Topoisomerase IV Catalysis and the Mechanism of Quinolone Action*

Topoisomerase IV is a bacterial type II topoisomerase that is essential for proper chromosome segregation and is a target for quinolone-based antimicrobial agents. Despite the importance of this enzyme to the survival of prokaryotic cells and to the treatment of bacterial infections, relatively little is known about the details of its catalytic mechanism or the basis by which quinolones alter its enzymatic functions. Therefore, a series of experiments that analyzed individual steps of the topoisomerase IV catalytic cycle were undertaken to address these critical mechanistic issues. The following conclusions were drawn. First, equilibrium levels of DNA cleavage mediated by the bacterial enzyme were considerably (>10-fold) higher than those observed with its eukaryotic counterparts. To a large extent, this reflected decreased rates of DNA religation. Second, the preference of topoisomerase IV for catalyzing DNA decatenation over relaxation reflects increased rates of strand passage and enzyme recycling rather than a heightened recognition of intermolecular DNA helices. Third, quinolones stimulate topoisomerase IV-mediated DNA cleavage both by increasing rates of DNA scission and by inhibiting religation of cleaved DNA. Finally, quinolones inhibit the overall catalytic activity of topoisomerase IV primarily by interfering with enzyme-ATP interactions.

Topoisomerase IV is a bacterial type II topoisomerase that is essential for proper chromosome segregation and is a target for quinolone-based antimicrobial agents. Despite the importance of this enzyme to the survival of prokaryotic cells and to the treatment of bacterial infections, relatively little is known about the details of its catalytic mechanism or the basis by which quinolones alter its enzymatic functions. Therefore, a series of experiments that analyzed individual steps of the topoisomerase IV catalytic cycle were undertaken to address these critical mechanistic issues. The following conclusions were drawn. First, equilibrium levels of DNA cleavage mediated by the bacterial enzyme were considerably (>10-fold) higher than those observed with its eukaryotic counterparts. To a large extent, this reflected decreased rates of DNA religation. Second, the preference of topoisomerase IV for catalyzing DNA decatenation over relaxation reflects increased rates of strand passage and enzyme recycling rather than a heightened recognition of intermolecular DNA helices. Third, quinolones stimulate topoisomerase IV-mediated DNA cleavage both by increasing rates of DNA scission and by inhibiting religation of cleaved DNA. Finally, quinolones inhibit the overall catalytic activity of topoisomerase IV primarily by interfering with enzyme-ATP interactions.
Type II topoisomerases alter the topological state of the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate DNA segment (1)(2)(3)(4)(5)(6). Eubacteria contain two distinct members of this enzyme class, DNA gyrase and topoisomerase IV (6). The most well characterized of the two, DNA gyrase, was discovered over 2 decades ago (7). It is unique among type II enzymes in that it is the only known topoisomerase (prokaryotic or eukaryotic) that is capable of introducing negative superhelical twists into relaxed DNA molecules (7). DNA gyrase plays critical roles in DNA replication, recombination, and transcription, as well as in the maintenance of genomic superhelical density (1,2,5,6,8,9).
The second prokaryotic type II enzyme, topoisomerase IV, is comprised of the products of the parC and parE genes (10). Although it was long known that ParC and ParE both were required for proper chromosome segregation in Escherichia coli (11,12), it was not realized until 1990 that these two polypeptides together constitute a novel type II topoisomerase (10). Consistent with its critical role in chromosome segregation, topoisomerase IV displays a prejudice for catalyzing intermolecular DNA strand passage events (i.e. catenation/decatenation reactions) as opposed to intramolecular events (i.e. DNA relaxation reactions) (13)(14)(15)(16). This prejudice notwithstanding, topoisomerase IV appears to relax DNA in vivo, and recent evidence suggests that this relaxation activity plays an important role in maintaining levels of DNA supercoiling in E. coli (17).
