 100 mM NaCl, used previously for analyzing poxvirus topoisomerases (24). The efficiency of DNA binding was found to be similar for each substrate under both conditions (data not shown).
Substrates a-k were next tested for covalent complex formation. Oligonucleotides were incubated with MCV topoisomerase, and then complexes were separated by SDS-polyacrylamide gel electrophoresis. Under these conditions, only covalently bound DNA strands migrated with the topoisomerase. An autoradiogram is shown in Fig. 2B and quantitated in Table I. The efficiency of covalent complex formation was drastically different for different DNAs, with substrate a showing 24% conversion of substrate to product (defined as 100% in Table I). Substrates b-k were much less active. Why the sequences in substrates h-k were not cleaved detectably in pUC19 is unclear, although cleavage at all sites except substrate a was inefficient, so cleavage at some of the weak sites may not have been detected. Overall, these data indicate that sequences outside the conserved pentamer can independently influence either DNA binding (for example, substrates e and k), or covalent complex formation (for example, substrates f and h).
To explore the importance of bases in the conserved pentanucleotide, mutants were constructed containing changes in the 5'-CCCTT-3' sequence (Table II; bases are numbered such that the T 5' of the scissile phosphate is +1). Altering base +5 (C to T) or +3 (C to T) had slight effects on binding, while altering base +1 (T to C) drastically reduced binding. For covalent complex formation, altering the base at +5 had only a modest effect, confirming that the consensus pentamer should include a C or T at this position. Altering base +3 reduced covalent complex formation 20-fold, while altering base +1 reduced covalent complex formation to less than 0.1% of the substrate a level. Evidently, the correct base at position +3 is most important for covalent complex formation after binding, while position +1 is important for binding. These results parallel findings in the vaccinia system (24).
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The ability to trap reaction intermediates by using different substrates provided a potential means for probing the reaction mechanism. As a first step, substrate sequences were mapped that were specifically important for covalent complex formation.
Sequence Determinants for Covalent Complex Formation--
To
identify the required sequences flanking the 5'-(C/T)CCTT-3'
pentanucleotide, flanking sequences were systematically exchanged between substrate a, the most active substrate, and substrate h (Table
III). Substrate h was chosen because it
contains the 5'-CCCTT-3' sequence found in substrate a, and substrate h
binds MCV topoisomerase almost as well as substrate a (72% of the
substrate a level). However, substrate h was impaired in forming the
covalent intermediate (0.2% of substrate a). Note that substrate h
differs from substrate a at only 12 out of 22 positions, facilitating
the identification of sequences important for covalent complex
formation. Independently replacing DNA to either side of the conserved
pentanucleotide of substrate h with substrate a sequences restored high
level covalent complex formation (Table III, substrates l and m). This indicates that both flanking regions must have disfavored nucleotides present for the substrate to display low cleavage activity.
Substitution of sequences in substrate a with shorter flanking
sequences of substrate h also yielded substrates active for cleavage
(substrate n and substrate o, base pairs substituted extending from 1
to
5 and +9 to +11, respectively). Replacement of base pairs +7 to
3 in substrate h with sequences from substrate a yielded a highly
active substrate (substrate p), indicating that at least one flanking
sequence was favorable. However, replacing base pairs
2 and
3 in
substrate p with sequences from substrate h yielded a substrate with
low activity (substrate q). This implicated base pairs
2 and
3 on
the right as important. On the left side, comparison of substrates m
and q indicates that bases at +9 to +11 must be important, since
disfavored nucleotides needed to be present on both sides of the
conserved pentanucleotide to impair function. To confirm these
findings, a derivative of substrate a containing base pairs from
substrate h at
2,
3, +9, and +10 was tested (substrate r). This
substrate displayed low activity for cleavage (3.1% of substrate a),
indicating that the four bases changed are indeed important for
cleavage activity. Changing only bases +9,
2, and
3 to match
substrate h in a substrate otherwise matching substrate a yielded a
substrate with high activity (substrate x), indicating that on the left
side base +10 is particularly important.
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Substrates l-q and substrate x all displayed noncovalent DNA binding
activity within 66% of substrate a, indicating that the altered
activities observed with mutant substrates were mainly due to changes
at postbinding steps. Evidently, in the context of substrate a, optimal
bases at positions +10, 2, and
3 are required for efficient
covalent complex formation but not for initial binding.
