From the Department of Biochemistry and Molecular Biology, New York
Medical College, Valhalla, New York 10595
To catalyze relaxation of supercoiled DNA, DNA
topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic
attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place
to change the linking number. This is followed by DNA religation during
which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for
the relaxation activity of type IA and type II DNA topoisomerases. A
number of conserved amino acids with acidic and basic side chains are
present near Tyr-319 in the active site of the crystal structure of the
67-kDa N-terminal fragment of Escherichia coli DNA
topoisomerase I. Their roles in enzyme catalysis were investigated by
site-directed mutation to alanine. Mutation of Arg-136 abolished all
the enzyme relaxation activity even though DNA cleavage activity was
retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the
mutants failed to complement chromosomal topA mutation in
E. coli AS17 at 42 °C, possibly accounting for the
conservation of these residues in evolution.
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INTRODUCTION |
DNA topoisomerases (for review, see Refs. 1-7) catalyze the
interconversion of different DNA topological isomers by first forming a
covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine
hydroxyl on the DNA phosphodiester linkage. After strand passage
through the break, religation involving nucleophilic attack of the
displaced DNA hydroxyl group on the phosphotyrosine linkage takes
place. Type IA and type II DNA topoisomerases are linked to the
5'-phosphoryl end of the cleaved DNA while type IB DNA topoisomerases
are linked to the 3'-phosphoryl end. Mg(II) is required for the
relaxation activities of both type IA and type II DNA topoisomerases
but not for the type IB enzymes. The detailed catalytic mechanism of
DNA cleavage and religation by topoisomerases remains to be elucidated.
The mechanism of the type IA and type II topoisomerase may share
similarities with other enzymes that also require Mg(II) for
nucleotidyl transfer activity.
Tyr-319 of Escherichia coli DNA topoisomerase I is the
catalytic residue that provides the hydroxyl group for forming the covalent intermediate with DNA. The three-dimensional structure of the
67-kDa N-terminal domain of this enzyme has been determined by x-ray
crystallography (8). In this structure, Tyr-319 is present in the
interface between domains I and III. It has been pointed out (8) that
the spatial arrangement of the three acidic residues Asp-111, Asp-113,
and Glu-115 in the active site region is similar to the acidic residues
that coordinate two divalent cations in the exonuclease catalytic site
of Klenow fragment (9). However, the structure observed has to undergo
additional conformational changes before there is sufficient space in
the active site region for DNA and possibly Mg(II) to bind. A number of
residues found in the active site, including Glu-9, Asp-111, Asp-113,
and Glu-115, and Arg-321, are strictly conserved among type IA DNA
topoisomerase sequences (Fig. 1). This
evolutionary conservation extends from the archaeal
(Methanococcus jannaschii) topoisomerase I to human topoisomerase III. There is a lower degree of conservation for Arg-136
which is also near the active site tyrosine. The acidic and basic
functional groups on these residues may be important for the enzyme
catalytic function. For DNA polymerases, two divalent metal ions
coordinated by carboxylates are essential for the enzyme activity
(10-14). One or two metal ion mechanisms utilizing carboxylates for
coordination have been proposed for the EcoRI and
EcoRV restriction endonucleases (15, 16). Certain bacterial
transposases and retroviral integrases have a conserved DDE motif that
has been proposed to be involved directly in catalysis (17-19). In a
mechanism proposed for the yeast site-specific recombinases Flp and R,
invariant arginines either facilitate the phosphoryl transfer by
stabilizing the leaving group or the partial charge on the phosphorus
in the transition state during the phosphodiester cleavage or exchange reactions (20). These recombinases do not require divalent ions for
their activity. Their mechanisms may also share some similarities with
topoisomerases. It should be noted that Mg(II) is not absolutely required for the DNA cleavage activity of E. coli DNA
topoisomerase I.

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Fig. 1.
