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INTRODUCTION |
Type I topoisomerases promote the relaxation of supercoiled DNA by
introducing a temporary break in one of the strands of the helix (1).
This function is important for a number of different cellular processes
such as DNA replication, transcription, and chromatin remodeling (1,
2). The type I topoisomerases have been divided into two subfamilies,
type IA and type IB. These two subfamilies share no sequence homology
and relax DNA by distinct mechanisms. The type IA subfamily was
originally identified in prokaryotes but is now known to include
topoisomerases III from eukaryotic organisms as well (3, 4).
Escherichia coli topoisomerase I is the prototype of the
type IA subfamily (1). Type IA topoisomerases relax only negatively
supercoiled DNA and require a region of single-stranded DNA as well as
Mg2+ for DNA relaxation.
The type IB topoisomerase subfamily includes eukaryotic topoisomerase I
and the topoisomerases that are encoded by poxviruses such as vaccinia.
The relaxation reaction catalyzed by a type IB topoisomerase is
initiated by DNA binding followed by nucleophilic attack of the
O-4 atom of the active site tyrosine on a phosphodiester bond in
the DNA. The phosphodiester bond energy is preserved during the
resulting cleaved state by a covalent linkage between the active site
tyrosine and the phosphate at the 3' end of the broken strand (2). DNA
is relaxed by the supercoil-driven rotation of the DNA helix downstream
of the nick around one or more bonds in the intact strand. Since
rotation of the DNA appears to be guided by contacts between the
protein and the DNA, the process has been referred to as "controlled
rotation" (5). The DNA is religated by a similar nucleophilic attack
by the 5'-hydroxyl at the end of the broken strand. After religation,
the enzyme can either re-cleave the DNA or dissociate from the DNA.
Unlike the type IA subfamily, members of the type IB family can relax both positively and negatively supercoiled DNA in the absence of a
metal ion cofactor, although Mg2+ and Ca2+ have
been shown to stimulate the relaxation activity (6-8).
Addition of a protein denaturant such as SDS or NaOH to eukaryotic
topoisomerase I reactions traps the enzyme molecules at the nicked
intermediate stage (2). This technique has facilitated the mapping of
break sites on duplex DNA fragments (9). A large number of SDS-induced
break sites have been characterized that define a weak consensus
sequence for cleavage of 5'-(A or T)(G, C, or A)(A or T)T-3' in
which the enzyme is covalently attached to the 3'-most thymidine
nucleotide (defined as the
1 residue) (9-13). A strong break site
has been identified within a 30-bp repeated hexadecameric sequence
derived from the rDNA spacers of Tetrahymena thermophilus,
which has been shown to be efficiently cleaved by all eukaryotic type I
topoisomerases examined (14, 15).
Human topoisomerase I is composed of 765 residues with a molecular mass
of 91 kDa (16). Sequence comparisons of cellular type IB eukaryotic
topoisomerases and limited proteolysis studies combined with the
crystal structure of the enzyme (5, 8, 17, 18) have defined four major
domains as follows: an NH2-terminal domain
(Met1-Gly214), a core domain
(Ile215-Ala635), a linker domain
(Pro636-Lys712), and a COOH-terminal domain
(Gln713-Phe765). The NH2-terminal
domain is poorly conserved, highly charged and unstructured, and
dispensable for activity in vitro (8, 17). It contains four
putative nuclear localization signals and has been shown to interact
with nucleolin (19) and perhaps other nuclear proteins as well (20).
Topo701 is a truncated form
of human topoisomerase I with a molecular mass of 70 kDa that lacks
residues 1-174 of the NH2-terminal domain and retains full
enzyme activity in vitro (8, 21). The core domain is highly
conserved and more protease-resistant than the other domains. The
crystal structure of human topoisomerase I indicates that the core
domain can be further divided into three subdomains as follows:
subdomains I and II that fold together to form the cap structure that
covers one side of the DNA, and subdomain III that contains all of the
active site residues with the exception of Tyr723 and that
cradles the DNA on the side opposite the cap (18). The linker domain
forms a coiled-coil structure that protrudes from the body of the
enzyme and connects the core to the highly conserved COOH-terminal
domain (5). The active site tyrosine (Tyr723) is located in
the COOH-terminal domain close to the scissile phosphate in the bound
DNA (18). Complete enzymatic activity can be reconstituted by mixing a
core fragment with a COOH-terminal domain fragment (22), thus the
linker region is dispensable for activity in vitro.
Although the details of catalysis for the eukaryotic topoisomerase I
reaction still remain to be elucidated, based on the crystal structure
of the non-covalent human topoisomerase I-DNA complexes (5, 23, 24),
both Arg488 and Lys532 appear to hydrogen-bond
to one of the nonbridging oxygens of the scissile phosphate
(O-1P atom), whereas His632 is within hydrogen
bonding distance of the other nonbridging oxygen (O-2P atom).
Arg590 is intermediate between the two nonbridging oxygens
and, in addition, is very close to the nucleophilic tyrosine O-4.
