(Received for publication, April 12, 1995; and in revised form, June 21, 1995)
From the
The binding of DNA topoisomerase III (Topo III) to a
single-stranded DNA substrate containing a strong cleavage site has
been examined. The minimal substrate requirement for Topo III-catalyzed
cleavage has been determined to consist of 7 bases: 6 bases 5` to the
cleavage site and only 1 base 3` to the site. Nuclease P1 protection
experiments indicate that the enzyme also binds to its substrate
asymmetrically, protecting 12 bases 5` to the cleavage site and
only 2 bases 3` to the cleavage site. A catalytically inactive mutant
of Topo III shows the same protection pattern as the active
polypeptide, indicating that Topo III is a site-specific binding
protein as well as a topoisomerase. Consistent with this view, an
oligonucleotide containing a cleavage site is a more effective
inhibitor and is bound more efficiently by Topo III than an
oligonucleotide without a cleavage site.
Escherichia coli has been shown to possess four DNA
topoisomerase activities. Two type I enzymes, DNA topoisomerase I (Topo
I) ()(1) and DNA topoisomerase
III(2, 3, 4) , have been purified and
characterized. In addition, two type II enzymes, DNA topoisomerase II
(DNA gyrase) (5) and DNA topoisomerase IV (Topo
IV)(6) , have also been purified and characterized. The role of
these enzymes in DNA metabolism has been defined by studies, both
genetic and in vitro. For example, DNA gyrase and Topo I have
been shown to be involved in the maintenance of superhelical density of
the E. coli chromosome(7) . In addition, there is also
evidence that DNA gyrase is involved in the terminal stages of DNA
replication (8) . Topo IV (9, 10) and Topo III (4, 11) have been shown to be potent decatenases in vitro, and it has been proposed that these enzymes are
involved in the separation of nascent daughter chromosomes during the
terminal stages of DNA replication. In addition, it has also been shown
that DNA gyrase, Topo IV, and Topo III can support DNA polymerase chain
elongation during the replication of plasmid DNA in
vitro(12) . The apparent redundant functions of these
enzymes emphasizes the importance of these enzymes in DNA metabolism.
A model termed ``sign inversion'' (13) has been proposed that describes a unified mechanism for both type I and type II bacterial topoisomerases. In this model, a topoisomerase binds to its substrate (either single-stranded DNA for a type I enzyme or double-stranded DNA for a type II enzyme), catalyzes a strand break (either single (type I) or double (type II)) and strand passage event, and then reseals the break. The ``sign inversion'' model was originally proposed to explain the mechanism of bacterial DNA gyrase (a type II DNA topoisomerase); however, it has been extended to explain the mechanism of E. coli DNA topoisomerase I, a type I enzyme. It has been proposed that in the case of Topo I, the strand passage event can be thought to invert the sign of the two strands of the helix(14) , whereas, the strand passage event catalyzed by E. coli DNA gyrase, a type II enzyme, inverts the sign of a wrap of the entire helix(13, 15) .
Although there is evidence supporting this model as a whole, there is very little information regarding the properties of the individual steps of topoisomerase catalysis; therefore, our laboratory has set out to perform a detailed characterization of the mechanism of DNA topoisomerase III. In this report, the binding of Topo III to its substrate is examined, and a model for Topo III-catalyzed cleavage of DNA is proposed.
Oligonucleotides, containing 5, 6, or 7 bases 5` of the cleavage site and 1 or 2 bases 3` to the cleavage site, were incubated with Topo III, and the cleavage products were resolved by electrophoresis through a polyacrylamide gel in the presence of urea (Fig. 1). When Topo III was incubated with a 9-base oligonucleotide containing 7 bases 5` of the major cleavage site, a major 7-base (corresponding to the previously identified cleavage site(16) ) and a minor 6-base cleavage product were observed (lane1). Incubation of Topo III with an 8-base oligonucleotide, containing 7 bases 5` and only one base 3` of the same major cleavage site, resulted in the production of identical cleavage products, although the 7-base cleavage product was diminished by 4.3-fold (lane3). This data clearly establishes the minimal 3`-sequence requirement for Topo III to be only 1 base.