Beyond their essential physiological functions, prokaryotic type II topoisomerases are the cytotoxic targets for quinolonebased antimicrobial agents (18 -25). One member of this drug class, ciprofloxacin, is the most active and broad spectrum antibacterial currently in clinical use (23,26,27). Quinolones act in a pernicious fashion. First, they dramatically increase levels of covalent enzyme-cleaved DNA complexes that are requisite but transitory intermediates in the catalytic doublestranded DNA passage reaction of type II enzymes (2,8,(21)(22)(23)25). As a result of this action, quinolones "poison" prokaryotic type II topoisomerases and convert them to potent cellular toxins that create double-stranded breaks in the genome of treated cells (2,22,27). Second, quinolones also inhibit the overall catalytic activity of the type II enzymes (20,21,28,29). In so doing, they further damage bacterial cells by depriving them of the critical functions performed by these ubiquitous enzymes.
Despite the importance of topoisomerase IV to the survival of prokaryotic cells and to the treatment of bacterial infections, relatively little is known about the details of its catalytic mechanism. Furthermore, the basis by which quinolones stimulate DNA cleavage or inhibit the overall catalytic activity of any bacterial type II topoisomerase remains an enigma. Therefore, a series of experiments that analyzed individual steps of the topoisomerase IV catalytic cycle was undertaken to address these critical mechanistic issues. Results provide insight into the underlying basis for the high levels of DNA cleavage generated by topoisomerase IV and the preference of the enzyme for intermolecular DNA substrates. In addition, they provide a framework for interpreting the actions of quinolones against prokaryotic type II topoisomerases.

EXPERIMENTAL PROCEDURES
Topoisomerase IV was purified from E. coli by the procedure of Peng and Marians (21). Human topoisomerase II␣ was expressed in Saccharomyces cerevisiae (30) and purified by the protocol of Kingma et al. (31). Drosophila melanogaster topoisomerase II was isolated from embryonic Kc cells as described by Shelton et al. (32). Ciprofloxacin was obtained from Sigma, and all other quinolones were synthesized at Pfizer Central Research by the procedure of Gilligan et al. (33). Quinolones were stored as 40 mM stock solutions in 0.1 N NaOH and then diluted to 8 mM with Tris-HCl, pH 7.9, immediately prior to use. Tris, ethidium bromide, and APP(NH)P 1 were obtained from Sigma; SDS and proteinase K were from Merck; ATP was from Amersham Pharmacia Biotech; histone H1 was from Boehringer Mannheim; and [␥-32 P]ATP was from Amersham Pharmacia Biotech. All other chemicals were analytical reagent grade.
Preparation of DNA Substrates-Negatively supercoiled pBR322 DNA was isolated from E. coli as described previously (34). Catenated pBR322 was prepared by incubating 5 nM negatively supercoiled pBR322 monomers with 100 nM Drosophila topoisomerase II, 1 mM ATP, and 15 g/ml histone H1 in 500 l of topo II assay buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 50 mM KCl, 0.1 mM EDTA, 5 mM MgCl 2 , and 2.5% glycerol) at 30°C for 60 min. The reaction was stopped by the addition of 75 l of stop solution (0.77% SDS, 77 mM EDTA), and the type II enzyme was digested with proteinase K (final concentration 80 g/ml) at 45°C for 30 min. Samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and resuspended in 15 l of water. As monitored by electrophoresis in agarose gels, Ն95% of the DNA product was catenated.
Catenated DNA (kDNA) was isolated from the kinetoplasts of Crithidia fasciculata as described (35). Monomeric (i.e. decatenated) kDNA was prepared by incubating 5 nM kDNA with 2 nM topoisomerase IV, 1 mM ATP, and 10 mM DTT in 500 l of topo IV assay buffer (40 mM Tris-HCl, pH 7.9, 80 mM potassium glutamate, 8 mM MgCl 2 , and 2.5% glycerol) at 37°C for 60 min. Reactions were stopped, and DNA samples were isolated as described above. As monitored by electrophoresis in agarose gels, Ն95% of the DNA product was monomeric.