Together, all of these residues likely constitute the active catalytic
center of the topoisomerase. Consistent with this view is the
observation that mutations leading to changes in the corresponding
amino acids in the structurally similar vaccinia topoisomerase at all
of these positions have adverse effects on the activity of the enzyme
(25-27). To define further the role of His632 in the
reaction catalyzed by human topoisomerase I and in particular to
explore the possibility that the side chain acts as a general acid-base
catalyst, we describe here the properties of a number of variants with
amino acid changes at this position.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
Single mutations were introduced
into the human topoisomerase I cDNA (16) using the uracil-DNA
method described by Kunkel et al. (28) or by using the
two-stage megaprimer PCR method (29). To make the H222Q, H367Q, and
H427Q mutations, the XhoI/NheI fragment of human
topoisomerase I was cloned in the pBluescript KS(
) vector and
transformed into E. coli CJ236
(dut-ung-) to obtain
the uracil-containing single-stranded DNA. After introducing the
mutations, the topo70 coding region (8) with or without the mutations
was cloned in the pGEX3 vector for the expression of the GST fusion
protein in E. coli. For the H632Q mutation, three
oligonucleotide primers were synthesized for use in the megaprimer PCR
method as follows: two flanking primers with restriction sites for
future cloning and a megaprimer with the single mutation. One of the
flanking primers and the megaprimer were used for the first stage PCR.
The first stage PCR product as well as the other flanking primer were
used for the second stage PCR. The second stage PCR product containing
the H632Q mutation was cloned in the pGEX-topo70 vector for GST fusion
protein expression and purification from E. coli.
A three-way ligation was used to generate the recombinant pFASTBAC1
plasmid (Life Technologies, Inc.) containing the topo70 coding region
with the H632Q mutation. The pFASTBAC1 vector was cut with
BamHI and EcoRI, and the large fragment was
combined with the HindIII-EcoRI fragment from
pGEX-topo70(H632Q) (above) containing the mutation and the
BamHI-HindIII fragment from pADH1Btopo70 (30).
After ligation and isolation of the desired pFASTBAC1 recombinant
plasmid (pFASTBAC1 topo70 H632Q), the Bac-to-Bac system (Life
Technologies, Inc.) was used to generate the recombinant baculovirus
expressing the topo70 H632Q protein according to the protocol provided
by the manufacturer. The PCR-based megaprimer method (29) was used to
generate PpuMI-NheI topoisomerase I-derived fragments containing the H632A, H632N, and H632W mutations. These fragments were used to replace the corresponding fragment in pFASTBAC1 topo70 H632Q to yield the additional mutant recombinant pFASTBAC1 derivatives. The generation of the recombinant baculovirus expressing WT topo70 was described previously (8). All mutations were confirmed by dideoxy sequencing of the region derived from the PCR fragment.
GST Fusion Protein Expression and Purification from E. coli--
E. coli cells containing a recombinant pGEX
plasmid (with the WT or mutant topo70 coding region) were cultured
overnight in LB medium containing 50 µg/ml ampicillin. The overnight
cultures were diluted into 10 volumes of LB with ampicillin and shaken vigorously (250 rpm) for 2 h at 37 °C until the
A600 reached ~0.5. Expression of the GST
topo70 fusion protein was induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside to 40 µg/ml. Following incubation for 2 h at 37 °C with shaking, the cells were harvested by centrifugation at 10,000 × g for 5 min and resuspended on ice in Sonication Buffer (500 mM
KCl, 10 mM Tris-HCl, pH 7.4, 1 mM DTT, 1 mM EDTA, 0.2% Triton X-100, 0.1 mM
phenylmethylsulfonyl fluoride). Samples were sonicated with 2-3 30-s
pulses on ice. Cell debris was removed by centrifugation at 20,000 × g for 30 min at 4 °C. Glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech) were added to the supernatant, and the
solution was rotated at room temperature for 1 h or at 4 °C
overnight. The beads were collected by centrifugation at 10,000 × g for 3 min and washed once with 10 mM Tris-HCl,
pH 7.4, 500 mM KCl, 1 mM DTT, 1 mM
EDTA, and twice with 10 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM DTT, 1 mM EDTA. The
GST fusion proteins were stored on the beads in the wash buffer at
4 °C until used in the relaxation assays.
Protein Expression and Purification from
Baculovirus-infected Insect Cells--
Recombinant bacmid DNAs
containing the coding region for the mutant forms of topo70 were
purified from E. coli DH10BAC cells and transfected into
insect SF9 cells to produce the recombinant baculovirus stocks. The
procedures for baculovirus infection and purification of WT topo70 have
been described previously (8). The purification of the mutant proteins
was similar except that the topo70 H632N and topo70 H632A proteins
eluted from the Mono S column at a slightly higher KPO4
concentration than WT topo70 (160 mM instead of 140 mM). The purified proteins were stored in 10 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM DTT and 50%
glycerol, and the purity was assessed by SDS-polyacrylamide gel
electrophoresis (Protogel from National Diagnostics).
