Background
Escherichia coli DNA topoisomerase I
is a representative example of type IA DNA topoisomerase
(for reviews, see refs [ 1 2 ] ). Its major biological role
in the bacterial cell is the removal of excessive negative
supercoils from DNA to maintain the DNA at optimal
superhelical density along with DNA gyrase [ 3 ] . The
enzyme has a molecular weight of 97 kDa and the active site
tyrosine responsible for DNA cleavage is found in the 67
kDa N-terminal transesterification domain. The structure of
this 67 kDa domain has been determined by X-ray
crystallography to be torus-like, indicating the need for
protein conformational change for strand passage to take
place after DNA cleavage [ 4 ] . Relaxation activity
requires the presence of the Zn(II) binding tetracysteine
motifs [ 5 ] found between the 67 kDa N-terminal domain
(Top67) and the 14 kDa C-terminal single-stranded DNA
binding domain (Figure 1). The three tetracysteine motifs
do not form a stably folded structure on its own, but when
combined with the 14 kDa C-terminal domain, forms a stably
folded 268 amino acid DNA binding domain (ZD domain) that
has higher affinity for single-stranded DNA than the 121
amino acid 14 kDa C-terminal region by itself [ 6 ] .
Recent sequence and structural analysis suggests that the
14 kDa domain is evolutionarily related to the three
tetracysteine motifs and belongs to the zinc ribbon family
[ 7 ] . The ZD domain in
E. coli topoisomerase I probably
evolved from a domain that binds five Zn(II)
originally.
Removal of negative supercoils from DNA by bacterial
type IA topoisomerase involves the following steps: (1)
binding of the enzyme to the junction of double-stranded
and single-stranded DNA [ 8 ] ; (2) cleavage of a
single-strand of DNA near the junction with cleavage
sequence preference of a cytosine in the -4 position to
form the covalent intermediate [ 9 10 ] ; (3)
conformational change of the covalent enzyme-DNA complex to
result in physical separation of the 5' phosphate
covalently linked to the active tyrosine, and the 3'
hydroxyl of the cleaved DNA; (4) passage of the
complementary single strand through the break; (5) enzyme
conformational change to bring the 5' phosphoryl end back
into the proximity of the 3' hydroxyl group of the cleaved
DNA; (6) religation of the phosphodiester bond. Although it
is known that the ZD domain can function as a DNA binding
domain, its exact role in these individual steps of removal
of a negative superhelical turn from DNA by
E. coli topoisomerase I remains to be
defined. Using purified 67 kDa transesterification domain
and 30 kDa ZD domain, results from experiments described
here provide new insight into the action of these two
individual domains in the enzyme mechanism.
Results
Partial restoration of relaxation activity from
mixing of Top67 and ZD domains
As reported previously [ 11 ] , the N-terminal
transesterification domain Top67 by itself did not
exhibit any relaxation activity when assayed with
negatively supercoiled plasmid DNA (Figure 2a). The 30 kD
C-terminal ZD domain also had no relaxation activity by
itself, as expected. Partial relaxation of the input
supercoiled DNA was detected when Top67 was mixed with
the ZD domain prior to addition of DNA. A ratio of 2 ZD
molecules added for each Top67 was found to be sufficient
for maximum relaxation activity, with no increase in
activity when higher ratio of ZD/Top67 was used (data not
shown). The specific activity observed under this
optimized condition (Figure 2a) was still about 10 fold
lower than that of the intact enzyme. Analysis of the
time course of relaxation with 6 pmoles of topoisomerase
I or top67 reconstituted with ZD (Figure 2b) showed that
negative supercoils were removed at a much slower rate by
the reconstituted activity.
