(Received for publication, June 5, 1995; and in revised form, August 17, 1995)
From the
The two type II topoisomerases in Escherichia coli, DNA gyrase and topoisomerase (Topo) IV, share considerable amino acid sequence similarity, yet they have distinctive topoisomerization activities. Only DNA gyrase can supercoil relaxed DNA, whereas during oriC DNA replication in vitro, only Topo IV can support the final stages of replication, processing of the late intermediate and decatenation of the daughter molecules. In order to develop an understanding for the basis of the differential activities of these two enzymes, we have initiated a characterization of Topo IV binding to DNA. We find that unlike gyrase, Topo IV neither constrains DNA in a positive supercoil when it binds nor protects a 150-base pair region of DNA from digestion with micrococcal nuclease. Consistent with this, DNase I footprinting experiments showed that Topo IV protected a 34-base pair region roughly centered about the topoisomerase-induced cleavage site. In addition, Topo IV preferentially bound supercoiled rather than relaxed DNA. Thus, the DNA binding characteristics of Topo IV are more akin to those of the type II eukaryotic enzymes rather than those of its prokaryotic partner.
Four DNA topoisomerases (Topos) ()have been
identified in Escherichia coli. Topo I (encoded by topA; Refs. 1 and 2) and Topo III (encoded by topB; (3) ) are type I enzymes. DNA gyrase and Topo IV are type II
enzymes. Both gyrase and Topo IV are composed of two subunits: GyrA and
GyrB (4, 5) and ParC and ParE(6) ,
respectively. DNA sequence analysis shows that parC and parE have significant homology with gyrA and gyrB, respectively. Whereas these two topoisomerases share
some common biochemical features, they can catalyze different reactions in vitro(7, 8) and appear to have different
functions in vivo(9) , although both enzymes are
essential for cell survival.
In an ATP-dependent fashion, gyrase can supercoil relaxed DNA, catenate and decatenate DNA rings, knot and unknot circular DNA, and convert positive supercoils directly to negative ones. Gyrase will relax negatively supercoiled DNA in the absence of ATP(10) . All Topo IV-catalyzed reactions require ATP. Topo IV will relax both positive and negative supercoils, knot and unknot DNA, and decatenate DNA rings (7, 11) .
During DNA replication in vitro, only Topo IV is capable of
supporting the terminal stages of replication, processing of the late
intermediate ()and decatenation of the daughter
molecules(8) . Both gyrase and Topo IV can support nascent
chain elongation during theta-type DNA replication in vitro(12) . Genetic analysis has suggested that both Topo IV
and gyrase are involved in chromosome
decatenation(6, 13) . This was supported by the study
of Bliska and Cozzarelli (14) showing that gyrase was
responsible for unlinking catenanes produced as a result of a
recombination event. On the other hand, Adams et al.(9) showed that pBR322 replication catenanes accumulated
at the nonpermissive temperature only in parC or parE strains, not in gyrA or gyrB strains.
In order to supercoil DNA, gyrase must be able to pass strands in one direction; otherwise only relaxation would occur. The mechanisms of gyrase-catalyzed reactions and the interaction of the enzyme with DNA has been studied extensively(15) . When bound to DNA, gyrase constrains about 150 bp of DNA about itself in a positive toroidal supercoil (16, 17, 18, 19) . This is consistent with the results of both nuclease protection experiments (17) and DNase I footprinting experiments(18, 19) . This ability of gyrase to order DNA locally with respect to the site of DNA cleavage during strand passage likely accounts for its supercoiling ability.