Alignment of type IA topoisomerase sequences
to demonstrate the conservation of amino acid residues
(highlighted) mutated in this study. ecI,
Escherichia coli topoisomerase I (33); hiI,
Hemophilus influenza topoisomerase I (34); bsI,
Bacillus subtilis topoisomerase I (Swiss Protein accession
number P39814); mtI, Mycobacterium tuberculosis
topoisomerase I (35); tmI, Thermotoga maritima
topoisomerase I (36); mgI, Mycoplasma genitalium
topoisomerase I (37); mpI, Mycoplasma pneumoniae
topoisomerase I (38); mjI, Methanococcus
jannaschii topoisomerase I (39); ecIII,
Escherichia coli topoisomerase III (40); hiIII,
Hemophilus influenza topoisomerase III (41);
ScIII, Saccharomyces cerevisiae topoisomerase III
(42); HsIII, human topoisomerase III (43); saR,
Sulfolobus acidocaldarius reverse gyrase (44);
mkR, Methanopyrus kandleri reverse gyrase (45);
mjR, Methanococcus jannaschii reverse gyrase
(39).
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To investigate the roles of the carboxylates and arginine residues in
the active site region of E. coli topoisomerase I, they were
altered by site-directed mutagenesis to alanines, abolishing the acidic
or basic functional groups. The mutant enzymes were expressed and
purified. Different enzymatic assays were carried out to determine how
the mutation affected the interaction of the topoisomerase enzyme with
DNA and/or Mg(II).
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EXPERIMENTAL PROCEDURES |
Materials--
All chemical reagents used were ultrapure or
Baker analyzed reagent grade. Solutions were prepared with water first
deionized with the Barnstead Nanopure system and then passed over a
Bio-Rad Chelex 100 resin (100-200 mesh sodium form) to remove any
remaining contaminating metal ions. Tubes, spectrophotometric cells,
and glassware for metal ion-sensitive experiments were first washed with 10 mM EDTA and then rinsed extensively with metal-free
water before use. Plasmid DNA was purified by cesium chloride
centrifugation.
Mutagenesis--
Plasmid pJW312, with the topA coding
region under the control of the lac promoter (21), was used
as the template for mutagenesis. The D111A, R136A, and R321A mutants
were constructed with the Chameleon site-directed mutagenesis kit from
Stratagene while the E9A, D113A, and E115A mutants were constructed
with the QuikChange site-directed mutagenesis kit, also from
Stratagene. The sequence of the oligonucleotides for the
mutations at the underlined positions were:
5'-CTTGTCATCGTTGCGTCCCCGGCAAA-3' (E9A),
5'-CTATCTCGCAACCGCCCTTGACCGCGAAGG-3' (D111A),
5'-CGCAACCGACCTTGCCCGCGAAGGGGAAGC-3' (D113A),
5'-CGACCTTGACCGCGCAGGGGAAGCCATTGC-3' (E115A),
5'-GCGCGCTATAGCGCAGTGGTGTTTAAC-3' (R136A), and
5'-TATATCACTTACATGGCTACCGACTCCAC-3' (R321A). The
mutants were identified by DNA sequencing of the plasmid DNA.
Enzyme Expression and Purification--
Wild-type E. coli DNA topoisomerase I enzyme was expressed from E. coli MV1190 cells transformed with plasmid pJW312. The R136A and
R321A mutants were expressed in E. coli AS17
(topAamPLL1(TcrsupDts),
from R. E. Depew, Northeastern Ohio University). The E9A, D111A, D113A, and E115A mutants were expressed in E. coli
GP200 (gyrA(Nalr) gyrB225
(topAcysB)204) (22). Enzyme purification was carried out
using the previously described procedures (23) with an additional hydroxyapatite chromatography step between the phosphocellulose and
single-stranded DNA-agarose columns.