Relaxation Assays--
Both a time course and a serial dilution
assay were used to determine the plasmid relaxation activity of WT and
mutant forms of topo70. The Reaction Buffer for both assays contained
10 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM EDTA, 1 mM DTT, and 50 µg/ml bovine serum
albumin. The substrate used for the relaxation assays was pBluescript
KSII(+) plasmid DNA (Stratagene) (final concentration 25 ng/µl). For
the time course assays, the reaction was carried out at an enzyme
concentration of 0.25 ng/µl in a final volume of 200 µl. At the
indicated times, 20-µl aliquots were removed, and the reaction was
stopped by the addition of 5 µl of Stop Buffer (2.5% SDS, 25 mM EDTA, 25% Ficoll 400, 0.08% bromphenol blue, 0.08%
xylene cyanol). For the serial dilution assay, the enzymes were 2-fold
serially diluted in Reaction Buffer, and the reactions were initiated
by the addition of 2 µl of the diluted enzyme to 18 µl of Reaction
Buffer containing DNA. The reactions were incubated at 37 °C for 30 min and stopped by the addition of 5 µl of Stop Buffer. The products
were analyzed by electrophoresis in a 0.8% agarose gel and visualized
with a UV illuminator after ethidium bromide staining.
Gel Shift Assay--
A 25-mer DNA oligonucleotide
(CL25, see Fig. 4) was 5' end-labeled by phosphorylation
with T4 polynucleotide kinase in the presence of
[
-32P]ATP (3000 Ci/mmol). To prepare the duplex
substrate, the labeled oligonucleotide was annealed to a 2-fold molar
excess of the complementary 25-mer (CP25, Fig. 4) by heating
to 94 °C for 1 min, followed by 10 min at 65 °C and then cooling
to 25 °C for another 10 min. The DNA binding assay was carried out
in 10 µl of Reaction Buffer containing the labeled duplex
oligonucleotide (0.5 nM) and 2-fold serial dilutions of the
topoisomerase yielding final concentrations ranging from 10 nM to 0.64 µM. The reactions were incubated
at room temperature for 15 min followed by the addition of 2.5 µl of
50% glycerol and analyzed by electrophoresis in a nondenaturing 6%
polyacrylamide gel at 4 °C. The running buffer contained 25 mM Tris-HCl and 162 mM glycine, pH 8.5. Due to
the high pI value of the topo70 protein (9.3), free protein and
protein-DNA complexes migrated to the cathode under these
electrophoresis conditions, and therefore only the unbound DNA entered
the gel. The amount of unbound oligonucleotide in the gel was
quantified by a PhosphorImager analysis using ImageQuant software
(Molecular Dynamics), and the dissociation constant
(Kd) was determined from the protein concentration
at which one-half of the total oligonucleotide was bound (31).
Suicide Cleavage Reactions--
A 14-mer DNA oligonucleotide
(CL14, Fig. 5) was 5' end-labeled as described above and
annealed with a 3-fold molar excess of the complementary CP25
oligonucleotide. The complementary strand was phosphorylated at the 5'
end to prevent self-religation. The two strands were annealed by
heating to 94 °C for 1 min followed by slow cooling to 25 °C for
2 h. The single-turnover suicide cleavage reactions were carried
out by incubating a 14-fold molar excess of the topoisomerase I protein
with the suicide cleavage substrate (20 nM) in Cleavage
Buffer (150 mM KCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM DTT) at 23 °C in a final
volume of 100 µl. A 10-µl sample of the reaction mixture prior to
the addition of protein was removed and used as the zero time point.
10-µl aliquots were removed at the indicated times and mixed with an equal volume of 1% SDS to stop the reaction. Samples were
ethanol-precipitated and resuspended in 10 µl of 1 mg/ml trypsin in
10 mM Tris-HCl, pH 7.4, 1 mM EDTA and digested
at 37 °C for 1 h to cleave all but a short peptide of the
enzyme from the covalent complex. The digested samples were analyzed by
electrophoresis through a 15% polyacrylamide-urea gel (SequaGel from
National Diagnostics). Uncleaved oligonucleotide migrates as 14-mer,
whereas the cleaved 12-mer oligonucleotide with the attached peptide
migrates slightly slower. The extent of covalent complex formation was
quantified using the PhosphorImager and ImageQuant software. The
percentage of cleaved oligonucleotide (Cl%) was determined based on
the end point cleavage values. The cleavage rate
(kcl) was determined by fitting the data from the
first three time points to the equation ln(100
Cl %) = 4.605
kclt (26, 32). The rate constant for religation (kr) was calculated
according to the following equation where KCR is the
equilibrium constant for the cleavage-religation reaction (see below),
kr = kcl/KCR.
Implicit in this calculation is the assumption that the rate of
cleavage with the suicide substrate is the same as the cleavage rate on
the completely duplex oligonucleotide. This assumption has been shown
to hold true for vaccinia topoisomerase (32).
To determine the effect of pH on the suicide cleavage rate, the
Cleavage Buffer was modified using the following buffers (50 mM) to the indicated pHs: NaMes, pH 6.0, 6.5, 7.2, and 7.4; Tris-HCl, pH 7.2, 7.6, 8.4, 8.9, and 9.4. The percent cleavage
was quantified as described above, and the logarithm of the cleavage
rate was plotted against the pH.
SDS-induced Cleavage Assay--
The 25-mer oligonucleotide CL25
was 5' end-labeled, gel-purified, and annealed to the complementary
25-mer strand (CP25) as described above. The SDS-induced cleavage
reactions were initiated by incubating a 14-fold molar excess of enzyme
(0.28 µM) with the DNA oligonucleotide (20 nM) in 10 µl of Reaction Buffer lacking bovine serum
albumin for the indicated times at 23 °C to allow the
cleavage-religation reaction to reach equilibrium. The cleaved covalent
complex was denatured by the addition of an equal volume of 1% SDS.