Both gyrase and Topo IV are targets for the quinolone and coumarin antibiotics(4, 5, 11, 20) , yet in E. coli, resistance to these antibiotics arises only via mutation of the gyrase genes (21, 22, 23, 24) . Thus, although gyrase and Topo IV seem quite similar, their cellular functions are different. We have initiated an investigation into the mechanisms of the Topo IV topoisomerization activities in order to illuminate the structural basis for the differences in gyrase and Topo IV function. We find that unlike gyrase, Topo IV neither wraps DNA about itself nor distorts the path of the helix significantly on binding. Instead, the enzyme appears to bind a region of 34 bp centered about the cleavage site. Again, unlike gyrase, Topo IV prefers to bind supercoiled rather than relaxed DNA.
Figure 5:
Binding of Topo IV to linear DNA is
competed preferentially by supercoiled DNA. A, standard filter
binding reaction mixtures containing Topo IV (150 fmol), linear
[P]pBSM13 DNA (6 fmol), and either no competitor
DNA or 0.75, 1.5, 3.0, 12.0, 24.0, or 48 fmol of unlabeled supercoiled
pBSM13 DNA or 3.0, 6.0, 12.0, 24.0, 48.0, or 96.0 fmol of unlabeled
linear pBSM13 DNA were incubated at 30 °C for 20 min before
filtering as described under ``Materials and Methods.'' The
data are presented as the percentage of binding of the
P-labeled linear DNA versus competitor. B and C, the molar excess of competitor required to compete
50% of the binding to
P-labeled linear DNA was calculated
for the linear and supercoiled competitors, respectively. r
, the percentage of DNA bound in the
absence of competitor; r, DNA bound in the presence of
competitor.
In order to assess whether Topo IV alters the path of the helix in a significant fashion when it is bound to DNA, Topo IV was bound to singly nicked form II DNA. The nick was sealed with DNA ligase, and the DNA was deproteinized and analyzed by electrophoresis through agarose gels. If binding of the enzyme constrains supercoils, they will become locked into the DNA upon covalent closure of the nick. They can then be observed easily by gel electrophoresis.
As reported previously for gyrase, binding of this enzyme to DNA resulted in the induction of supercoils after closure of the nick (Fig. 1, lanes 1-3). The photonegative of the stained gel was traced densitometrically to determine the shift in the position, compared with that in the absence of gyrase, of the center of the distribution of the topoisomers formed in the presence of gyrase. In this way, we could calculate that 0.5 superhelical turns were introduced to the DNA per bound gyrase tetramer. This is similar to the stoichiometry determined previously(16) . Although not shown here, it has been determined previously that the superhelical turns induced by gyrase binding are positive(16) .
Figure 1: Topo IV does not constrain supercoils when bound to DNA. Singly nicked form II DNA (210 fmol) was incubated with either no topoisomerase (lanes 1 and 4), DNA gyrase (lanes 2 and 3), or Topo IV (lanes 5-8) for 20 min at 23 °C. E. coli DNA ligase was then added, and the reactions were continued for 1 h. Reaction products were then processed as described under ``Materials and Methods'' and analyzed by electrophoresis through a 1.5% agarose gel containing 14 µg/ml chloroquine phosphate.
The binding of Topo IV to the DNA resulted in a slight shift in the pattern of topoisomers toward a more positive distribution. This corresponded to the induction of 0.06 superhelical turns/Topo IV tetramer (Fig. 1, lanes 4-8). This was the case even at Topo IV to DNA ratios 4-fold higher than the ratio where gyrase-induced supercoiling was very obvious. Thus, it seemed unlikely that Topo IV was wrapping DNA about itself as gyrase does. Instead, it is possible that Topo IV unwinds duplex DNA somewhat upon binding.
To confirm that Topo IV was not wrapping DNA about itself, we determined the extent of DNA protected from micrococcal nuclease digestion by Topo IV binding. As established originally for nucleosomes (27) , micrococcal nuclease will cut only in the spacer region between bound proteins. Thus, if a protein wraps DNA about itself, it should protect a significant region of the DNA from digestion by the nuclease. This is clearly observed for DNA gyrase. As reported previously(17) , under protein to DNA ratios equivalent to one gyrase tetramer/200 bp of DNA, gyrase protected DNA in the size range of 110-160 bp from micrococcal nuclease digestion (Fig. 2A, lane 2). This is consistent with the ability of gyrase to wrap DNA about itself. No such protection was evident at a similar ratio of Topo IV to DNA (Fig. 2A, lane 3).