Top67 and ZD domains have comparable binding
affinities to single-stranded DNA but significantly
different affinities for double-stranded DNA
The gel mobility shift assay was used to compare the
binding affinities of Top67 and the ZD domain to a 5'
end-labeled single-stranded oligonucleotide 35 base in
length. As shown in Figure 3a, these two domains had
similar affinities for binding to the single-stranded
substrate. The half maximal binding values based on the
average of results from three different experiments were
0.02 μM for Top67 and 0.04 μM for the ZD domain. However,
with the same oligonucleotide in a duplex form (Figure
3b), Top67 exhibited much higher affinity (half maximal
binding value = 0.07 μM) than the ZD domain (half maximal
binding value > 5 μM).
Top67 can recognize cleavage sites preferred by E.
coliDNA topoisomerase I
Previous studies have shown that
E. coli DNA topoisomerase I
cleavage of single-stranded DNA occurs with selectivity
for sites with the C nucleotide base at the - 4 position
[ 9 10 ] and that the enzyme preferentially cleaves at
junctions of double-stranded and single-stranded DNA [ 8
] . Several different 5'-end labeled substrates were
prepared and used in cleavage assays to compare the
cleavage sites selected by Top67 versus topoisomerase I.
The results showed that with single-stranded substrates,
Top67 also preferred cleavage sites with a C nucleotide
base at the -4 position as reported for most of the type
IA topoisomerases [ 12 ] . There were some differences
from topoisomerase I in the relative distribution of
cleavage products among the potential cleavage sites
(Figure 4a,4b). Top67 appeared to be more
non-discriminatory in selection of the possible cleavage
sites with the C nucleotide in the -4 position. Addition
of the ZD domain had no effect on the cleavage
selectivity of Top67. A substrate with both
single-stranded and double-stranded regions was
constructed to mimic such junction in negatively
supercoiled DNA. Top67 and topoisomerase I recognised the
same cleavage site on this substrate (Figure 4c). Maximal
yield of cleavage products was obtained for both Top67
and topoisomerase I within seconds after mixing of the
enzyme and DNA so any potential difference in cleavage
rates between the Top67 and topoisomerase I is unlikely
to account for the difference in relaxation
efficiency.
Top67 cleavage sites are religated upon addition of
high salt and Mg 2+
To test the religation capability of Top67, a 5'-end
labeled oligonucleotide 61 base in length was first
incubated with the enzyme in low ionic strength buffer to
allow formation of the cleaved complex. Sodium chloride
concentration was then increased to 1 M to induce
reversal of cleavage and dissociation of the enzyme from
the DNA. We observed that more complete and consistent
reversal of cleavage was obtained with both topoisomerase
I and Top67 if a low concentration of Mg 2+(4 mM) was
also added with the NaCl. This is consistent with an
early observation of dissociation of the enzyme-DNA
complex in high salt upon addition of Mg 2+ [ 13 ] . It
has also been reported [ 14 ] that addition of Mg 2+was
apparently not required for observation of this reversal
of cleavage. However, it is possible that some enzyme
preparations may contain bound Mg 2+and the low
concentration of bound Mg 2+might have been sufficient
for reversal of cleavage, as postulated previously to
explain the data [ 14 ] . The results of this cleavage
reversal experiment (Figure 5) indicated that the ZD
domain was not required for efficient reversal of
cleavage and Top67 could carry out religation of cleaved
DNA. Again the reversal of cleavage was complete for both
Top67 and topoisomerase I within seconds after the
addition of high salt and Mg 2+even when the reactions
were carried out on ice (data not shown) so the lack of
relaxation activity by Top67 is unlikely to be due to
deficiency in religation.