Samples were precipitated by ethanol and digested in 10 µl of the
trypsin solution described above for 1 h at 37 °C. Samples were
electrophoresed through a 15% polyacrylamide-urea gel. The primary
cleavage product, a 12-mer with a short attached peptide as well as a
small amount of a secondary cleavage product (10-mer with peptide),
were well resolved from the input 25-mer allowing the percent cleavage
(%Cl) to be determined using the PhosphorImager. The value of
KCR was calculated from the equation
KCR = %Cl/(l00
%Cl).
Cleavage Specificity Analysis--
The cleavage specificities of
the WT and mutant topo70 enzymes were analyzed as described previously
(22) except that the 136-bp NheI fragment from plasmid
pBendWest that contains the preferred site for topoisomerase I cleavage
was gel-purified prior to the 3' end-labeling step. The cleavage
reactions were initiated by mixing 2.8 pmol of the WT or mutant enzymes
with 20 fmol of labeled DNA in a 10-µl reaction containing 50 mM KCl, 1 mM EDTA, 1 mM DTT, 10 mM Tris-HCl, pH 7.4, and, where indicated, 50 µM camptothecin (CPT). After incubation for 1 h at
23 °C, 2 µl of 5% SDS was added to induce topoisomerase
I-mediated DNA breakage. The resulting breakage products were
ethanol-precipitated and, after electrophoresis in a 10%
polyacrylamide-urea gel, analyzed using the PhosphorImager.
Structure Analysis and Modeling Studies--
Bond distances and
the effects of replacing amino acids on protein structure were
determined using the Swiss-PDB Viewer (version 3.51) software (Glaxo
Wellcome Experimental Research) (33).
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RESULTS |
Site-directed Mutagenesis of Conserved Histidine Residues in Human
Topoisomerase I--
Depending on whether it is protonated or not, a
histidine side chain is an ideal candidate to act either as general
acid or a general base during the cleavage and religation
transesterification reactions catalyzed by human topoisomerase I (34).
The solution of the crystal structure of human topoisomerase I provided
support for this view as the highly conserved His632 was
found to be relatively close to the 5'-oxygen of the leaving deoxyribose sugar (5, 18). To test this possibility directly, we used
site-directed mutagenesis to change His632 to glutamine;
like histidine, glutamine can participate in hydrogen bonding, but
unlike histidine it is unable to act as a general acid-base catalyst.
As controls, we independently changed each of the other three invariant
histidines in topoisomerase I proteins (His222,
His367, His406) to glutamine as well.
The histidine to glutamine mutations were introduced into the topo70
form of human topoisomerase I, and the mutant proteins were expressed
in E. coli as GST fusions. Although human topoisomerase I is
unstable when expressed in E. coli, we found previously that sufficient protein is present in crude extracts to permit reliable enzyme assays (35). For these experiments, we purified the four GST
fusion proteins using glutathione-Sepharose 4B beads and carried out
plasmid relaxation assays using equal amounts of protein as determined
by SDS-polyacrylamide gel electrophoresis. The effects of the mutations
on enzyme activity were analyzed by a standard serial dilution DNA
relaxation assay, and the results are shown in Fig.
1. Only a trace amount of enzyme activity
was detected for the H632Q mutant protein when the undiluted sample was
used in the assay (Fig. 1, lane 28). However, the activities
of the H222Q, H367Q, and H406Q topo70 proteins were nearly
indistinguishable from that of WT topo70, demonstrating that the change
of a highly conserved histidine residue did not invariably abolish the
activity of the enzyme. Apparently, these three amino acids are
conserved for reasons having nothing to do with the catalytic activity
of the protein. Overall, these results confirm the essential nature of
His632 in the topoisomerase I reaction as predicted by the
crystal structure (5, 18).

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Fig. 1.
Effects of mutating conserved histidine
residues on the plasmid relaxation activity of human topoisomerase
I. Equal quantities of WT topo70 (WT, lanes
2-9), H222Q (lanes 10-15), H367Q (lanes
16-21), and H406Q (lanes 22-27) mutant proteins (~5
ng) were 2-fold serially diluted and incubated with 0.5 µg of
supercoiled plasmid DNA in Relaxation Buffer for 30 min at 37 °C. A
control reaction without added enzyme is shown in lane 1.
Since little activity was detected for the H632Q protein even with the
undiluted sample, only the assay for the highest protein concentration
tested is shown (lane 28). The products of the relaxation
reactions were visualized by ethidium bromide staining after agarose
gel electrophoresis.
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Effects of H632N, H632A, and H632W Mutations on Relaxation
Activity--
We extended our analysis of the role of
His632 by replacing this residue with asparagine, alanine,
or tryptophan. The WT and four mutant topo70 proteins (H632Q, H632N,
H632A, and H632W) were expressed from recombinant baculoviruses and
purified from infected Sf9 insect cells. All of the proteins
were stable in insect cells and were purified to near homogeneity (Fig.
2).

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Fig. 2.