Figure 1: Determination of the minimum substrates required for Topo III-catalyzed cleavage of single-stranded DNA. DNA cleavage experiments were performed using oligonucleotides containing either 7+2 bases (lanes1 and 2; where 7+2 indicates 7 bases 5` and 2 bases 3` of the cleavage site, respectively), 7+1 bases (lanes3 and 4), 6+2 bases (lanes5 and 6), 6+1 bases (lanes7 and 8), and 5+2 bases (lanes9 and 10). Reactions contained either 1 pmol of Topo III (lanes1, 3, 5, 7, 9) or no enzyme (lanes2, 4, 6, 8, 10). The reaction products were separated through a 25% polyacrylamide gel containing 50% (w/v) urea as described under ``Materials and Methods.''
To establish the 5`-minimal sequence requirement for Topo III-catalyzed cleavage, the enzyme was incubated with an 8-base oligonucleotide containing 6 bases 5` of the major cleavage site. This reaction resulted in the production of the 6-base cleavage product; however, the expected 5-base minor cleavage product (observed in the reaction containing the oligonucleotide with 7 bases 5` of the cleavage site) was absent (compare lane3 with lane5). This result suggested that the minimal 5`-sequence requirement for cleavage was 6 bases. This conclusion was confirmed by incubating Topo III with a 7-base oligonucleotide containing the predicted minimal site of 6 bases 5` of the cleavage site (lane7) and with a 7-base oligonucleotide containing only 5 bases 5` of the cleavage site (lane9). A 6-base cleavage product was observed in the case of the former (although the cleavage was reduced 9.3-fold relative to the substrate containing 6 bases 5` and 2 bases 3` of the cleavage site (lane5) and reduced 4.8-fold relative to the substrate containing 7 bases 5` and 1 base 3` of the cleavage site (lane3)), but no 5-base cleavage product was observed for the latter oligonucleotide. A summary of the cleavage experiments performed with multiple oligonucleotides is presented in Fig. 2.
Figure 2: Summary of the minimal substrate requirement for Topo III-catalyzed cleavage of single-stranded DNA. Topo III was incubated with the indicated oligonucleotides. The cleavage reactions were processed as previously described(16) , and the results are summarized here. A (+) indicates cleavage, and(-) indicates no cleavage. The total length of the oligonucleotide, as well as the length both 5` and 3` of the cleavage site, are indicated. The minimal substrate is indicated by the rectangle below the DNA sequence.
To further establish whether the minimal substrate was an actual characteristic of the enzyme rather than a peculiarity of the substrate, the minimal substrate requirement for Topo III-catalyzed cleavage was determined for two other independent cleavage sites. The minimal substrate requirement was identical using the different oligonucleotide substrates (data not shown).
Figure 3: Topoisomerase III protects a 14-base region, surrounding its cleavage site, from nuclease digestion. Nuclease P1 protection reactions were performed as described under ``Materials and Methods.'' The reaction products were resolved through a 25% polyacrylamide gel containing 50% (w/v) urea. The reactions contained no Topo III (lane2) or 0.1 pmol (lane1), 0.25 pmol (lane3), 0.5 pmol (lane4), 1 pmol (lane5), or 2 pmol (lane6) of Topo III. The topoisomerase III-induced cleavage product was obtained by incubation in the absence of P1 nuclease (lane1). The solidbar indicates the position of the Topo III-induced footprint.
Figure 4: Comparison of the DNA protection patterns generated by active Topo III and a catalytically inactive mutant polypeptide. Nuclease P1 protection reactions were performed with either Topo III (lanes1-6) or Topo III-phe328 (lanes7-12) as described under ``Materials and Methods'' and separated through a 15% polyacrylamide gel in the presence of 50% (w/v) urea. Reactions contained no topoisomerase (lanes1 and 7) or 0.25 pmol (lanes2 and 8), 0.5 pmol (lanes3 and 9), 1 pmol (lanes4, 6, 10, and 12), or 2 pmol (lanes5 and 11) of topoisomerase. Nuclease P1 was either included (lanes1-5 and 7-11) or absent (lane6 and 12) from the reaction. The cleavage product present in lanes2-6 is the major Topo III cleavage site present in the oligonucleotide.