Figure 2:
Protection of DNA from micrococcal
nuclease digestion by Topo IV and gyrase. A, pBSM13 DNA (60
fmol) labeled with [P]dAMP by nick translation
was incubated at 30 °C for 20 min with either no topoisomerase (lane 1) or 1 pmol of either gyrase (lane 2) or Topo
IV (lane 3). Micrococcal nuclease (4 units) was then added to
the reaction mixtures containing topoisomerase, and the incubation was
continued for an additional 20 min. Reaction products were then
processed as described under ``Materials and Methods'' and
analyzed by electrophoresis through a 7% polyacrylamide gel. Size
markers were MspI-digested pBR322 DNA. B, identical
to the experiment shown in A except that micrococcal nuclease
(6 units) was added to all reaction mixtures and 5 pmol of
topoisomerase was used.
At 5-fold higher ratios of topoisomerase to DNA, the same pattern of protected DNA was evident for gyrase (Fig. 2B, lane 2), whereas Topo IV protected a wide range of DNA varying in size between the limit products of the micrococcal nuclease digestion to about 700 bp (Fig. 2B, lane 3). The wide size range of DNA protected by Topo IV under these conditions is most likely indicative of the binding to the DNA of multiple Topo IV tetramers close enough together to exclude access of micrococcal nuclease to the DNA.
In
order to determine how large a region of DNA was bound by Topo IV, we
performed DNase I footprinting. The substrate was a 276-bp DNA fragment
made by PCR using plasmid pTH101 as a template. We had determined that
this region of DNA had one major Topo IV cleavage site that could be
observed in the absence of quinolones (data not shown). By 5` P labeling each primer separately, we were able to easily
observe the Topo IV footprint on each DNA strand.
The results of the DNase I footprinting analysis (Fig. 3) showed that like all known type II topoisomerases, the Topo IV cleavage sites on the top (Fig. 3D) and the bottom (Fig. 3D) strands were staggered by 4 nt (Fig. 3C). Topo IV protected from DNase I digestion about 34 nt of DNA on each strand roughly centered about the cleavage site (Fig. 3, A-C). Because the cleavage site is staggered, this results in slightly asymmetric protection of the duplex from nuclease digestion. Thus, it seems that the mode of Topo IV binding to DNA is distinct from that of gyrase and is similar to that of the eukaryotic type II topoisomerases(28, 29) .
Figure 3: DNase I footprinting of Topo IV bound to DNA. A, the 276-bp PCR DNA fragment (65 fmol) labeled at the 5`-end of the XhoI primer was treated with 0, 4, 2, or 1 ng DNase I in the absence of Topo IV (lanes 1-4, respectively) or with 0, 16, 8, or 4 ng DNase I in the presence of Topo IV (7.0 pmol) (lanes 5-8, respectively) for 30 s at 30 °C and processed and analyzed as described under ``Materials and Methods.'' The sequence ladder (G, A, T, and C) was made using the XhoI oligonucleotide as a primer on pTH101 DNA. The arrow indicates the position of the Topo IV cleavage site. B, the 276-bp PCR fragment (50 fmol) labeled at the 5`-end of the SmaI primer was treated with 0, 2, 1, or 0.5 ng of DNase in the absence of Topo IV (lanes 1-4, respectively) or 0, 8, 4, or 2 ng DNase I in the presence of Topo IV (4.0 pmol) (lanes 5-8, respectively) for 30 s at 30 °C and processed and analyzed as described under ``Materials and Methods.'' The sequence ladder (G, A, T, and C) was made using the SmaI oligonucleotide as a primer on pTH101 DNA. The arrow indicates the position of the Topo IV cleavage site. Topo IV cleavage in A and B was in the absence of quinolones and is thus not very prominent. Quinolone-stimulated cleavage occurs at precisely the same location and is very prominent (D). C, schematic of the region on the DNA protected from DNase I digestion by Topo IV. The arrows denote the point of cleavage by Topo IV.