SDS-polyacrylamide gel electrophoresis of
purified WT and mutant proteins with changes at residue 632. The topo70 forms of the indicated proteins were expressed using
the baculovirus expression system and purified as described under
"Experimental Procedures." The purified proteins (~4 µg each)
were analyzed by electrophoresis in a 10% SDS-polyacrylamide gel. The
markers in the leftmost lane had molecular masses of 26.6, 36.5, 48.5, 58, 84, and 116 kDa, respectively.
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The effects of the mutations on enzyme activity were analyzed by
standard plasmid DNA relaxation time course assays with a molar ratio
of DNA to enzyme of 4:1 (Fig. 3,
panel A). WT topo70 completely relaxed the DNA within 2 min
under these conditions, whereas all four mutant enzymes exhibited
slowed relaxation kinetics. Complete relaxation by the H632Q enzyme was
not observed until ~40 min, and thus the H632Q protein appeared to be
~20-fold less active than the WT enzyme. H632N had less activity than
H632Q, failing to relax completely the supercoiled DNA even after 40 min. Very little relaxing activity was detectable for the H632A and
H632W proteins. We estimate the activity of the H632A and H632W
proteins to be more than 1000-fold reduced compared with WT topo70.

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Fig. 3.
Plasmid relaxation assays for WT topo70 and
mutant topoisomerases I. Reaction mixtures containing 0.5 µg of
supercoiled plasmid DNA in Relaxation Buffer without (panel
A) and with 10 mM Mg2+ (panel
B) were incubated at 37 °C. The reactions were initiated by the
addition of enzyme. At the indicated times, aliquots (20 µl) were
withdrawn and mixed immediately with Stop Buffer. The zero time point
was taken prior to the addition of enzyme. Reaction products were
analyzed by agarose gel electrophoresis.
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Supercoiled DNA relaxation under conditions of limiting topoisomerase I
is stimulated ~10-fold in the presence of 10 mM
Mg2+, likely as a result of an increase in the dissociation
rate of the enzyme from the DNA (8, 32). It follows that the
rate-limiting step for DNA relaxation by the WT topoisomerase I under
the normal assay conditions is enzyme dissociation (32). This effect
can be seen in Fig. 3 (panel B), where the addition of 10 mM Mg2+ to the WT topo70 reaction increased the
rate to a value too fast to measure (reaction complete in <5 s).
However, the presence of Mg2+ in the reactions for the
His632 mutant proteins had no effect on the relaxation
rates (Fig. 3, panel B), suggesting that enzyme chemistry
rather than enzyme dissociation was the rate-limiting step for all of
the mutant enzymes. Moreover, in the presence of 10 mM
Mg2+, the differences between the estimated activity for WT
topo70 and the activities of the mutant proteins were magnified. H632Q was at least 100-fold less active than WT topo70, whereas H632N was
more than 200-fold less active than the WT enzyme.
DNA Binding as Measured by Gel Shift Assay--
To test whether
the observed reduction in relaxing activity for the mutant proteins
resulted from a reduced affinity of the enzymes for DNA, a native gel
mobility shift assay was used to compare the DNA binding properties of
the mutant proteins with WT topo70. Because topo70 has a pI value of
9.3, the enzyme is positively charged under standard electrophoresis
conditions (pH 8.5) and fails to enter the gel. In addition, binding of
a negatively charged duplex DNA oligonucleotide (CL25:CP25) (Fig.
4, panel A) only
partially neutralizes the charge on the protein and thus the
protein-DNA complexes also remain in the well of the gel. Consequently,
in this gel shift assay only the unbound duplex oligonucleotide enters
the gel. From the reduction in the amount of free oligonucleotide in
the gel with increasing concentrations of protein, it was possible to
compare the substrate binding properties of the various forms of the
protein (Fig. 4, panels B and C). Under these
conditions, the protein concentration at which the amount of unbound
oligonucleotides was reduced by a factor of 2 is equal to the
Kd (31). The binding profiles revealed that the
affinity of the mutant proteins for DNA substrate was about the same as
that of the WT enzyme with Kd values of ~50
nM (Fig. 4, panel C). These results
demonstrated that the reduction in relaxing activity for the mutant
proteins did not result from a defect in DNA binding.

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Fig. 4.
Analysis of DNA binding by nondenaturing gel
electrophoresis. Reaction mixtures containing the indicated 5'
end-labeled duplex 25-mer DNA (CL25:CP25) (panel A, large
arrow shows major cleavage site; small arrow indicates
minor cleavage site) and either WT topo70 or mutant topoisomerase I
proteins were analyzed by nondenaturing gel electrophoresis. The amount
of unbound oligonucleotide remaining in the gel with increasing protein
concentration is shown in panel B for the WT, H632Q, and
H632A topo70 proteins. For each enzyme, the leftmost lane
shows the total unbound oligonucleotide in the absence of added
protein. The free oligonucleotide was quantified and plotted as a
function of protein concentration for all five proteins (panel
C).
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Suicide Cleavage Assay--
A suicide oligonucleotide substrate
containing the topoisomerase I cleavage sequence ACTT was used to
examine the effect of the mutations on cleavage under single-turnover
conditions. The 5' end-labeled 14-mer scissile strand (CL14) was
annealed to the CP25 complementary strand to produce a 14-bp duplex
with an 11-base 5' single-stranded extension (Fig.