DNA Cleavage-DNA cleavage assays in the absence or presence of quinolones were performed as described by Corbett et al. (36). Briefly, to monitor levels of pre-strand passage DNA cleavage, 5 nM negatively supercoiled pBR322 DNA was incubated with 7.5 nM topoisomerase IV and 10 mM DTT in 20 l of topo IV cleavage buffer (40 mM Tris-HCl, pH 7.9, 3 mM MgCl 2 , and 2.5% glycerol) at 37°C for 8 min. Alternatively, assays contained either 75 nM Drosophila topoisomerase II (in topo II assay buffer) or 150 nM human topoisomerase II␣ (in 50 mM Tris-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl 2 , 5 mM ␤-mercaptoethanol, 0.5 mM EDTA, and 2.5% glycerol) and were incubated at 30°C for 6 min or 37°C for 10 min, respectively. Assays that monitored post-strand passage DNA cleavage also contained 1 mM APP(NH)P. In all cases, DNA cleavage reactions were stopped by the addition of SDS (1% final concentration) followed by EDTA (15 mM final concentration). Samples were digested with proteinase K as described above. Following the addition of 2 l of loading buffer (30% sucrose, 0.5% bromphenol blue, and 0.5% xylene cyanole FF in 10 mM Tris-HCl, pH 7.9), DNA products were resolved by electrophoresis in 1% agarose gels in 40 mM Tris acetate, pH 8.3, 2 mM EDTA, and 0.5 g/ml ethidium bromide. DNA bands were visualized by UV light, photographed through Kodak 23A and 12 filters with Polaroid type 665 film, and quantitated by scanning negatives with an E-C apparatus model EC910 densitometer in conjunction with Hoefer GS-370 Data System software. The intensity of bands in the negative was proportional to the amount of DNA present. Double-stranded DNA breaks were monitored by the conversion of negatively supercoiled plasmid to linear molecules.
DNA Religation-Reactions were carried out by the procedure of Robinson and Osheroff (37). DNA cleavage/religation equilibria were established as described in the preceding section in the presence or absence of 20 M ciprofloxacin. Pre-or post-strand passage DNA religation assays were carried out in the absence of a nucleoside triphosphate or in the presence of 1 mM APP(NH)P, respectively. Religation was initiated by shifting the temperature from 37 to 75°C and stopped at various times up to 150 s by the addition of SDS (1% final concentration). Samples were processed and analyzed by agarose gel electrophoresis as described in the preceding section. The apparent first order rate of religation was determined by quantifying the loss of linear DNA.
ATP Hydrolysis-Reactions were performed at 37°C as described by Corbett et al. (36) in 20 l of topo IV assay buffer in the presence of 7.5 nM topoisomerase IV, 50 nM DNA (either catenated or monomeric pBR322 plasmid, or catenated or monomeric kDNA), 1 mM ATP, ϳ10 Ci of [␥-32 P]ATP, 10 mM DTT, and 0 to 20 M ciprofloxacin. Samples (3 l) were removed at various times up to 10 min and spotted onto thin layer cellulose plates impregnated with polyethyleneimine (J. T. Baker Inc.). Following chromatography in freshly made 400 mM NH 4 HCO 3 , levels of ATP hydrolysis were monitored by quantifying the release of inorganic phosphate using a Molecular Dynamics PhosphorImager.
Decatenation Reactions-Assays contained 5 nM kDNA, 2 nM topoisomerase IV, 1 mM ATP, 10 mM DTT, and 0 to 50 M ciprofloxacin and were incubated in 20 l of topo IV assay buffer at 37°C for 15 min. Reactions were terminated by the addition of 3 l of stop solution and digested with proteinase K as above. DNA products were resolved by electrophoresis in 1% agarose gels in 100 mM Tris borate, pH 8.3, 2 mM EDTA, stained with 1 g/ml ethidium bromide, and visualized as described above. The percent decatenation was quantified by determining the appearance of monomeric circular DNA molecules.

