Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlA(S81F) and GrlA(S81Y) quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg(2+) bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg(2+) concentrations to support DNA cleavage by GrlA(S81F) topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the enzyme-quinolone-DNA ternary complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg(2+) bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically relevant quinolone resistance mutations in bacterial type II topoisomerases.     

Quinolones inhibit DNA religation mediated by Staphylococcus aureus topoisomerase IV. Changes in drug mechanism across evolutionary boundaries

Quinolones are the most active oral antibacterials in clinical use and act by increasing DNA cleavage mediated by prokaryotic type II topoisomerases. Although topoisomerase IV appears to be the primary cytotoxic target for most quinolones in Gram-positive bacteria, interactions between the enzyme and these drugs are poorly understood. Therefore, the effects of ciprofloxacin on the DNA cleavage and religation reactions of Staphylococcus aureus topoisomerase IV were characterized. Ciprofloxacin doubled DNA scission at 150 nM drug and increased cleavage approximately 9-fold at 5 microM. Furthermore, it dramatically inhibited rates of DNA religation mediated by S. aureus topoisomerase IV. This inhibition of religation is in marked contrast to the effects of antineoplastic quinolones on eukaryotic topoisomerase II, and suggests that the mechanistic basis for quinolone action against type II topoisomerases has not been maintained across evolutionary boundaries. The apparent change in quinolone mechanism was not caused by an overt difference in the drug interaction domain on topoisomerase IV. Therefore, we propose that the mechanistic basis for quinolone action is regulated by subtle changes in drug orientation within the enzyme.drug.DNA ternary complex rather than gross differences in the site of drug binding.     

Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage

Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the −1 position, i.e. immediately 5′ of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria.     

Quinolone action against human topoisomerase IIalpha: stimulation of enzyme-mediated double-stranded DNA cleavage

Several important antineoplastic drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. These compounds act by two distinct mechanisms. Agents such as etoposide inhibit the ability of topoisomerase II to ligate enzyme-linked DNA breaks. Conversely, compounds such as quinolones have little effect on ligation and are believed to stimulate the forward rate of topoisomerase II-mediated DNA cleavage. The fact that there are two scissile bonds per double-stranded DNA break implies that there are two sites for drug action in every enzyme-DNA cleavage complex. However, since agents in the latter group are believed to act by locally perturbing DNA structure, it is possible that quinolone interactions at a single scissile bond are sufficient to distort both strands of the double helix and generate an enzyme-mediated double-stranded DNA break. Therefore, an oligonucleotide system was established to further define the actions of topoisomerase II-targeted drugs that stimulate the forward rate of DNA cleavage. Results indicate that the presence of the quinolone CP-115,953 at one scissile bond increased the extent of enzyme-mediated scission at the opposite scissile bond and was sufficient to stimulate the formation of a double-stranded DNA break by human topoisomerase IIalpha. These findings stand in marked contrast to those for etoposide, which must be present at both scissile bonds to stabilize a double-stranded DNA break [Bromberg, K. D., et al. (2003) J. Biol. Chem. 278, 7406-7412]. Moreover, they underscore important mechanistic differences between drugs that enhance DNA cleavage and those that inhibit ligation.     

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.     

DNA cleavage and religation by human topoisomerase II alpha at high temperature.

A common DNA religation assay for topoisomerase II takes advantage of the fact that the enzyme can rejoin cleaved nucleic acids but cannot mediate DNA scission at suboptimal temperatures (either high or low). Although temperature-induced DNA religation assays have provided valuable mechanistic information for several type II enzymes, high-temperature shifts have not been examined for human topoisomerase IIalpha. Therefore, the effects of temperature on the DNA cleavage/religation activity of the enzyme were characterized. Human topoisomerase IIalpha undergoes two distinct transitions at high temperatures. The first transition occurs between 45 and 55 degrees C and is accompanied by a 6-fold increase in the level of DNA cleavage at 60 degrees C. It also leads to a loss of DNA strand passage activity, due primarily to an inability of ATP to convert the enzyme to a protein clamp. The enzyme alterations that accompany the first transition appear to be stable and do not revert at lower temperature. The second transition in human topoisomerase IIalpha occurs between 65 and 70 degrees C and correlates with a precipitous drop in the level of DNA scission. At 75 degrees C, cleavage falls well below amounts seen at 37 degrees C. This loss of DNA scission appears to result from a decrease in the forward rate of DNA cleavage rather than an increase in the religation rate. Finally, similar high-temperature alterations were observed for yeast topoisomerase II and human topoisomerase IIbeta, suggesting that parallel heat-induced transitions may be widespread among type II topoisomerases.     