Relaxation Activity Assay--
Wild-type and mutant enzymes were
serially diluted and assayed for relaxation activity in 20 µl with
0.5 µg of negatively supercoiled plasmid DNA, 10 mM
Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/ml gelatin, and 6 mM MgCl2 unless indicated otherwise. Incubation was at 37 °C for 30 min. The reactions were stopped by the addition of 5 µl of 50% glycerol, 50 mM EDTA, and 0.5% (v/v)
bromphenol blue. After electrophoresis in a 0.7% agarose gel with TAE
buffer (40 mM Tris acetate, pH 8.1, 2 mM EDTA),
the DNA was stained with ethidium bromide and photographed over UV
light.
Cleavage of 5'-End-labeled Single-stranded DNA--
Plasmid
pT7-1 DNA (from U. S. Biochemical Corp.) was cleaved with
EcoRI and then labeled at the 5' end with
[
-32P]ATP and T4 polynucleotide kinase. The labeled
DNA was denatured to single strand and incubated with the wild-type and
mutant enzymes, and then sodium hydroxide was added to trap the
covalent complex and cleavage of the DNA (24). After electrophoresis in
a 6% DNA sequencing gel, the 5'-end-labeled DNA cleavage products were visualized by autoradiography.
Covalent Complex with Oligonucleotide Substrate--
The
single-stranded oligonucleotide 5'-CAATGCGCT-3', containing the
sequence of a strong cleavage site (24), was labeled at the 3' end with
[
-32P]dATP and terminal deoxynucleotidyl transferase.
After incubation of 0.15 µg of the labeled oligonucleotide with 0.4 µg of the enzyme in 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 20 mM potassium phosphate at 37 °C
for 5 min, the reaction was stopped with the addition of 1% SDS. The
enzyme was separated from the non-covalently bound oligonucleotides by
electrophoresis in a 10% SDS-polyacrylamide gel. The labeled covalent
complex was visualized by autoradiography of the dried gel.
Gel Mobility Shift Assay--
The single-stranded 36-mer
5'-TAACCCTGAAAGATTATGCAATGCGCTTTGGGCAAA-3' sequence (24) that has the
same strong cleavage site as the 9-mer used in the covalent complex
formation was labeled at the 5' end with T4 polynucleotide kinase and
[
-32P]ATP. The reaction mixture (10 µl) contained 1 pmol of labeled oligo, 20 mM Tris-HCl, pH 8, 100 µg/ml
bovine serum albumin, 12% glycerol, and 0.5 mM EDTA. After
incubation at 37 °C for 5 min, the reaction mixtures were applied to
a 6% polyacrylamide gel (19:1) with 0.5 × Tris borate-EDTA
buffer. Electrophoresis was at 2 V/cm for 1.5 h. The gel was then
dried and visualized by autoradiography. Quantitation was carried out
using the Molecular Dynamics PhosphorImager.
Intrinsic Tryptophan Fluorescence Measurements--
Fluorescence
measurements were performed with the Perkin-Elmer LS-5B spectrometer
with excitation at either 280 or 295 nm at either 42 °C or room
temperature (~25 °C). The spectral bandwidths were 3 and 10 nm,
respectively, for excitation and emission. The wild-type or mutant
topoisomerase I was present at 0.1 mg/ml in 20 mM potassium
phosphate, pH 7.5, 0.1 M KCl, 0.2 mM
dithiothreitol. All the measurements were corrected for the spectrum of
the buffer used.
Equilibrium Dialysis to Determine Mg(II) Binding
Stoichiometry--
1 ml of topoisomerase I (0.4 mg/ml) was dialyzed
against 400 ml of buffer (20 mM potassium phosphate, pH
7.5, 0.1 M KCl, 0.2 mM dithiothreitol, and 400 µM MgCl2) at room temperature for 7 h.
The enzyme and dialysis buffer samples were submitted to Quantitative Technologies, Inc., NJ, for Mg(II) content analysis using the inductively coupled plasma method.