Topoisomerase IV Catalysis
The DNA Cleavage/Religation Reaction Mediated by Topoisomerase IV-The double-stranded DNA cleavage/religation cycle is central to the catalytic functions of all type II topoisomerases (1,2,4,38). During the cleavage step of this cycle, these enzymes form a covalent intermediate with the newly created 5Ј-DNA termini generated by scission (1, 2, 4, 38 -40). The DNA cleavage/religation equilibrium established by type II topoisomerases can be monitored by quantitating levels of this covalent cleavage complex (39,41).
During their catalytic cycles, type II topoisomerases undergo two separate DNA cleavage/religation reactions (1,4,37,38,42). The first of the two occurs prior to (and is requisite for) the DNA strand passage reaction step. This "pre-strand passage" reaction can be examined by monitoring DNA cleavage/religation in the absence of ATP (binding of which triggers strand passage) (43). The second reaction occurs subsequent to the DNA strand passage event. This "post-strand passage" reaction can be examined by monitoring DNA cleavage/religation in the presence of a nonhydrolyzable ATP analog such as APP(NH)P, which triggers strand passage but does not allow enzyme recycling (42,43).
Reflecting the fact that type II topoisomerases need to religate cleaved DNA substrates in order to complete their catalytic cycles, the DNA cleavage/religation equilibria of these enzymes generally lie heavily toward religation (1,38,44). Levels of DNA cleavage tend to increase somewhat (ϳ2-4-fold) following strand passage, due primarily to a decrease in the apparent first order rate of post-strand passage DNA religation (at least for the eukaryotic type II enzymes) (37,38,42).
Although the DNA cleavage/religation reactions of topoisomerase IV are essential to its physiological functions, relatively little is known about these enzyme-mediated events. Therefore, three approaches were utilized to describe further these critical reaction steps. In the first, equilibrium levels of pre-strand passage DNA scission generated by E. coli topoisomerase IV were determined. In the absence of ATP, the predominance of religation was far less pronounced for topoisomerase IV than it was for previously characterized type II enzymes. As seen in Fig. 1, equilibrium levels of DNA scission for the bacterial enzyme were dramatically higher (ϳ200-and ϳ40-fold, respectively) than those observed for two other nongyrase type II topoisomerases, human topoisomerase II␣ and Drosophila topoisomerase II. In fact, at equilibrium, ϳ8% of topoisomerase IV appears to be in a covalent DNA cleavage complex as compared with significantly less than 1% for other type II enzymes examined.
In the second approach, equilibrium levels of post-strand passage DNA cleavage were determined. As seen in Fig. 2, levels of scission were (for the most part) equal to or less than those observed in the absence of a nucleoside triphosphate when either APP(NH)P or ATP (which yields an average of preand post-strand passage equilibria) were included in reactions. This result is in marked contrast to previous findings with other non-gyrase type II topoisomerases (37,38,42).
In the last approach, DNA religation reactions mediated by topoisomerase IV were characterized. As seen in Fig. 3, prior to DNA strand passage, the apparent first order rate of religation mediated by topoisomerase IV was nearly an order of magni-tude slower than that previously reported for Drosophila topoisomerase II (0.028 s Ϫ1 versus 0.248 s Ϫ1 ) (37). Furthermore, unlike the Drosophila enzyme, religation rates did not drop (and actually increased slightly) following strand passage. (Apparent first order rates of post-strand passage DNA religation were 0.035 s Ϫ1 and 0.095 s Ϫ1 for the E. coli and Drosophila enzymes, respectively.) Taken together, these results strongly suggest that differences in DNA cleavage/religation equilibria observed for topoisomerase IV and the eukaryotic type II topoisomerases are due (at least in part) to differences in DNA religation rates.
Preference of Topoisomerase IV for Intermolecular DNA Strand Passage-Topoisomerase IV displays a pronounced preference for catalyzing in vitro strand passage reactions in which the two DNA helices are on separate molecules (i.e. decatenation as opposed to relaxation reactions) (13)(14)(15)(16). However, the mechanistic basis that underlies this preference for intermolecular strand passage is unknown.