Figure 5: Inhibition of Topo III-catalyzed relaxation of supercoiled DNA. Upper panel, DNA cleavage assay of 22-base oligonucleotides. An oligonucleotide containing (lanes1-4) or lacking (lanes5-8) a strong Topo III cleavage site was incubated with no Topo III (lanes1 and 5) or 0.01 pmol (lanes2 and 6), 0.1 pmol (lanes3 and 7), or 1 pmol (lanes4 and 8) of Topo III. Reaction products were separated through a 25% polyacrylamide gel in the presence of 50% (w/v) urea as described under ``Materials and Methods.'' Lower panel, superhelical DNA relaxation assay of Topo III in the presence of increasing levels of oligonucleotide. DNA relaxation assays were performed in the presence (lanes2-6 and 8-12) or absence (lanes1 and 7) of oligonucleotides that either contained no Topo III cleavage site (lanes2-6) or contained a strong Topo III cleavage site (lanes8-12). The oligonucleotide/substrate ratios used in the reactions were: 4 (lanes2 and 8), 20 (lanes3 and 9), 100 (lanes4 and 10), 500 (lanes5 and 11), and 1000 (lanes6 and 12). OC, open circle (nicked or gapped circular) DNA; SC, negatively supercoiled circular DNA.
The 22-base oligonucleotides consisted of a subset of sequence derived from the 45-base oligonucleotide (shown in Fig. 2), which either contained (bases 9-30) or did not contain (bases 1-22) the cleavage site. The presence or absence of the cleavage site was confirmed by incubating the 22-base oligonucleotides with increasing levels of Topo III (Fig. 5, toppanel). It is clear that only one of the oligonucleotides contains a strong cleavage site.
Each oligonucleotide was then assessed for its ability to serve as an inhibitor of Topo III-catalyzed relaxation of negatively supercoiled DNA (Fig. 5, lowerpanel). Both oligonucleotides can serve an inhibitor of Topo III catalysis (presumably by acting as an alternate substrate). At oligonucleotide/substrate ratios of up to 20:1, the oligonucleotide containing the cleavage site (lanes8-12) was approximately five times more effective as an inhibitor than an oligonucleotide without such a site (lanes2-6). However, with greater oligonucleotide/substrate ratios, the oligonucleotide containing the cleavage site appeared to be 10-20 times more effective as an inhibitor than an oligonucleotide without a cleavage site (a 1000-fold excess of the oligonucleotide without the cleavage site (lane6) inhibits to a significantly lesser extent than a 100-fold excess of the oligonucleotide containing a cleavage site (lane10)).
Figure 6: Oligonucleotide gel mobility shift assay of oligonucleotides containing or lacking a strong Topo III cleavage site. Oligonucleotides that either contained (lanes1-5 and lanes11-15) or lacked (lanes6-10) a strong Topo III binding/cleavage site were incubated with no Topo III (lanes1, 6, 11) or 0.4 pmol (lanes2, 7, 12), 1.2 pmol (lanes3, 8, 13), 3.6 pmol (lanes4, 9, 14), or 10.8 pmol (lanes5, 10, 15) of Topo III. The reactions were processed and resolved through a 10% polyacrylamide gel. The position of the stable topoisomerase-oligonucleotide complex is indicated. The amount of the topoisomerase-oligonucleotide complex was determined as described under ``Materials and Methods.'' The positions of the unbound 45-base and 22-base oligonucleotides are indicated.
Interestingly, the 45-base oligonucleotide (which contained the same cleavage site as the 22-base oligonucleotide) was bound far more efficiently than either of the 22-base oligonucleotides (compare lanes2-5 with 7-10 and 12-15). The decrease in binding efficiency observed with decreasing oligonucleotide length also appears to affect the amount of unbound oligonucleotide observed in this analysis. There is a concomitant increase in the amount of stable oligonucleotide-topoisomerase complex with the decrease of unbound oligonucleotide with the 45-base oligonucleotide substrate (lanes2-5); however, this is not observed for the smaller substrates. This is presumably due to the fact that unstable complexes formed with the smaller substrates disintegrate and reform during electrophoresis, resulting in a smear of products throughout the lane.