DNase I Footprinting of MCV Topoisomerase--
How much of the DNA
surrounding the 5'-(T/C)CCTT-3' sequence does MCV topoisomerase cover
when bound? Are the +10, 2, and
3 sequences likely to be contacted
by bound enzyme? In previously reported DNase I footprinting studies,
vaccinia topoisomerase protected base pairs +13 to
13 on the cleaved
strand and from +13 to
9 on the noncleaved strand (9). To determine
where MCV topoisomerase binds on substrate DNAs, a footprinting
experiment was carried out on a 61-mer duplex DNA fragment derived from
substrate a (Fig. 3A). MCV
topoisomerase was incubated with oligonucleotide radiolabeled uniquely
at one 5'-end, and DNase I was added to cleave exposed DNA. Reaction
products were treated with proteinase K and SDS, denatured, separated
on DNA sequencing-type gels, and visualized by autoradiography.
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Treatment of the labeled substrate with DNase I produced a partial
digestion pattern (Fig. 3B, lanes 2 and 6). Digestions were repeated in the presence of MCV
topoisomerase. DNase I cleavage products were mapped by comparison with
labeled DNA size markers. MCV topoisomerase protected bases +11 to 15
on the cleaved strand (Fig. 3B, lanes
7 and 8) and bases +8 to
11 on the noncleaved strand (Fig. 3B, lanes 3 and
4). Bases +9 to +11 on the noncleaved strand were slightly
enhanced for DNase I cleavage. Exact boundaries of the protected region
are somewhat ambiguous due to the weak DNase I cleavage at some
sequences. In addition, bands were seen on the cleaved strand that did
not depend on the addition of DNase I, corresponding to DNA cleavage by
topoisomerase-mediated covalent complex formation (data not shown). The
heterogeneity of this product (Fig. 3B, bracket)
probably results from incomplete removal of protein fragments by
proteinase K. No topoisomerase-dependent cleavage product
was observed when the nonscissile strand was labeled, as expected (7,
9).
Probing the Location of Topoisomerase-DNA Interactions Using
Inosine Substitutions--
To investigate protein-DNA contacts at +10,
2, and
3, oligonucleotides containing inosine substitutions were
synthesized and tested. Changing T:A to I:C base pairs leaves the minor
groove side of the DNA matching that of T:A, while the major groove
side matches G:C (25). Thus, if major groove contacts at a T:A pair are
important, then substitution of an I:C pair should be harmful to the
interaction. Conversely, if the essential contacts are in the minor
groove, then the I:C substitution should be tolerated.
DNA substrates were synthesized that contained inosine substitutions in
the context of substrate r, which matches the highly active substrate
substrate a except at positions +10, +9, 2, and
3 and so displays
weak activity. Since substrates that contain one favorable and one
unfavorable flank are active (Table III), it is expected that
conversion of a disfavored flanking sequence to a favored flanking
sequence will restore activity. MCV topoisomerase bound to all seven
DNA substrates efficiently (Table IV),
allowing the relative activities in covalent complex formation to be
compared. All I:C replacements at +10,
2, and
3 failed to restore
covalent complex formation (Table IV, substrates s-u). These data are
consistent with a model in which important contacts at the positions
+10,
2,
3 are made at least in part in the major groove.
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As a control, the effect of an inosine substitution were tested within the conserved pentamer (substrate a 3T, Table II). This substrate, containing a T substituted for C at position +3 in the 5'-CCCTT-3' pentamer, was reduced 25-fold relative to the wild type substrate for covalent complex formation (Table II). Substituting a C:I pair at +3 boosted activity 5-fold (substrates v and w, Table IV). This finding suggested that the recognition of position +3 is at least in part in the major groove, although activity of this substrate was still down 5-fold from substrate a, indicating a possible component of minor groove recognition also. This result was consistent with the previous finding that positions +2 and +1 are contacted in the major groove by vaccinia virus topoisomerase (24) but that the enzyme wraps around the helix in this region (18).