The ability of topoisomerase IV to distinguish between interand intramolecular DNA substrates may be explained by two distinct (but not mutually exclusive) mechanisms. Either the enzyme preferentially recognizes and binds DNA molecules that contain intermolecular crossovers or it catalyzes higher rates of DNA strand passage with intermolecular substrates.
Two experiments were carried out to distinguish between these possibilities. In the first, the ability of topoisomerase IV to recognize intermolecular substrates was assessed by a DNA cleavage assay. Topoisomerase IV displayed virtually no difference in its propensity to cleave negatively supercoiled monomeric (i.e. intramolecular substrate) or catenated (i.e. intermolecular substrate) pBR322 molecules (Fig. 4). If anything, levels of DNA scission were slightly decreased with catenated substrates. Since the efficiency of cleavage within a given DNA substrate reflects (to a large extent) levels of enzyme-DNA binding (45), this result indicates that the strong decatenation activity of topoisomerase IV is not due to a heightened recognition of substrates with intermolecular DNA helices.
In the second experiment, the effects of inter-versus intramolecular DNA substrates on the ATPase activity of topoisomerase IV were assessed. ATP hydrolysis represents the penultimate step in the topoisomerase IV catalytic cycle and triggers enzyme recycling (3,38). As seen in Fig. 5, the rate of ATP hydrolysis was Ͼ4-fold higher in the presence of kDNA networks than it was with monomeric kDNA molecules. A more dramatic increase was observed when catenated pBR322 molecules were compared with monomeric pBR322 substrates. These data strongly suggest that the preferential decatenase activity of topoisomerase IV results from higher rates of strand passage and enzyme recycling with intermolecular substrates.

Quinolone Mechanism
Prokaryotic type II topoisomerases are the cytotoxic targets for a clinically important class of antibacterials known as quinolones (18 -21, 23-25). One member of this drug class, ciprofloxacin (shown in Fig. 6), is widely prescribed and displays potent activity against a spectrum of pathogenic bacteria (23,26,27). In Gram-negative species, DNA gyrase appears to be the primary target of quinolone-based agents, whereas topoisomerase IV serves as a secondary target and contributes to drug efficacy (10,18,19,(23)(24)(25)29). Beyond the effects of quinolones on enzyme-mediated DNA cleavage, it has been suggested that the ability of these drugs to block the catalytic activity of topoisomerase IV also contributes to their cytotoxicity (20,21,28,29). Finally, in Gram-positive bacterial species, topoisomerase IV rather than DNA gyrase appears to be the primary cellular target of most quinolones (23, 46 -50).
Although quinolone-based drugs play an important role in the treatment of bacterial infections, virtually nothing is known regarding the mechanism by which they stimulate DNA cleavage mediated by prokaryotic type II topoisomerases or block their overall catalytic activities. As a first step toward defining the basis of quinolone action, the effects of ciprofloxacin on topoisomerase IV were delineated.
Stimulation of Topoisomerase IV-mediated DNA Cleavage by Ciprofloxacin-Consistent with previous reports (25,29,51,52), ciprofloxacin was a potent enhancer of DNA scission mediated by topoisomerase IV (Fig. 7). Levels of DNA cleavage doubled at a drug concentration of ϳ500 nM (Table I) and plateaued in the 10 -20 M range. In addition, the concentration dependence of ciprofloxacin-induced DNA cleavage was identical in the absence or presence of APP(NH)P (Fig. 7), indicating that quinolone efficacy is not dependent on the DNA strand passage state of topoisomerase IV.
No previous reports have determined the mechanism by which antibacterial agents stimulate DNA cleavage mediated by prokaryotic type II topoisomerases. However, a body of literature indicates that anticancer drugs targeted to eukaryotic type II topoisomerases stimulate DNA cleavage in one of two primary fashions. They act either by inhibiting the ability of the enzyme to religate cleaved DNA molecules, or by increasing rates of enzyme-mediated DNA cleavage (37,(53)(54)(55)(56)(57)(58).