Effects of antineoplastic drugs on the post-strand-passage DNA cleavage/religation equilibrium of topoisomerase II.

The post-strand-passage DNA cleavage/religation equilibrium of Drosophila melanogaster topoisomerase II was examined. This was accomplished by including adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analogue which supports strand passage but not enzyme turnover, in assays. Levels of post-strand-passage enzyme-mediated DNA breakage were 3-5 times higher than those generated by topoisomerase II prior to the strand-passage event. This finding correlated with a decrease in the apparent first-order rate of topoisomerase II mediated DNA religation in the post-strand-passage cleavage complex. Since previous studies demonstrated that antineoplastic drugs stabilize the pre-strand-passage cleavage complex of topoisomerase II by impairing the enzyme's ability to religate cleaved DNA [Osheroff, N. (1989) Biochemistry 28, 6157-6160; Robinson, M.J., & Osheroff, N. (1990) Biochemistry 29, 2511-2515], the effects of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide on the enzyme's post-strand-passage DNA cleavage complex were characterized. Both drugs stimulated the ability of topoisomerase II to break double-stranded DNA after strand passage. As determined by two independent assay systems, m-AMSA and etoposide stabilized the enzyme's post-strand-passage DNA cleavage complex primarily by inhibiting DNA religation. These results strongly suggest that both the pre- and post-strand-passage DNA cleavage complexes of topoisomerase II serve as physiological targets for these structurally disparate antineoplastic drugs.     

Mode of action of topoisomerase II-targeting agents at a specific DNA sequence. Uncoupling the DNA binding, cleavage and religation events.

Methods of uncoupling the DNA binding, cleavage and religation reactions of topoisomerase II were employed to investigate the influence of topoisomerase II-directed drugs on the individual steps in the enzyme's catalytic cycle. A special DNA substrate containing a major topoisomerase II interaction site, which can be cleaved by the enzyme in the absence of any concomitant religation, was used to examine the effect of topoisomerase II-directed agents upon the DNA cleavage reaction. The experiment demonstrated that the topoisomerase II targeting agent Ro 15-0216 stimulates the DNA cleavage reaction extensively, whereas the traditional topoisomerase II inhibitor, mAMSA, has only a minor effect on this reaction. Topoisomerase II trapped in the cleavage complexes can religate to the 3' hydroxyl end of another DNA strand. Using this religation assay, it was demonstrated that the major effect of mAMSA is an inhibition of the enzyme's religation reaction, whereas Ro 15-0216 has no effect on this reaction. Recently, considerable attention has been given to drugs preventing topoisomerase II from introducing DNA cleavages. In the present paper the initial non-covalent DNA binding reaction of topoisomerase II was investigated under conditions excluding enzyme-mediated DNA cleavage. This demonstrated that the anthracycline, aclarubicin, prevents topoisomerase II from performing its initial non-covalent DNA binding reaction and thereby abolishes the DNA cleavage reaction of the enzyme. The results presented here demonstrate that profound differences exist in the mode of action of different agents targeting topoisomerase II, and that the enzyme can be affected by such agents at both its DNA binding, cleavage and religation subreactions.     