Assessing the Kinetic Steps Influenced by Different Sequences Using
5'-Bridging Phosphorothiolates--
The activating sequences at +10,
2, and
3 could have increased recovery of the covalent complex by
either of two means. The forward rate of initial cleavage might have
been increased, or the rate of resealing the covalent intermediate
might have been decreased. To distinguish between these possibilities,
reactions were studied using suicide substrates containing 5'- bridging phosphorothiolate linkages at the site of strand cleavage and religation (Fig. 4A). The
5'-bridging sulfur atoms in such substrates are good leaving groups, so
covalent intermediates form efficiently. However, sulfur is a poor
nucleophile at phosphorous, so attack of the 5'-sulfur atom on the
phosphotyrosine linkage is inhibited. Thus, the religation step is
blocked, allowing the forward rate of initial cleavage to be studied in
isolation (23, 26, 27).
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The optimal sequence (substrate a) was compared with substrate y, which
is identical to substrate a but contains disfavored bases at +10, +9,
2, and
3. Derivatives of substrate a and substrate y containing
5'-bridging phosphorothiolates were synthesized and compared with the
normal forms containing bridging oxygens (Table V). The incorporation of the sulfur atom
was confirmed by demonstrating that the phosphorothiolate bond was
cleaved by incubation with heavy metal (Fig. 4B,
lanes 3-6), while the normal oxygen-containing substrate was insensitive as expected (Fig. 4B,
lanes 1 and 2).
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Substrate a and substrate y were then assayed in the presence and
absence of the 5'-bridging phosphorothiolate at the scissile bond. DNA
binding assayed by gel retardation revealed little difference between
substrate a (Fig. 4C, lanes 2-4),
substrate y (lanes 6-8), and the
phosphorothiolate derivatives (lanes 10-12 and
14-16). Assays of covalent complex formation revealed that
substrate a and substrate a-thio formed covalent complexes efficiently
(Fig. 4D, lanes 2-5 and
12-18), while substrate y and substrate y-thio were
similarly reduced in activity (lanes 7-10 and
20-26). Quantitation (Fig. 4E; Table V) revealed
that the initial rate for substrate a was 14 times faster than that for
substrate y. Similarly, the initial rate for substrate a-thio was 16 times faster than that for substrate y-thio. Thus, the substitution of
a 5'-bridging phosphorothiolate in substrate y did not lead to an
accumulation of covalent complex, indicating that the base changes in
substrate y specifically inhibit initial covalent complex formation
without stimulating religation.
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DISCUSSION |
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Our studies indicated that MCV topoisomerase recognizes a larger site than the previously described 5'-(C/T)CCTT-3' motif. Sequence contacts to the major groove outside the conserved pentamer were found to be selectively important for catalysis in the noncovalent protein-DNA complex. These data emphasize the importance of a reaction step between initial binding and covalent complex formation, a point also raised in structural studies of type 1B topoisomerases and related enzymes (26-30).
DNA Binding by Poxvirus Topoisomerases--
DNase I footprinting
studies of MCV topoisomerase and vaccinia topoisomerase (9) revealed
that the enzyme bound to a recognition site spanning roughly 20 base
pairs centered on the 5'-(C/T)CCTT-3' motif. The additional base pairs
identified as important here, at +10, 2, and
3, are within the
region bound on the cleaved strand, but the +10-position may not be
contacted on the uncleaved strand. This differed slightly from results
with vaccinia, in which the footprint was larger on the uncleaved
strand for the same substrate, indicating possible subtle differences
in the complexes. Previously, vaccinia topoisomerase substrates were found to have differential impairments in catalysis and binding, but
the locations of base pairs important for each were not clarified. Data
presented here for MCV represent the first elucidation of important
sequences outside of the 5'-(C/T)CCTT-3' motif and their role in
postbinding activation of catalysis.
The vaccinia virus topoisomerase catalytic domain (residues 81-314)
has been visualized by x-ray crystallography, and a model of the DNA
complex has been built that satisfies several biochemical and
structural constraints (18). In this model, the catalytic domain spans
positions +10 to 9 with respect to the scissile phosphate, with
varying degrees of engagement of the DNA in this region. The
amino-terminal domain binds the conserved 5'-(C/T)CCTT-3' motif in the
major groove. The catalytic domain lies on the other side of the DNA
near the scissile phosphate (Fig. 5).