Topo IV binding to supercoiled
(form I), relaxed (form I`), and linear (form III) pBR322 DNAs was
compared (Fig. 4). The form I` DNA was prepared by treatment of
form I DNA with E. coli Topo I. The resultant preparation
contained no detectable form I DNA and about 5% form II (nicked) DNA.
The form III DNA was prepared by digestion of form I DNA with the EcoRI restriction endonuclease. K was
calculated according to Riggs et al.(31) .
Figure 4:
Binding of Topo IV to supercoiled and
relaxed DNA. A, standard filter binding reaction mixtures
containing either form I (), form I` (
), or form III
(
) [
H]pBR322 DNA (all at 2 nM), and
the indicated concentrations of Topo IV were incubated at 30 °C for
20 min before filtering as described under ``Materials and
Methods.'' Background in all cases was <1% of input DNA
retained on the filters. B, the initial phase of the binding
curves shown in A are reproduced in exploded
view.
Topo IV
bound to form I, I`, and III DNAs with K values of
0.6 nM, 3.3. nM, and 9.3 nM, respectively.
Because these DNAs were topological isomers, the different affinities
of Topo IV for them can only be attributed to their different
topological states. Thus, as predicted, unlike gyrase, Topo IV clearly
bound supercoiled DNA preferentially to relaxed DNA. This was confirmed
by a competition binding experiment.
Topo IV binding to form III 5`
[P]pBSM13 DNA was competed by either unlabeled
form I or form III pBSM13 DNA (Fig. 5A). Calculation of
the amount of competitor needed to reduce binding to the
[
P]-labeled DNA by 50% (Fig. 5, B and C) showed that an 18-fold higher molar excess of
linear compared with supercoiled DNA was required. This was in good
agreement with the nearly 16-fold difference in K
determined for binding of Topo IV to form I and III DNAs (Fig. 4).
Whereas it was clear that Topo IV bound supercoiled DNA better than relaxed DNA, it also seemed that of the two types of relaxed DNA used in the binding experiments, Topo IV bound form I` DNA roughly 3-fold better that form III DNA. Because the only difference between these two DNA forms is that the latter has ends and the former does not, we considered whether the difference in binding affinities could be accounted for as a result of Topo IV molecules sliding off the linear form. A similar explanation was raised to account for the reduced binding affinity of the Drosophila type II enzyme to form III DNA compared with form I DNA(32) .
Experiments from
Wang's lab (33, 34) suggest that the eukaryotic
type II topoisomerases are possessed of an annular DNA binding site,
and Sekiguchi and Shuman (35) have shown that the vaccinia type
I enzyme binds DNA circumferentially. Thus, we prepared
[S]Topo IV and measured its binding to form I`
and form III DNAs by gel filtration. At very low concentrations of Topo
IV (<5 nM), we observed 50% more Topo IV bound to form I`
compared with form III. However, this difference was lost at higher
concentrations (data not shown). Thus, whereas sliding of Topo IV off
of form III DNA may account for some of the observed binding
differences, it cannot account for the full effect. It is of course
possible that the EcoRI cleavage used to generate the form III
DNA disrupted a high affinity biding site.
E. coli has two type II topoisomerases, DNA gyrase and the recently discovered Topo IV. Even though these enzymes share considerable amino acid sequence similarity(6) , they support different reactions during DNA replication in vitro(8) and appear to behave distinctively in vivo(9) .