To determine the mechanistic basis for the enhancement of topoisomerase IV-mediated DNA cleavage by quinolones, the effects of ciprofloxacin on the rate of DNA religation generated by the enzyme were characterized (Fig. 8). Under conditions in which ciprofloxacin stimulated DNA scission ϳ4 -5-fold, the drug decreased the apparent first order rate of pre-strand passage religation from 0.028 s Ϫ1 to 0.018 s Ϫ1 and post-strand passage religation from 0.035 s Ϫ1 to 0.016 s Ϫ1 . Since this inhibition cannot fully account for the levels of cleavage stimulation observed, it is proposed that ciprofloxacin acts both by increasing rates of DNA scission and by inhibiting the ability of topoisomerase IV to religate cleaved DNA.
Sensitivity of Topoisomerase IV to the C-7 Quinolone Ring Substituent-Of the DNA cleavage-enhancing agents described to date, quinolones are the only drugs that display potent activity toward both bacterial and eukaryotic type II topoisomerases (23-25, 59 -62). A distinguishing feature, however, between bacterial DNA gyrase and eukaryotic topoisomerase II is their response to the nature of the substituent at the C-7 position of the quinolone ring (56, 60 -63). Whereas the sensitivity of DNA gyrase is only mildly increased (ϳ2.5-fold) by the substitution of an aromatic hydroxyphenyl ring for an aliphatic piperazine ring (converting ciprofloxacin to CP-115,955, Fig. 6), the sensitivity of human topoisomerase II␣ is dramatically enhanced (ϳ180-fold) by this ring modification (Table I). Similar trends are observed for other quinolones containing a hydroxyphenyl group at C-7.
To determine the response of topoisomerase IV to aromatic substituents in the C-7 quinolone ring position, the sensitivity of the enzyme toward a panel of quinolones containing a C-7 hydroxyphenyl ring was characterized (see Fig. 6). As seen in Table I, topoisomerase IV was relatively insensitive to this distinguishing modification. Thus, while its decatenation (and lack of supercoiling) activity aligns topoisomerase IV "enzymatically" with the eukaryotic type II topoisomerases, its insensitivity to aromatic C-7 quinolone ring substituents aligns the enzyme "pharmacologically" with bacterial DNA gyrase.
Inhibition of Topoisomerase IV Catalysis by Ciprofloxa-cin-In addition to the DNA cleavage-enhancing properties of quinolones, these drugs also inhibit the catalytic DNA strand passage activity of topoisomerase IV (Fig. 9). It has been suggested that this inhibition contributes to the cytotoxicity of quinolone-based drugs to E. coli by blocking chromosome segregation (20,21,28,29). However, the mechanistic basis for this potentially lethal drug effect has yet to be described. The inhibition of topoisomerase IV-catalyzed DNA decatenation by ciprofloxacin can be explained, at least in part, by the ability of the drug to interfere with enzyme-mediated DNA religation (see Fig. 8). However, quinolones may also affect topoisomerase IV at several additional steps of the catalytic cycle of the enzyme. Previous studies on the mechanism of action of antineoplastic agents targeted to eukaryotic type II topoisomerases indicate that many of these DNA cleavageenhancing drugs (including quinolones) disrupt interactions between the enzyme and its ATP cofactor (64 -66). Therefore, to further our understanding of quinolone inhibition, the effects of ciprofloxacin on the ATPase activity of topoisomerase IV were assessed (Fig. 10).