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RESULTS |
Expression and Purification of the Topoisomerase I
Mutants--
After site-directed mutagenesis of the plasmid pJW312 to
produce the desired alanine substitutions, expression of the mutant proteins in E. coli strain AS17 was examined by SDS-gel
electrophoresis of the total soluble extract followed by Coomassie Blue
staining of the gel. The expression level of the Arg-136 mutant was
comparable with that of the wild-type topoisomerase I while expression
of the Arg-321 mutant was detectable but lower than the wild type. Bands corresponding to the Glu-9, Asp-111, Asp-113, and Glu-115 mutants
could not be identified in AS17 extracts (data not shown). Expression
of these three mutant enzymes were then carried out in E. coli strain GP200. Detectable levels of expression of these mutant
enzymes allowed the purification of the mutant enzymes to homogeneity
(Fig. 2).

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Fig. 2.
SDS gel of purified E. coli DNA
topoisomerase I wild-type (WT) enzyme and mutants with the
indicated residues changed to alanine.
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Effect of the Mutations on Enzyme Activities--
Purified mutant
enzymes were diluted serially and compared with the wild-type
topoisomerase I for relaxation of negatively supercoiled plasmid DNA in
relaxation buffer containing 6 mM MgCl2 (Fig. 3A). The results showed
that the Arg-136 mutant was totally inactive in the relaxation assay.
No relaxation activity was observable even with 400 ng of the enzyme, a
50-fold excess over the amount needed to observe relaxation by the
wild-type enzyme. The other mutants had varying degrees of loss of
relaxation activity. Examination of the effect of dilutions and time
course of relaxation (Fig. 3B) showed that the Glu-9 mutant
had the greatest reduction in catalytic activity (>90% reduction).
The Glu-115, Asp-113, and Arg-321 mutants had about 80-90% reduction
in activity. The Asp-111 mutant was closest to the wild-type enzyme in
activity (<50% reduction).

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Fig. 3.
Relaxation activity of the E. coli DNA topoisomerase I mutants. A, comparison of the
effect of serial dilutions on enzyme activity. The dilution factors of
1, 10, 50, 100, and 200 correspond to 400, 40, 8, 4, and 2 ng of enzyme
being added to the 30 min relaxation reaction. C, control,
no enzyme added; S, supercoiled DNA; R,
completely relaxed DNA. B, comparison of the time course of
relaxation activity using 100 ng (dilution factor of 4) of each
enzyme.
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The E. coli strain AS17 does not grow at 42 °C because of
the temperature sensitivity of the suppressor for the chromosomal topAam mutation. The pJW312 plasmid carrying the
wild-type topoisomerase I gene can complement this chromosomal mutation
(21). Each one of the active site mutations tested here was found to
abolish this in vivo complementation even though they showed
varying degrees of loss of in vitro relaxation (Table
I).
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Table I
In vivo complementation of the topAam mutation in E. coli AS17
by the plasmic encoded topoisomerase I mutants
Plasmid pJW312 encoding either the wild-type or mutant topoisomerase I
was transformed into AS17 containing a pMK161q plasmid (21). Serial
dilutions of cultures of the transformants (grown overnight at
30 °C) were plated on LB plates with ampicillin and incubated
overnight. The ratio of viable colonies obtained at 42 °C versus
30 °C is shown here.
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The inability of the Glu-9, Glu-111, Asp-113, and Glu-115 mutants to
complement efficiently in E. coli AS17 may be due to their
low level of expression in this E. coli strain. To evaluate the in vivo activities of these mutants, the
thermosensitivities of GP200 expressing these mutants were examined
(Table II). Loss of topA
activity in E. coli can lead to a lower rate of survival when the temperature was raised to 52 °C (reviewed in Ref. 25). The
rate of survival of E. coli GP200 was increased ~50-fold
when plasmid encoded wild-type topA activity was present
(Table II). The survival rates of GP200 transformed with plasmid
encoding the Glu-9, Glu-111, Asp-113, and Glu-115 mutants correlated
with their in vitro activity, with the Glu-111 mutant
conferring close to wild-type thermoresistance and the Glu-9 mutant
conferring the least amount of thermoresistance.