Both enzymes can support nascent chain
elongation during oriC DNA replication reconstituted in
vitro with purified proteins(8, 12) , although
only Topo IV can support the terminal stages of replication, processing
of the late intermediate and decatenation of the linked
daughter molecules(8) . Gyrase, but not Topo IV, has been
implicated as the enzyme responsible for supporting chain elongation in vivo(36) , although conclusions based on the
effects on DNA replication of the quinolone antibiotics (36) must now be considered questionable because both gyrase (4, 5) and Topo IV (7, 11) are
sensitive to these drugs.
Mutations that display a par phenotype can be mapped to both the gyrase (37, 38) and Topo IV genes (6, 39, 40, 41) and incompletely segregated nucleoids have been observed in gyrB mutant strains at the nonpermissive temperature(13) . On the other hand, Adams et al.(9) demonstrated that catenated pBR322 daughter molecules arise at the nonpermissive temperature only in parC and parE strains, not in gyrA or gyrB strains.
In order to better appreciate the basis for the differential action of these two topoisomerases, we have investigated the interaction between Topo IV and DNA. Binding of gyrase to DNA is distinctive. The enzyme wraps roughly 150 bp of DNA about itself in a positive toroidal supercoil(15) . It has been proposed that this ordering of the DNA across the surface of the DNA cleavage site facilitates the vectorial strand passage required for supercoiling(42) . DNA bound to Topo IV in a manner more reflective of a eukaryotic type II topoisomerase than of gyrase.
Topo IV protected a small region of 34 bp from attack by DNase I when bound to DNA. Given that the Stokes radius of Topo IV is 65 Å(11) , it is highly unlikely that the enzyme wraps DNA about itself. This is supported by the observation that the binding of Topo IV to DNA followed by the subsequent closure of the DNA into a ring did not result in the induction of positive supercoils, as was the case for gyrase(16) . Thus, the size of the binding site of Topo IV on DNA is similar to that of the eukaryotic type II enzyme, which has an identical Stokes radius and protects 25-28 bp of DNA from nuclease digestion(28) . In spite of the different mode of binding to DNA, Topo IV and gyrase binding sites appear to be dictated by similar sequence features because they overlap considerably(11) . Perhaps the different modes of DNA binding accounts for the observed difference in site preference(11) .
Filter binding studies showed that Topo IV bound supercoiled DNA preferentially compared with relaxed. This is opposite to the preference shown by DNA gyrase (30) but similar to the preference shown by the eukaryotic enzyme (32) and is in keeping with the inability of Topo IV to supercoil DNA. Thus, the net result of the interaction between closed circular DNA and Topo IV is the removal of turns of the duplex about itself. This, not surprisingly, has apparently resulted in the evolution of an enzyme that preferentially binds its substrate.
The difference in binding site size between Topo IV and gyrase and the preferential binding of Topo IV to superhelical DNA helps to explain their differential action during the terminal stages of oriC plasmid DNA replication in vitro. The inability of gyrase to process the late intermediate or to decatenate the linked daughter molecules may derive partially from its large DNA binding site and preference for relaxed DNA. Gyrase is probably excluded from binding ahead of the replication forks in the late intermediate where there is only about 200 bp of unreplicated parental DNA available. This exclusionary feature would be considerably less severe for Topo IV. Likewise, because the intermolecular linkages between catenated daughter molecules are similar to supercoils in that they impart writhe to the helix(43, 44) , Topo IV binding to these replication products should be considerably more stable than that of DNA gyrase.
Interestingly, the difference in substrate binding preference exhibited between Topo IV and gyrase confounds an explanation for the observation that in laboratory strains of E. coli, resistance to nalidixic acid maps only to gyrA(22, 23) and gyrB(24) . Because the chromosome is supercoiled, one might expect Topo IV rather than gyrase to be bound preferentially and to serve as the primary target for the drug. Thus, it is clear that considerable genetic and biochemical analysis of these two topoisomerases is required in order to understand the complex interactions between them and their role in the cell.