Ciprofloxacin was a potent inhibitor of ATP hydrolysis (IC 50 Ϸ7 M; where IC 50 indicates drug concentration required to inhibit catalysis by 50%). Since this IC 50 value is similar to that observed for inhibition of decatenation (IC 50 Ϸ12 M, Fig. 9), it is likely that obstruction of topoisomerase IV-ATP interactions by quinolones contributes significantly to the inhibition of overall catalytic activity by these drugs. DISCUSSION Topoisomerase IV is ubiquitous among bacteria and is essential for chromosomal segregation in prokaryotic species (10 -12). Despite its importance to the bacterial cell, considerably less is known regarding its catalytic mechanism than that of its sister prokaryotic enzyme, DNA gyrase, or its eukaryotic counterparts.
Of the type II topoisomerases examined to date, E. coli topoisomerase IV generates (by far) the highest levels of DNA scission at equilibrium. This prolific cleavage activity is due in large part to decreased rates of DNA religation. It is not clear how E. coli cells tolerate a topoisomerase that has the potential to create such a large number of breaks in the genetic material. However, it may be related to the fact that topoisomerase IV specifically acts behind replication forks during DNA synthesis, decreasing the probability that transient DNA cleavage com-    plexes would be converted to permanent chromosomal breaks (29). Alternatively, the DNA cleavage activity of topoisomerase IV may be under some form of regulatory control in vivo.
In common with other non-gyrase type II topoisomerases, topoisomerase IV displays a considerable preference for DNA decatenation over relaxation in vitro (13)(14)(15)(16). However, given its role in regulating physiological levels of DNA supercoiling (17), topoisomerase IV must be able to distinguish nucleic acids that contain intermolecular DNA crossovers from those that are predominantly intramolecular in nature by a mechanism that does not obviate its ability to relax chromosomal DNA.
Results of the present study suggest an answer to this apparent paradox. Data indicate that topoisomerase IV recognizes inter-and intramolecular crossovers with equal efficiency but catalyzes higher rates of strand passage with intermolecular DNA substrates. By regulating the DNA decatenation/ relaxation switch at the point of strand passage rather than recognition, topoisomerase IV is able to interact with both catenated and supercoiled substrates and therefore can carry out either reaction as dictated by the cell.
In addition to its essential cellular functions, topoisomerase IV is a target for quinolone antibacterial agents (10, 23, 25, 29, 46 -50). Results suggest that these drugs stimulate topoisomerase IV-mediated DNA scission both by increasing rates of cleavage and by decreasing rates of religation. Furthermore, it appears that quinolones inhibit the catalytic activity of the enzyme primarily by interfering with topoisomerase IV-ATP interactions. Since ATP binding is not required for cleavage (and has little effect on the reaction mediated by topoisomerase IV), this inhibitory mechanism robs the cell of the essential catalytic functions of topoisomerase IV without impacting the ability of quinolones to convert the enzyme into a potent cellular toxin.
Quinolones exhibit the broadest spectrum of action of all DNA cleavage-enhancing drugs known to target type II topoisomerases (23-25, 59 -62). Members of this drug class display potent activity against type II enzymes ranging from bacterial to mammalian species. Furthermore, the enzyme interaction domain for quinolones appears to be conserved throughout evolution (65,(67)(68)(69). The above notwithstanding, a characteristic that distinguishes eukaryotic type II topoisomerases from their prokaryotic counterparts is their dramatic increase in sensitivity to quinolones that contain aromatic groups in the C-7 ring position. As observed previously for DNA gyrase (56,60,61,63), topoisomerase IV was relatively insensitive to the C-7 substituent. This response to quinolones clearly marks topoisomerase IV as a member of the prokaryotic family, despite the fact that the catalytic activity of the enzyme appears to be more closely aligned with the eukaryotic type II topoisomerases than it is with bacterial DNA gyrase.
Topoisomerase IV is critical to the survival of all bacterial species (10,12,13). It plays fundamental roles in many aspects of DNA metabolism and appears to be an emerging target for antibacterial therapy (10, 23, 25, 29, 46 -49). Despite its importance, it remains one of the least well characterized of all the type II topoisomerases. Results of the present study increase our understanding of the mechanism of action of this essential enzyme and further define interactions between topoisomerase IV and quinolone-based therapeutics.