The ZD domain is not required for catenation of
double-stranded DNA circles
E. coli topoisomerase I can
catalyze catenation of double-stranded DNA circles if the
molecules contain single-strand scissions [ 15 16 ] . To
test if the Top67 can carry out double-stranded DNA
passage at enzyme cleavage sites across from the DNA
nicks, the yield of DNA catenanes were compared with that
obtained with topoisomerase I. In contrast to the
relaxation activity, the catenating activity of Top67
shown in figure 6was as efficient as that of full-length
topoisomerase I, and the addition of the ZD domain had no
effect (Figure 6a). The rate of catenane formation for
Top67 alone was similar to that of topoisomerase I
(Figure 6b). This catenation activity observed with
topoisomerase I and Top67 was unlikely to be due to
contaminating topoisomerase III activity since it was not
observed with the ZD domain purified under almost
identical procedures and a site-directed mutant with
substitution of the active site Tyr319 by phenylalanine
also did not exhibit this activity (Figure 6a).
Discussion
There are two homologous type IA topoisomerases present
in
E. coli . Topoisomerase III has
potent DNA decatenating activity for resolution of plasmid
DNA replication intermediates, but much weaker relaxation
activity than topoisomerase I [ 17 ] . To exhibit maximal
relaxation activity, topoisomerase III requires high
temperature (52°C) along with low magnesium and monovalent
ion [ 17 18 ] . In contrast,
E. coli topoisomerase I was not
active in the
in vitro assay for resolution of
plasmid DNA replication intermediates [ 19 ] . Removal of
the C-terminal 49 amino acids from the 653 amino acid
topoisomerase III protein resulted in drastic reduction of
catalytic activity [ 20 ] . Fusion of the carboxyl-terminal
312 amino acid residues of
E. coli topoisomerase I, which
includes the entire ZD domain, onto the 605 N-terminal
amino acids of topoisomerase III generated a hybrid
topoisomerase that has relaxation activity resembling
topoisomerase III along with weak decatenating activity [
21 ] . Although preferring single-stranded DNA as binding
substrate, topoisomerase I had been shown to also bind
double-stranded DNA [ 22 ] , but there is no data available
to indicate which domain in the enzyme is responsible for
this interaction.
The experiments described here measured directly the
interaction of the ZD domain with both double-stranded and
single-stranded DNA substrates. ZD domain was found to bind
to single-stranded DNA, but not double-stranded DNA, with
high affinity. This result indicated that with regard to
the mechanism of
E. coli topoisomerase I, the ZD
domain was likely to function as a single-stranded DNA
binding domain instead of having double-stranded DNA
binding function as previously suggested [ 21 ] . Even
though Zn(II) binding transcription factors that recognise
specific double-stranded DNA are well represented in
eukaryotes [ 23 24 ] , there are also numerous examples of
Zn(II) coordination being required for interaction with
single-stranded nucleic acid or damaged DNA with
single-strand characteristics [ 24 25 26 27 ] .
The effect of removal of the ZD domain on the individual
step of enzyme action was also investigated using Top67.
The results indicated that Top67 was effective in binding
to both double-stranded and single-stranded DNA. As a
result, Top67 could position itself in the absence of ZD
domain at the junction of double- and single-stranded DNA
for subsequent DNA cleavage, as previously observed for
intact topoisomerase I [ 8 ] . Reversal of DNA cleavage
also took place readily with Top67 upon addition of 1 M
NaCl and 4 mM MgCl
2 . The ZD domain also was not required
for selectivity of a cytosine in the -4 position relative
to the cleavage sites.
Despite its ability to recognise the DNA substrate and
carry out DNA cleavage-religation, Top67 by itself cannot
catalyze change of linking number in the relaxation of
supercoiled DNA. The single-strand DNA substrate designated
for the ZD domain in the catalytic mechanism of the enzyme
may be the strand of DNA complementary to the strand first
cleaved by the enzyme to form the covalent complex. This
interaction with the passing strand of DNA would not be
needed for the first two steps of enzyme mechanism up to
the formation of the covalent complex. Our results showed
that adding the purified ZD domain partially restored the
relaxation activity. Therefore the ZD domain can supply the
function that is missing in Top67 even when the two domains
are not covalently linked. However, the resulting
relaxation activity is much less efficient than that of the
intact enzyme, suggesting that coordinated actions of the
two domains are required for efficient removal of negative
supercoils from DNA. The requirement of specific
protein-protein interactions between the two domains could
also account for the weak relaxation activity observed for
the hybrid topoisomerase with ZD linked to topoisomerase
III sequence [ 21 ] .