5). Upon cleavage and formation of the
covalent complex, the dinucleotide AG at the 3' end of the scissile
strand is released, preventing religation. Treatment of the cleavage
products with trypsin leaves only a small topoisomerase I-derived
peptide covalently attached to the 3' end of the 12-mer cleavage
product. The resulting complex can be resolved from the residual
uncleaved oligonucleotide by electrophoresis in a polyacrylamide-urea gel. WT topo70 or the mutant proteins were incubated with the suicide
substrate, and the amount of cleaved product (after normalization to
the plateau value for the WT enzyme) was plotted against the time of
incubation (Fig. 5). Suicide cleavage by the WT enzyme at the earliest
time point (5 s) reached more than 20% of the final cleavage value and
was complete by ~5 min. Based on the results from three independent
analyses, we estimated the cleavage rate (kcl) for
WT topo70 to be 0.036 s
1, which is only a
factor of 2 slower that the value determined for the vaccinia
topoisomerase (32). The cleavage rates for the H632Q and H632N mutant
enzymes were approximately 2 orders of magnitude slower than that of
the WT enzyme, with kcl values of 3.8 × 10
4 and 2.0 × 10
4 s
1,
respectively (Fig. 5 and Table I). The
cleavage rates for the H632A and H632W proteins were just detectable
above the background with estimated kcl values of
6 × 10
6 and 2.3 × 10
6 s
1,
respectively (Fig. 5 and Table I). Thus, the effects of the various
changes at position 632 on the cleavage rates quantitatively parallel
the reductions in the rates of relaxation described above.

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Fig. 5.
Suicide cleavage assays. WT topo70 and
the mutant topo70 enzymes were incubated with the partially duplex
suicide substrate (CL14:CP25) shown at the top of the figure
(recognition sequence shown in bold; arrow marks cleavage
site), and the reactions were stopped with SDS at a series of time
points. After trypsin treatment, the amount of suicide cleavage was
quantified by electrophoresis in a urea-polyacrylamide gel. The results
were normalized, and the percent cleavage values were plotted for each
of the enzymes.
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Cleavage-Religation Equilibrium--
Since the rates of religation
were too fast to obtain reliable measurements, we instead measured the
equilibrium cleavage value (KCR) for the WT and the
H632Q and H632N mutant topo70 proteins, and we combined these values
with the cleavage rates to estimate the rates of religation. We
measured KCR using SDS-induced cleavage of the
duplex oligonucleotide shown in Fig. 4, panel A, under
conditions of excess enzyme and at a substrate concentration such that
all of the oligonucleotide should be enzyme-bound (22). To ensure that
equilibrium had been reached, two different time points (1 and 2 h) were assayed in each case. The cleavage percentage (%Cl) of the
labeled scissile strand was determined by urea-polyacrylamide gel
electrophoresis (Fig. 6) followed by
quantitation using the PhosphorImager, and KCR was
calculated as described under "Experimental Procedures" (Table I).
The KCR for the H632N mutant was only slightly
reduced relative to WT topo70, but a distinct shift toward religation was apparent for the H632Q mutant protein. The magnitudes of the reductions in the KCR values for both mutant
proteins as compared with the WT enzyme are less than what one would
expect if the only effect of the mutations was to reduce the rates of cleavage. Assuming that the cleavage rates determined with the partially single-stranded suicide substrate (kcl)
reflect the true cleavage rates on the completely duplex
oligonucleotide used for the measurements of KCR,
the rates of religation (kr) can be calculated using
the equation KCR = kcl/kr. Although this assumption
has not been verified for the human enzyme, it has been shown to hold
true for the vaccinia topoisomerase (32). The calculated values of kr for WT topo70 and the H632N and H632Q mutant
proteins are included in Table I. The extents of cleavage and the
cleavage rates for the H632A and H632W proteins were substantially
reduced below those of the other mutants, precluding a reliable measure of the KCR values (data not shown).

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Fig. 6.
SDS-induced cleavage of duplex
oligonucleotide. The duplex substrate shown in Fig. 4, panel
A, containing 5' end-labeled CL25 was incubated with WT topo70 and
mutant proteins for either 1 or 2 h, and the reactions were
stopped by the addition of SDS. After treatment with trypsin, the
cleavage products containing a covalently bound peptide were analyzed
by electrophoresis in a urea-polyacrylamide gel. The slowest migrating
band corresponds to the uncleaved oligonucleotide (indicated at
right). The positions of the major and minor cleavage
products (see Fig. 4) are indicated by the long and
short arrows, respectively.
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DNA Cleavage Specificity--
The reduced efficiency of the
SDS-induced DNA breakage by the mutant proteins could, in principle, be
due to the sequence of the particular duplex oligonucleotide substrate
used in the assays. For example, WT topo70 and the mutant proteins may
have different preferences for nucleotide sequence at the sites where they bind and cleave the DNA. To address this possibility, we examined
the pattern of SDS-induced DNA breakage by both WT topo70 and the
mutant enzymes using a 3' end-labeled 136-bp DNA that contains multiple
topoisomerase I cleavage sites, including the strong cleavage site from
the rDNA of T. thermophilus (14, 22). The reduction in
cleavage for the mutant proteins relative to WT topo70 followed the
same order as described above for the magnitude in the shift in the
KCR (WT > H632N > H632Q > H632A
H632W) (Fig. 7,
CPT). Although the cleavage pattern for the H632N mutant
was very similar to that of the WT enzyme, there was insufficient cleavage with the other mutant proteins to make the comparison.