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Table II
Survival of E. coli GP200 and transformants after 1 hr exposure to
52 °C
Exponential phase cultures in LB medium were shifted from 37 °C to
52 °C for 1 h. The viable counts before and after the
temperature shift were determined by serial dilutions in cold PBS
buffer and incubation overnight on LB plates with ampicillin. The
survival rate is the ratio of the viable count obtained after the
exposure to high temperature versus that before the temperature shift.
The average of results from three or more independent experiments are
shown here.
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The Arg-136 Mutant Could Cleave DNA and Form the Covalent
Complex--
The cleavage activities of the mutant enzymes were
examined using 5'-end-labeled single-stranded DNA. Even though the
Arg-136 mutant was totally inactive in the relaxation assay, the amount of cleaved DNA formed was comparable with that from the wild-type enzyme (Fig. 4). For the other mutants,
the amounts of cleavage products observed were decreased, with the
Glu-9 mutant having the lowest cleavage activity, so that the reduction
of relaxation activity seen with these other mutants might be due to
the decrease in the amount of cleaved complex formed by these mutants.
The Asp-111 mutant had the same DNA cleavage efficiency as the
wild-type enzyme.

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Fig. 4.
Cleavage of 5'-end-labeled single-stranded
DNA by wild-type and mutant E. coli topoisomerase I
enzymes. The cleavage reactions were analyzed by electrophoresis
in a 6% DNA sequencing gel. C, no enzyme added.
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A 3'-end-labeled oligonucleotide substrate was used to form a covalent
complex with the enzyme. At 5 min after the addition of the enzyme to
the oligonucleotides, the amounts of the covalent complex observed for
the wild-type and the Arg-136 mutant were identical
(Fig. 5). The amount of covalent complex
formed by the Asp-111 mutant was also close to that of the wild type
while the other mutant enzymes gave lower levels of labeled covalent
complexes under the experimental conditions employed. This experiment
also demonstrated that the Arg-136 mutation affected a step in the enzyme relaxation mechanism that took place after DNA cleavage. This
could be the strand passage or the DNA religation step. The inter-molecular strand transfer activity of the Arg-136 mutant was
compared with the wild type enzyme using the covalent complex formed
with this 3'-end-labeled oligonucleotide substrate and HindIII-digested
DNA as acceptor molecules (26). The
result from one experiment is shown in
Fig. 6. Data from several experiments indicated that the inter-molecular religation activity is about the
same for the wild-type and the Arg-136 mutants. The efficiency of this
reaction is low for both the wild-type and Arg-136 mutant enzymes. It
may or may not reflect the relative activity of the intra-molecular
religation that takes place during relaxation of supercoiled DNA.

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Fig. 5.
Covalent complexes formed between a
3'-end-labeled oligonucleotide and wild-type or mutant E. coli topoisomerase I enzyme. The complexes were separated
from the oligonucleotide substrate by electrophoresis in a 10% SDS
gel. C, no enzyme was added.
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Fig. 6.
Inter-molecular strand transfer by the
wild-type topoisomerase I and the R136A mutant enzyme. The
reaction conditions were as described in Ref. 27, with the enzymes
first incubated with the 3'-end-labeled oligonucleotide used in the
covalent complex formation assay. HindIII-digested DNA
(0.5 µg) was then added as the acceptor molecule. C, no
enzyme added.
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Effect of the Glu-9, Asp-113, Glu-115, and R321 Mutations on
Non-covalent DNA Binding--
The decreased amount of cleaved complex
formed by some of these mutants could be due to the effect of the
mutation on non-covalent binding to DNA. The gel mobility shift assay
was used to evaluate the non-covalent topoisomerase-DNA complex
formation. The results (Fig. 7) showed
that the Arg-321 and Glu-9 mutants had about 50% of the DNA binding
activity as that of the wild-type enzyme. The Glu-115 mutant had
slightly less binding activity (40%) while the Asp-113 mutant had the
lowest DNA binding activity (25%).

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Fig. 7.