This proposed role for the ZD domain in interacting with
the passing single-strand of DNA is also supported by the
observation that there is no difference between Top67 and
intact topoisomerase I in the formation of catenanes. This
reaction involves passage of another double-stranded DNA
circle, instead of the complementary DNA strand through the
break generated by DNA cleavage so the ZD domain would not
be expected to play any significant role. High
concentration of DNA substrate is required to favor
formation of catenanes catalyzed by topoisomerase I, and
the enzyme also has to be present in higher concentration
compared to the relaxation reaction. The double-stranded
DNA-binding activity in
E. coli topoisomerase III required
for highly efficient decatenation activity is attributed to
a 17-amino-acid residue with no counterpart in
E. coli topoisomerase I [ 28 29 ] .
It may be required for interaction with the passing
double-strand of DNA in the decatenation mechanism. The
presence of this decatenation loop instead of the Zn(II)
binding ZD domain in topoisomerase III may account for the
dominance of the decatenation activity over the relaxation
activity.
Based on these results, we propose a model for the
relaxation of supercoiled DNA by
E. coli topoisomerase I (Figure 7)
modified from previous versions that have a number of
common features but differ most significantly in the role
of the Zn(II) binding domain [ 2 4 21 29 30 ] . In this
model, the subdomains in Top67 is responsible for
interacting with the G-strand of DNA both upstream and
downstream of the cleavage site. The ZD domain interacts
with the passing single-strand DNA to be transported
(T-strand). After cleavage of the DNA gate strand which
becomes covalently linked to Tyr319 on Top67 (step 2),
protein conformational change involving both Top67 and the
ZD domain increases the distance between the covalently
bound 5' phosphate and non-covalently bound 3' hydroxyl of
the cleaved DNA gate strand while the passing DNA strand
(T-strand) is guided through the "gate" via interaction
with the ZD domain (step 3) to lead to change in linking
number. A second enzyme conformational change positions the
cleaved DNA ends for religation (step 4). The ZD domain can
still interact with the T-strand of DNA even when not
linked to Top67 in the same polypeptide, but efficiency of
catalysis is reduced as a result, probably due to loss of
coordinated action by the two domains. The presence of the
ZD domain may enhance the transition of Top67 from a closed
conformation to a more open conformation so that strand
passage can take place through the "DNA gate". Previous
data showed that although Zn(II) binding is not absolutely
required for formation of the cleaved complex, it increased
the amount of cleaved complex that can be isolated [ 31 ] .
When linked to Top67, the ZD domain also has some influence
on the cleavage site selections. It has previously been
observed that a mutation in the Zn(II) binding motif can
affect the cleavage site selectivity of topoisomerase I [
32 ] even though Top67 by itself can recognize both the
cytosine in the -4 position and the junction of single- and
double-stranded DNA. To gain further details for this model
of enzyme action, we are characterizing the protein-protein
interactions between the Top67 transesterification domain
and the ZD domain, as well as the protein conformational
changes that can take place when the enzyme interacts with
DNA substrate.
The hyperthermophilic topoisomerase I from
Thermotoga maritima has been shown to
coordinate one Zn(II) with a unique tetracysteine motif
Cys-X-Cys-X
16 -Cys-X-Cys but Zn(II) binding is not
required for relaxation activity [ 33 ] . The sequence of
this unique tetracysteine motifs is somewhat different from
those present in other type IA topoisomerases in that the
other tetracysteine motifs always had at least two amino
acids separating the pairs of cysteines (Cys-X
2-11 -Cys), instead of just one amino
acid (Cys-X-Cys) in
T. maritima topoisomerase I [ 33 ] .
Therefore the structure and function of the single Zn(II)
binding motif in
T. maritima may differ from the
multiple Zn(II) binding motifs in
E. coli topoisomerase I. Direct
interaction between DNA and the
T. maritima Zn(II) binding motif has
not been demonstrated. It has been suggested that the
mechanisms of these two enzymes may be different [ 33 ] .