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Fig. 7.
Cleavage specificities of WT and mutant
topo70 proteins. A 3' end-labeled 136-bp DNA fragment was
incubated with the indicated proteins in the absence ( CPT)
or presence of 50 µM camptothecin (+CPT), and
the reactions were stopped with SDS. The products were analyzed by
electrophoresis in a urea-polyacrylamide gel. The positions of the
uncleaved 136-bp DNA (uncleaved DNA) and the 94-bp fragment produced by
BamHI digestion are indicated with arrows on the
right and left sides of the gel, respectively.
The most intense band in the WT topo70 (WT) lanes
corresponds to cleavage at the strong T. thermophilus rDNA
site that produces a fragment 107 bp in length (indicated by
arrow at left).
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CPT is a topoisomerase I poison that enhances SDS-induced cleavage by
slowing the religation phase of the reaction (36-38). By including CPT
in the reactions, DNA cleavage was increased to a level that permitted
a determination of the cleavage specificity of the other mutant
proteins in relation to that of WT topo70. Since CPT alters the
specificity of the WT enzyme somewhat (39, 40), it was important to
determine the cleavage patterns of both the WT and mutant topo70
enzymes in the presence of the drug. In the presence of CPT, the
cleavage patterns for the H632A and H632Q enzymes were again very
similar to the WT topo70 pattern (Fig. 7, +CPT). Although
only minimal cleavage was detected for H632W, the observed cleavage
products also co-migrated with the WT topo70 cleavage products. These
results indicate that the reduced cleavage observed for the mutant
proteins was not due to an alteration in sequence specificity.
Effect of pH on the Cleavage Rate--
Since the crystal structure
of human topoisomerase I shows that His632 is proximal to
the 5'-bridging oxygen of the scissile phosphate, we suggested
previously that the histidine side chain might possibly serve as a
general acid to donate a proton to the leaving 5'-hydroxyl as cleavage
occurs (5). If His632 were to act as a general acid,
deprotonation of the imidazole ring with increased pH should reduce the
rate of the cleavage reaction for WT topo70, but a similar increase in
pH should have no effect on cleavage by the H632Q mutant enzyme. To
test this prediction, we measured the cleavage rates of both the WT and the H632Q mutant topo70 proteins at the following pH values: 6.0, 6.5, 7.2, 7.4, 7.6, 8.4, 8.9, and 9.4. As shown in Fig.
8, the activity of WT topo70 decreases
slightly over the pH range from 7.6 to 9.4, but the response of the
H632Q mutant enzyme was very similar. Thus, it appears unlikely that
His632 acts as a general acid to donate a proton to the
leaving 5'-oxygen.

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Fig. 8.
Effect of pH on suicide cleavage rate for WT
and H632Q topo70 proteins. The rate of suicide cleavage was
measured as described above for Fig. 5, and the logarithm (base 10) of
the rate was plotted against the pH.
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DISCUSSION |
Given the structural similarity of vaccinia topoisomerase to core
subdomains I and III of human topoisomerase I (24), it is of interest
to compare the properties of human topoisomerase I mutant proteins with
changes at position 632 with vaccinia variants containing mutations at
the corresponding His265 residue (26). Unlike with the
human enzyme, changing His265 in the vaccinia enzyme to
either glutamine or asparagine resulted in only a slight reduction in
enzyme activity (approximately 3-fold). Similar to the H632A mutation,
a H265A mutation in the vaccinia enzyme had a major effect on enzyme
activity, but the magnitude of the reduction for vaccinia topoisomerase
was only 100-fold compared with the almost 4 orders of magnitude
observed here for the human enzyme. Although this particular histidine
residue is clearly essential for the transesterification reaction
catalyzed by the two enzymes, changes at this position appear to
perturb the corresponding active sites somewhat differently.
In all of the available crystal structures of human topoisomerase I in
noncovalent complexes with DNA (5, 18, 23), the N-
2 atom of
His632 is positioned within hydrogen-bonding distance of
the nonbridging O-2P atom of the scissile phosphate. It was
hypothesized previously (5) that this interaction contributes to
catalysis by stabilization of the pentavalent transition state
intermediate. The 6000-fold reduction in kcl on
changing His632 to alanine is consistent with this
hypothesis and represents strong support for an essential role of
His632 in the cleavage reaction.