Effect of the mutations on the formation of
non-covalent complex as assayed by the gel mobility shift assay using a
5'-end-labeled 36-mer.
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The Glu-9 and Glu-115 Mutations Altered the Mg(II)
Binding--
The mutant enzymes were tested for Mg(II) binding by
equilibrium dialysis against buffer containing 0.4 mM
MgCl2. The Mg(II) binding stoichiometry was determined by
inductively coupled plasma analysis (Table
III). At this Mg(II) concentration, each
molecule of the wild-type enzyme has been found to bind around 2 Mg(II) (27). A higher Mg(II) binding stoichiometry was observed for the
Arg-136 and Arg-321 mutants. The replacement of the positively charged
arginine residues by an alanine might make available an additional
Mg(II) binding site in the enzyme, but this higher Mg(II) binding did
not compensate for the mutation in the enzyme. The Asp-111 mutant had
only a slight reduction in the amount of bound Mg(II) when compared
with the wild-type enzyme. The Glu-9 and Glu-115 mutants had the
greatest reduction in the binding of Mg(II).
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Table III
Mg(II) binding stoichiometry after equilibrium dialysis against buffer
with 0.4 mM MgCl2 for wild-type and mutant enzymes
determined by ICP analysis
The average of results from two independent experiments is shown here.
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Change in Topoisomerase I Fluorescence from Mutation--
The
fluorescence spectra of the wild-type and mutant topoisomerase I
mutants were first compared at room temperature
(Fig. 8 and Table
IV). The Glu-9 and Glu-115 mutants were
found to have an ~30% drop in maximal fluorescence intensity,
indicating a change in the protein conformation influencing the
environment of the tryptophan residues in the enzyme. The decreases in
fluorescence intensities were to a lesser extent for the Arg-136 and
Arg-321 mutants (around 20%) while the Asp-111 and Asp-113 mutants had an ~25% drop in maximal fluorescence intensity. The fluorescence measurements were repeated at 42 °C. The wild-type enzyme
fluorescence was not affected significantly by the temperature shift.
In contrast, the fluorescence intensity of the Arg-321 mutant decreased
by more than 40%, indicating lower stability. The Glu-9 mutant had a
much smaller decrease in fluorescence intensity at the higher temperature (<14%) while the fluorescence intensities of the
other mutants did not change significantly.

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Fig. 8.
Fluorescence emission spectra of wild-type
and mutant E. coli DNA topoisomerase I proteins at
25 °C. Excitation was at 295 nm. The spectra from
top to bottom correspond to the wild-type and the
Arg-136, Arg-321, Asp-111, Asp-113, Glu-9, and Glu-115 mutants.
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Table IV
Effect of the mutations on the relative intrinsic fluorescence
intensity of E. coli DNA topoisomerase I (at 335 nm)
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DISCUSSIONS |
The site-directed mutagenesis study described here aimed at
elucidating the function of several strictly conserved acidic or basic
amino acid residues found at the proximity of the active site
nucleophile Tyr-319. For Arg-136, mutation to alanine abolished relaxation activity totally. Mutations of the other residues produced enzymes with reduced but observable in vitro activities.
Therefore, the strict conservation in evolution did not necessarily
correlate with absolute requirement of the residue for in
vitro activity. Nevertheless, all the mutants tested failed to
complement E. coli AS17 for growth at 42 °C. This might
at least partly be due to effect of the mutations on the stability and
thus expression level of the enzyme in E. coli AS17. The
Arg-136 mutant was the only one among the active site mutants tested
here found to have an expression level in E. coli AS17
comparable with that of the wild-type enzyme. The other mutant proteins
were accumulated at a reduced or unobservable level. In contrast,
mutations in the Zn(II) binding domain resulted in mutant proteins that
were accumulated at higher levels than the wild-type enzyme (27). In
the E. coli strain GP200 where the Glu-9, Asp-111, Asp-113,
and Glu-115 mutants were stably expressed, these mutants were able to
confer some degree of thermoresistance to compensate for the absence of
chromosomal topA activity.