Direct interaction between the enzyme and the passing
strand may not be necessary for the
T. maritima topoisomerase I activity.
The relaxation and decatenation activities of
T. maritima topoisomerase I appear to
be significantly more efficient than those of the
E. coli topoisomerase I [ 33 ] .
Based on their primary sequences, a number of bacterial
topoisomerase I enzymes do not appear to coordinate any
Zn(II) with tetracysteines motifs while other type IA
topoisomerase has up to 4 tetracysteine motifs [ 7 ] . The
topoisomerase I from
Mycobacterium smegmatis has been
demonstrated biochemically not to bind Zn(II) [ 34 ] . In
contrast, mutation disrupting the fourth Zn(II) motif of
Helicobacter pylori topoisomerase I
abolished enzyme function
in vivo [ 35 ] . Therefore there may
be significant differences in the mechanisms of type IA
topoisomerases from different organisms with respect to
requirement of Zn(II) binding for relaxation activity.
There is also another possible explanation for the
varied number of tetracysteine motifs and requirement of
Zn(II) for relaxation activity found in different type IA
topoisomerases. The 14 kDa C-terminal region of
E. coli topoisomerase I has been
classified based on its structure to be in the Zn-ribbon
superfamily [SCOP release 1.50, 7] even though it does not
bind Zn(II). It also has high affinity for binding to
single-stranded DNA on its own when separated from the
three tetracysteine motifs [ 36 ] . Based on the structural
and DNA-binding properties of the
E. coli topoisomerase I 14 kDa
domain, one can conclude that it is possible for a
subdomain in topoisomerase I to lose the Zn(II) binding
cysteines during evolution and still maintains the
Zn-ribbon structure and single-strand DNA binding
properties [ 7 ] .
Finally, the
in vivo catalytic activities of
eukarytotic type IA topoisomerases, the topoisomerase III
from various higher organisms may be related to their
sequences. The transesterification domains of these
eukaryotic enzymes have high degrees of identity to
E. coli DNA topoisomerase III [ 7 37
] . However, the decatenation loop is not present in the
eukaryotic topoisomerase III sequences and to date the
decatenation activity has not been demonstrated for these
enzymes. The number of potential Zn(II) binding cysteine
motifs range from none in
S. cerevisiae DNA topoisomerase III
to four highly conserved tetracysteine motifs in the beta
family of the topoisomerase III enzymes [ 38 ] . The Zn(II)
domain formed by these tetracysteine motifs may be required
for interaction with single-strand DNA in removal of
hypernegative supercoils [ 39 ] or disruption of early
recombination intermediates between inappropriately paired
DNA molecules [ 40 ] .
Conclusions
We have shown that the ZD domain of
E. coli DNA topoisomerase I is not
required for the substrate recognition and DNA
cleavage-religation action of the enzyme. We propose that
the ZD domain interacts with the passing single-strand of
DNA in the relaxation of negatively supercoiled DNA by this
enzyme.
Materials and methods
Enzyme and DNA
E. coli DNA topoisomerase I and the
ZD domain were expressed and purified as described [ 6 41
] . To express the 67 kDa N-terminal transesterification
domain (Top67), a stop codon at amino acid 598 was
introduced into plasmid pJW312 [ 42 ] used for
topoisomerase I expression by site-directed mutagenesis
employing the Chameleon-Mutagenesis kit from Stratagene.
Top67 was expressed and purified with the same procedures
as topoisomerase I.