Although the crystal structure of human topoisomerase I appears to rule
out a role for His632 as a general base in the activation
of the nucleophilic tyrosine (5, 18), the proximity of this histidine
to the 5'-oxygen of the leaving sugar (~3.9 Å) suggests that in
addition to stabilizing the transition state through hydrogen bonding
to the nonbridging oxygen, this same residue might also act as a
general acid catalyst and protonate the leaving oxygen on the sugar
(5). Substituting a glutamine for His632 provides one test
of this hypothesis. When glutamine is modeled in place of
His632, all of the allowable rotamers are within
hydrogen-bonding distance of the scissile phosphate nonbridging O-2P
atom (~3.0 Å compared with 2.8 Å for histidine), and therefore some
transition state stabilization by the H632Q mutant enzyme would still
be expected. However, unlike histidine, a glutamine side chain is
unable to act as a general acid. The ~100-fold reduction in the rates
of relaxation and suicide cleavage for the H632Q mutant enzyme could be
explained by the loss of general acid catalysis while retaining some
interaction with the nonbridging phosphate oxygen. A second test of the
possible general acid character of His632 is to compare the
pH dependence of catalysis by WT topo70 and the H632Q mutant topo70.
For His632 to donate a proton to the leaving 5'-oxygen, the
amino acid side chain must be protonated. Since the normal
pKa of histidine side chains in proteins ranges from
5 to 8 (41), at pH values >8 it would be predicted that the rate of
cleavage by the WT enzyme should be substantially reduced and that the
rate for the H632Q mutant would not exhibit a similar pH dependence.
However, the pH profile of the activity of the WT enzyme parallels that
of the H632Q mutant enzyme with only minimal loss of activity at the
higher pH values. An unlikely explanation that we cannot completely rule out is that the minimal effect of increasing pH on the WT reaction
rate results from an activation of the tyrosine nucleophile by
production of a phenolate anion that is just offset by loss of the
general acid character of His632 by titration of the
histidine. Overall, it seems most likely that the active site
His632 is not acting as a general acid in the
transesterification cleavage reaction and that its major role is to
stabilize the pentavalent transition state through an interaction with
the nonbridging oxygen of the scissile phosphate. Based on the pH
profile of the enzyme (26) and a variety of kinetic analyses (42, 43),
a similar conclusion has been reached for the vaccinia topoisomerase.
In a recent report, it was suggested that Lys167 in
vaccinia topoisomerase acts as a general acid to protonate the leaving
5'-oxygen during the cleavage reaction (44). This suggestion is
plausible given the relative pKa values for the two
groups, but in the absence of a crystal structure of the vaccinia
enzyme with bound DNA, the exact role of this amino acid in catalysis
by the vaccinia enzyme remains uncertain. The corresponding amino acid in human topoisomerase I (Lys532) is hydrogen-bonded to the
nonbridging O-1P atom of the scissile phosphate as well as to the O-2
atom of the pyrimidine base on the
1 nucleotide (23).
Lys532 in the human enzyme is ~4 Å away from the leaving
5'-oxygen, a distance that is consistent with a role as a general acid
during the cleavage reaction. Further experimentation is required to define fully the role of Lys532 in the catalytic mechanism
of human topoisomerase I.
As mentioned above, modeling studies indicate that glutamine in place
of histidine at position 632 is still within hydrogen-bonding distance
of the scissile phosphate O-2P atom, but a similar analysis shows that
asparagine at position 632 is >4.4 Å away from either nonbridging
oxygen and therefore apparently not positioned properly to participate
in transition state stabilization. Thus it is surprising that the
kcl values for both H632Q and H632N are reduced to
approximately the same extent relative to WT topo70 (~100-fold). One
explanation is that despite the predicted close proximity of the
glutamine replacement, the detailed geometry is not ideal for
stabilization of the transition state. Alternatively, the asparagine
side chain in place of His632 may contribute to catalysis
in ways other than through a direct interaction with the scissile phosphate.
Our original interest in testing the effects of the H632W mutation was
that Trp315 of the structurally homologous Cre recombinase
(45) superimposes on His632 of the human enzyme (24). Like
histidine and glutamine, tryptophan can, in principle, participate in
catalysis through hydrogen-bonding interactions with the scissile
phosphate, and this interaction probably explains its active site role
for the Cre recombinase. However, we find that replacing
His632 in human topoisomerase I with tryptophan reduces
both relaxation and suicide cleavage activities to nearly undetectable
levels. The most likely explanation for this observation is that within the context of the topoisomerase I active site, a tryptophan residue at
position 632 is not only nonfunctional as a hydrogen bond donor but
also disrupts the overall architecture of the active site such that
virtually all activity is lost. Consistent with this suggestion is the
finding that when a tryptophan residue is modeled into human
topoisomerase I at position 632, the amino acid side chain of the only
rotamer that is within hydrogen-bonding distance of the scissile
phosphate sterically clashes with Ser719.
Although it seems likely that the catalytic chemistry of religation is
simply the reverse of cleavage (32), it remains possible that the
relative contributions by groups on the enzyme to transition state
stabilization may be slightly different in the two cases (46). If
His632 is identically hydrogen-bonded to the O-2P atom of
the scissile phosphate in the transition state for both the cleavage
and religation reactions, then it would be expected that the glutamine
and asparagine mutations at this position would have similar effects on
the rates of the two reactions. Within the observed experimental error, the results presented in this study are compatible with this
possibility (Table I). However, the observation that His632
is ordered in the crystal structure containing noncovalently bound DNA,
but is disordered and not visible in the covalent complex (18),
suggests that the detailed architecture of the active site may be
different in the two cases. An understanding of the events that occur
on the pathway of religation, including conformational changes in the
enzyme, will be required to decide between these possibilities.