A 13-base oligonucleotide (a subsequence of the 45-base oligonucleotide) that did not show any evidence of interaction with Topo III (by footprint analysis) was also used as a substrate in a gel mobility shift assay. In accord with previous results, Topo III was capable of interacting with this substrate; however, the amount of stable topoisomerase-oligonucleotide complex was much lower than that formed with either the 22-base oligonucleotide or the 45-base oligonucleotide (data not shown). This length effect may be indicative of the mechanism by which Topo III locates its binding/cleavage site during catalysis.
The minimum substrate requirement for DNA topoisomerase
III-catalyzed cleavage has been examined and found to consist of only 7
bases, asymmetrically encompassing a known cleavage site of the enzyme.
Oligonucleotides containing as little a 1 base 3` of the cleavage site
were cleaved by Topo III, establishing the minimal 3` requirement for
the enzyme. An oligonucleotide containing 6 bases 5` of the cleavage
site and 2 bases 3` of the cleavage site was also cleaved by Topo III;
however, an oligonucleotide containing 5 bases 5` and 2 bases 3` of the
site was not cleaved by the enzyme, establishing a 5`-sequence
requirement of 6 bases. When a Topo III-induced cleavage assay was
performed using the minimal substrate, cleavage was observed; however,
it was reduced 9-fold when compared to the efficiency of cleavage
of an oligonucleotide containing one more base 3` of the minimal
sequence and reduced
5-fold when compared to an oligonucleotide
containing one more base 5` of the minimal sequence.
The minimal sequence requirement for E. coli Topo I has previously been determined to be 7 or 8 bases(18, 19) , but the substrates used in these studies were homopolymers and not an actual cleavage site of the enzyme. In any event, it appears likely that the minimal substrate requirement for the two enzymes is similar. It is unclear, however, if Topo I will show the same asymmetric sequence requirement for cleavage as Topo III.
Nuclease P1 footprinting experiments indicate that the enzyme also binds asymmetrically to its substrate relative to its cleavage site. Topo III was shown to protect a region of the substrate from 12 bases 5` to the cleavage site to 2 bases 3` of the site. An identical footprint was observed using a polypeptide incapable of cleaving the substrate, illustrating that catalysis is not a requirement for cleavage site recognition. Topo III, therefore, is a cleavage site-specific binding protein as well as topoisomerase.
The highly asymmetric sequence requirement of Topo III is consistent with the known catalytic mechanism of prokaryotic topoisomerases. These enzymes cleave DNA by making a nucleophilic attack 5` to the phosphate in the phosphodiester backbone. In this mechanism, the topoisomerase becomes covalently linked to the DNA 3` to the cleavage site via an enzyme-bridged phosphotyrosine linkage (reviewed in (7) ). Since the enzyme is covalently bound to the 3`-DNA fragment, it is not surprising that there may be only a minimal sequence requirement in this region. In fact, these experiments suggest that the enzyme may only require a single nucleotide to serve as a receptor for the nucleophilic attack. To prevent free rotation about the strand scission, however, the enzyme must have considerable noncovalent interactions with the 5`-fragment. This is again consistent with the observed minimal substrate requirement of at least 6 bases 5` to the cleavage site.
The binding properties of Topo III are also reminiscent of a site-specific binding protein. First, an oligonucleotide containing a Topo III cleavage site is more efficiently bound by the enzyme than an oligonucleotide without a site. Second, an oligonucleotide containing a cleavage site is a better inhibitor of Topo III-catalyzed relaxation of negatively supercoiled DNA than an oligonucleotide lacking a cleavage site. Although gel mobility shift experiments indicate that substrates containing a cleavage site are bound more efficiently by the enzyme, Topo III is also capable of interacting with substrates that do not contain a cleavage site. These properties suggest a model for Topo III-catalyzed cleavage of DNA (Fig. 7).