The oligonucleotides were custom synthesized by
Genosys. The single-strand substrates and the top strand
of the duplex substrates were labeled at the 5' termini
with T4 polynucleotide kinase and γ 32P-ATP. The labeled
oligonucleotides were purified by electrophoresis in a 12
or 15% sequencing gel. After elution from the gel slice,
the labeled single-stranded oligonucleotides were
desalted by centrifugation through a Sephadex G10 spin
column.
The duplex or heteroduplex substrates were prepared by
mixing the labeled top strand with 4 fold excess of the
unlabeled bottom strand, heating at 80°C for three
minutes, cooling to room temperature and purified by
electrophoresis in a 20% non-denaturing polyacrylamide
gel with TBE buffer.
Plasmid pJW312 DNA used in relaxation assay was
purified by CsCl centrifugation. Phage PM2 DNA was
extracted from infected
Pseudoalteromonas espejiana cells [
43 ] and PM2 DNA with one or more single-chain scissions
used in the catenation assay was prepared as described [
44 ] .
DNA relaxation assay
Top67 and the ZD domains at different concentrations
were mixed and incubated at 37°C for 10 min before
addition to the 0.3 μg of supercoiled plasmid DNA in 20
μl of 10 mM Tris-HCl pH 8.0, 2 mM MgCl
2 , 0.1 mg/ml gelatin. After
incubation at 37°C for up to 1 h, the reaction was
stopped by addition of 50 mM EDTA and electrophoresed in
a 0.7% agarose gel and visualized by ethidium bromide
staining as described [ 45 ] .
Gel mobility shift assay
The proteins were mixed with the 1 pmole of the
labeled DNA substrates in 10 μl of 20 mM Tris-HCl pH 8.0,
100 μg/ml BSA, 12% glycerol and 0.5 mM EDTA. The samples
were incubated at 37°C for 5 min and then loaded onto a
6% polyacrylamide gel and electrophoresed with buffer of
45 mM Tris-borate pH 8.3, 1 mM EDTA. Electrophoresis was
carried out at room temperature at 2 V/cm for 2 h. After
drying of the gel, bands corresponding to the
protein-bound oligonucleotides and unbound
oligonucleotides were visualized by autoradiography,
excised and counted in a Scintillation counter for
quantitation.
DNA cleavage assay
The cleavage assays were carried out with 1 pmole of
5' 32P-end labeled DNA substrate and 5-10 pmoles of
topoisomerase I or Top67 in 10 μl of the buffer used for
the gel mobility shift assay. After incubation at 37°C
for up to 20 min, an equal volume of 90% formamide, 10 mM
KOH, 0.25% bromophenol blue and 0.25% xylene cyanol was
added to stop the reactions. The samples were analyzed by
electrophoresis in a 12% sequencing gel followed by
autoradiography.
Salt and Mg 2+induced reversal of cleavage
The conditions were modified from those described
previously [ 14 ] . The cleavage reactions were incubated
at 37°C for 5 min and then divided into three aliquots.
The cleavage products were trapped in one aliquot by the
addition of SDS to 1%. NaCl (1 M) alone or NaCl with MgCl
2 (4 mM) were added to the other
aliquots followed by further incubation at 37°C for up to
30 min before the addition of SDS. The products were
analyzed as described for the cleavage reactions.
Catenation of nicked DNA circles
The catenation reaction was carried out with 1.4 μg of
nicked PM2 phage DNA in 20 μl of 10 mM Tris-HCl, pH 8.0,
0.1 mM EDTA, 10 mM KCl, 10 mM MgCl
2 . After incubation at 37°C for up
to1 h, the reactions were stopped with the addition of 1%
SDS and 50 mM EDTA. The products were analyzed as
described for the relaxation assay.
Authors' contributions
Author 1 (A.A.) carried out all the experiments except
the catenation assay. Author 2 (Y.T.) conceived of the
study, participated in its design and coordination and
carried out the catenation assay. All authors read and
approved the final manuscript.