Although the structure of the MCV topoisomerase has not been studied by x-ray crystallography, the high degree of sequence identity between the
enzymes (54%) and functional similarity described here suggests that
the vaccinia model probably describes MCV in some detail.
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Postbinding Steps Potentiating Catalysis by Type 1B Topoisomerases and Related Enzymes-- Vaccinia virus topoisomerase and several related proteins have been proposed to require a protein conformational change to potentiate covalent complex formation. In the structure of the catalytic domain of vaccinia virus topoisomerase, solved in the absence of DNA substrate, the tyrosine residue that acts as the nucleophile in forming the covalent intermediate was not correctly positioned to attack the scissile phosphate. A conformational change is thus necessary to activate the enzyme for covalent complex formation (18). Mutational studies have also supported a model in which a postbinding isomerization takes place prior to covalent complex formation (31).
Structural studies and sequence alignments indicate that the eukaryotic
type 1B topoisomerases are members of the larger family of
-integrase-related proteins. Several
-integrase family protein structures, solved in the absence of DNA, also contain active site
tyrosines in what appear to be inactive conformations. In XerD, the
active site residues are apparently buried in the protein interior
(28), while in the
-integrase catalytic domain, the active site
tyrosine is part of a disordered loop (30). For human type 1B
topoisomerase, comparison of structures of catalytic domain fragments
in both covalent and noncovalent complexes reveal clear although modest
rearrangements of active site residues (26, 27).
In the case of the phage P1 Cre protein, a site-specific recombinase of
the -integrase family, an x-ray structure is available of a
recombination intermediate in which two of four Cre monomers have
formed covalent complexes (29). In these structures, the positions of
the active site tyrosines differ substantially, with the tyrosine in
the two noncovalently bound complexes moved away from the scissile
phosphate by a conformational change. Evidently, conformational
transitions following the first pair of strand exchanges bring the
second pair of active site tyrosines into alignment at the proper time
for catalysis. These findings taken together emphasize the ubiquity of
postbinding conformational changes and the potential of this step as a
regulatory input.
DNA Binding and Catalysis by Poxvirus Topoisomerases--
In our
study of catalysis by MCV topoisomerase, an intermediate step between
DNA binding and covalent complex formation appears to be rate-limiting,
at least for some substrates. Correct DNA contacts may increase the
rate of this intermediate step, potentially involving changes that
bring the active site tyrosine into proper position. We hypothesize
that the noncovalent complexes formed by MCV topoisomerase on poor
substrates such as substrate y mimic the normal noncovalent
intermediate. According to this view, the suboptimal base pairs at +10,
2, and
3 obstruct the postbinding conformational change.
Conversely, correct engagement of the key flanking sequences may help
the catalytic domain to adopt the active conformation. In support of
this model, we demonstrated using 5'-bridging phosphorothiolate
substrates that the step activated by optimal flanking sequences was
initial cleavage after DNA binding.
The activating contacts are probably made at least in part by the
catalytic domain. Studies of inosine substitutions specify the side of
the helix toward the catalytic domain as the probable region contacted
(Fig. 5). Furthermore, a catalytic domain fragment of MCV topoisomerase
is sensitive to base sequence +10, 2, and
3, supporting this aspect
of the model.3 In the model
of the vaccinia catalytic domain structure docked on straight B-DNA
(18), these flanking sequences may not be extensively engaged,
indicating that the protein or DNA may need to bend to make these
contacts (Fig. 5). A previous study of the vaccinia topoisomerase
emphasized the role of the amino-terminal and linker domains in
activating catalysis (18). However, in any model, conformational
changes must ultimately position the active site tyrosine for cleavage,
implying that changes must propagate to the catalytic domain as
suggested here.
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ACKNOWLEDGEMENTS |
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We thank members of the Bushman and Burgin laboratories for discussion and comments on the manuscript and A. Bocksruker for help preparing the manuscript.
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FOOTNOTES |
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* This work was supported by University of California Universitywide AIDS Research Program Grant R97-SAL-088 (to F. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Scholar of the Leukemia Society of America. To whom correspondence should be addressed: The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037.
2 A. Burgin, manuscript in preparation.
3 Y. Hwang, A. Burgin Jr., and F. Bushman, unpublished data.
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ABBREVIATIONS |
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The abbreviation used is: MCV, molluscum contagiosum virus.
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REFERENCES |
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