Figure 7: Model of DNA topoisomerase III-catalyzed cleavage of single-stranded DNA. The proposed mechanism of Topo III-mediated cleavage of DNA is presented. The reaction has been separated into three distinct steps (A-C). Briefly, Topo III (represented by the oval) binds randomly to single-stranded DNA. The enzyme then diffuses or tracks (arrows) along its substrate until it encounters a binding/cleavage site (diagonallyfilledrectangle). The enzyme forms a stable complex with its cleavage site through interactions within the active site of the enzyme and then catalyzes a transient break in the single-stranded DNA (the triangle represents the active site tyrosine residue). A detailed explanation of the model is given in the text.
In the first stage of interaction (which we term generalized binding), Topo III binds randomly along its substrate molecule (Fig. 7A). This is consistent with the finding that Topo III can bind to substrates with and without cleavage sites. This model would predict a length dependence of the binding reaction since, in a nonspecific binding interaction, a longer oligonucleotide provides a larger target site per mole of substrate than a smaller oligonucleotide. Once bound to the substrate, the enzyme diffuses, or tracks, along the DNA until it encounters a binding/cleavage site.
Locating a specific binding site via a nondirectional tracking process has been ascribed to Topo III because there is no evidence of a nucleoside triphosphate hydrolysis-driven directional search by these enzymes. In principle, a directional search is a more efficient mechanism to locate sequences that are relatively distant from one another, but the location of both Topo III and Topo I cleavage sites has been found to be, on average, only 10-20 nucleotides apart(4, 20) . The relative abundance of these sites may not require a directional search.
Although the tracking-mediated search proposed in this model is consistent with our experimental results, kinetic experiments will be required to elucidate the actual mechanism by which Topo III locates its binding/cleavage site. Oligonucleotide mobility shift assays only give information on equilibrium binding; therefore, the length effect observed in our experiments, while consistent with the model, is only indicative of the more stable binding of Topo III to the longer oligonucleotide than to the shorter oligonucleotide.
In the second stage of the reaction (which we term sequence recognition and stabilization), the enzyme recognizes and forms a stable complex with its cleavage site (Fig. 7B). In this stage, interactions with amino acids near or within the active site of the enzyme stabilize the enzyme-DNA complex and properly position the substrate within the active site (as illustrated by the ability to generate a nuclease P1 footprint). Positioning of the substrate within the active site is independent of the generalized binding of the substrate since truncations within the carboxyl terminus that drastically reduce the enzyme's binding affinity (>99%) do not alter cleavage site selection by the enzyme(16) .
In the final stage of the reaction (which we term asymmetric cleavage), the enzyme cleaves the substrate (Fig. 7C). During this step of catalysis, the majority of noncovalent interactions occur with DNA 5` to the cleavage site, with only minimal interactions occurring 3` to the site.
The model above has been derived using data obtained from studies using small, single-stranded DNA oligonucleotides as model substrates. These observations can be extended to physiologically relevant reactions catalyzed by topoisomerase III upon double-stranded substrates. Topo III is a single-stranded DNA binding protein with a relatively low affinity for double-stranded DNA(16) ; therefore, Topo III-catalyzed relaxation of negatively supercoiled DNA would require the enzyme to recognize and bind to single-stranded regions present in a negatively supercoiled, predominantly double-stranded molecule. The presence of single-stranded regions within a covalently closed DNA substrate is influenced by the superhelical density of the molecule, the local base composition within the molecule, and the reaction conditions. The optimum reaction conditions for Topo III-catalyzed relaxation activity (i.e. low magnesium optimum and stimulation by high temperatures(4) ) favor the formation of single-stranded DNA and are consistent with this interpretation.
In the case of the decatenation of multiply interlinked plasmid DNA molecules, the enzyme would bind to small single-stranded regions present in the replicated DNA. These regions would be a consequence of lagging strand DNA synthesis, particularly from the removal of the small RNA primers used to initiate DNA synthesis. This is also consistent with the finding that the decatenation of isolated plasmid DNA dimers by Topo III is dramatically stimulated by the presence of small gaps within the substrate(4) .
In this report, we have addressed only a half-reaction in the total scheme of topoisomerase catalysis. There is very little known about the next step of catalysis, strand passage. We are in the process of trying to isolate mutants that affect the strand passage reaction in the hope of being able to further dissect the mechanism of topoisomerase III.