In the crystal structure of the 67-kDa N-terminal fragment, Glu-9,
Asp-111, Asp-113, Glu-115, and Arg-321 are part of an extensive network
of hydrogen bonds and salt bridges that are responsible for the
domain-domain interactions (8). Mutation of these residues to alanines
might abolish some of these interactions and destabilize the protein
structure. This potential effect of the mutation on the enzyme
structure and stability is consistent with the protein fluorescence
data shown in Table IV, with the structure of Arg-321 mutant
particularly sensitive to the increase in temperature.
Mg(II) is required for the relaxation of DNA by E. coli DNA
topoisomerase I. It is not required for the cleavage of single-stranded DNA although the rate of cleavage of small oligonucleotide substrates can be enhanced by the presence of Mg(II) (28, 29). Mutations of Glu-9
and Glu-115 reduced the Mg(II) binding stoichiometry significantly
after equilibrium dialysis against buffer containing 0.4 mM
MgCl2. This might have resulted from one of the multiple coordination sites for Mg(II) in the enzyme being removed or replaced by a ligand of lower affinity to Mg(II). The effects of the mutations of Glu-9, Asp-111, Asp-113, and Glu-115 to alanine on the relaxation activity were less severe than those observed for mutations of carboxylates proposed to be Mg(II) coordination sites involved in
nucleotidyl transfer in other enzyme systems (11, 14, 30, 31). It is
possible that for E. coli DNA topoisomerase I, water or a
DNA phosphate could substitute for the mutated carboxylate in Mg(II)
coordination in the relaxation reaction to provide partial enzymatic
activity, especially at Mg(II) concentration significantly above the
minimal concentrations needed for relaxation activity. The effect of
mutation of Asp-111 on the enzyme activity and Mg(II) binding was
significantly smaller than mutations of the other carboxylates, so it
is unlikely that Asp-111 is a catalyticly important residue.
The Glu-9, Asp-113, Glu-115, and Arg-321 mutants also had reduced DNA
binding activity. This could be due to the participation of the residue
in direct protein-DNA interaction which is a plausible role for
Arg-321. Alternatively, the reduced DNA binding could be due to the
effect of the mutation on the protein folding. It also cannot be ruled
out that the lower Mg(II) binding stoichiometries observed for the
Glu-9 and Glu-115 mutants might be due to the effect of the mutations
on the protein structure and not due to loss of a Mg(II) coordination
site. A Mg(II) binding site distinct from the catalytic center has been
proposed for the EcoRV restriction endonuclease (32).
E. coli DNA topoisomerase I may also have a second Mg(II)
binding site away from the active site region that is required for
relaxation activity because of its effect on the protein conformational
changes that take place during the relaxation reaction cycle (28). The
carboxylates at the active site may be conserved for their roles in
protein structure instead of catalytic functions. The magnitude of the
effect of a single mutation of these catalytically non-essential
residues on the overall protein stability may depend on the E. coli strain background, but nevertheless be of sufficient
significance to account for the evolutionary conservation.
The steps of DNA cleavage and religation may or may not involve the
same catalytic residues in the activation of nucleophiles as well as
the stabilization of transition states and leaving groups. Presently
there is a lack of data to address this question, and it remains
unclear what these catalytic residues may be. Among the mutants tested,
the Glu-9 mutant had the greatest loss of DNA cleavage activity with
only a modest reduction in non-covalent DNA binding. Besides a possible
role in binding Mg(II), this residue might be involved in the catalytic
step of DNA cleavage. In contrast, the Arg-136 mutant had normal DNA
cleavage activity but no relaxation activity. It might be needed in the
DNA strand passage step since it could perform the inter-molecular
rejoining of DNA.
There is also a lack of information on the orientation of the DNA
substrate when it binds to the active site. Additional biochemical data
and/or structural information of the enzyme-DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of
catalysis by the enzyme.
We thank Dr. James C. Wang for
communications and discussions of results.