N-acetyl-p-benzoquinone imine, the toxic metabolite of acetaminophen, is a topoisomerase II poison

Although acetaminophen is the most widely used analgesic in the world, it is also a leading cause of toxic drug overdoses. Beyond normal therapeutic doses, the drug is hepatotoxic and genotoxic. All of the harmful effects of acetaminophen have been attributed to the production of its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Since many of the cytotoxic/genotoxic events triggered by NAPQI are consistent with the actions of topoisomerase II-targeted drugs, the effects of this metabolite on human topoisomerase IIalpha were examined. NAPQI was a strong topoisomerase II poison and increased levels of enzyme-mediated DNA cleavage >5-fold at 100 microM. The compound induced scission at a number of DNA sites that were similar to those observed in the presence of the topoisomerase II-targeted anticancer drug etoposide; however, the relative site utilization differed. NAPQI strongly impaired the ability of topoisomerase IIalpha to reseal cleaved DNA molecules, suggesting that inhibition of DNA religation is the primary mechanism underlying cleavage enhancement. In addition to its effects in purified systems, NAPQI appeared to increase levels of DNA scission mediated by human topoisomerase IIalpha in cultured CEM leukemia cells. In contrast, acetaminophen did not significantly affect the DNA cleavage activity of the human enzyme in vitro or in cultured CEM cells. Furthermore, the analgesic did not interfere with the actions of etoposide against the type II enzyme. These results suggest that at least some of the cytotoxic/genotoxic effects caused by acetaminophen overdose may be mediated by the actions of NAPQI as a topoisomerase II poison.     

Mutational analysis of Escherichia coli topoisomerase IV. II. ATPase negative mutants of parE induce hyper-DNA cleavage

ParE is the ATP-binding subunit of topoisomerase IV (Topo IV). During topoisomerization, the ATP-binding and hydrolysis cycle must be coordinated with the cycle of DNA cleavage and religation. We have isolated three dominant-negative mutant alleles of parE that encode ParE proteins that fail to hydrolyze ATP when reconstituted with ParC to form Topo IV. ParE G110S Topo IV and ParE S123L Topo IV failed to bind ATP at all, whereas ParE T201A could bind ATP. All three mutant Topo IV proteins exhibited an elevated level of spontaneous DNA cleavage that could be associated with a decreased rate of DNA resealing. In ParE T201A Topo IV, this defect appeared to result from an increased likelihood that the tetrameric enzyme would fall apart after DNA cleavage. Thus, while ATP is not required for DNA cleavage, the properties of these mutant enzymes suggests that ATP-hydrolysis informs DNA religation.     

Cobalt enhances DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in cleavage reactions. Cobalt(II) stimulated cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.     

The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase

We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.     

New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site.

A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 mM, while the religation reaction seemed unaffected by concentrations as high as 3 M-NaCl. The divalent cations Mg2+, Ca2+ and Mn2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu2+ and Zn2+ inhibited both reactions. Furthermore, the effect of the anti-neoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation.     

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.     

Exocyclic DNA lesions stimulate DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

DNA adducts are mutagenic and clastogenic. Because of their harmful nature, lesions are recognized by many proteins involved in DNA repair. However, mounting evidence suggests that lesions also are recognized by proteins with no obvious role in repair processes. One such protein is topoisomerase II, an essential enzyme that removes knots and tangles from the DNA. Because topoisomerase II generates a protein-linked double-stranded DNA break during its catalytic cycle, it has the potential to fragment the genome. Previous studies indicate that abasic sites and other lesions that distort the double helix stimulate topoisomerase II-mediated DNA cleavage. Therefore, to further explore interactions between DNA lesions and the enzyme, the effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. When located within the four-base overhang of a topoisomerase II cleavage site (at the +2 or +3 position 3' relative to the scissile bond), 3,N(4)-ethenodeoxycytidine, 3,N(4)-etheno-2'-ribocytidine, 1,N(2)-ethenodeoxyguanosine, pyrimido[1,2-a]purin-10(3H)-one deoxyribose (M(1)dG), and 1,N(2)-propanodeoxyguanosine increased DNA scission approximately 5-17-fold. Enhanced cleavage did not result from an increased affinity of topoisomerase IIalpha for adducted DNA or a decreased rate of religation. Therefore, it is concluded that these exocyclic lesions act by accelerating the forward rate of enzyme-mediated DNA scission. Finally, treatment of cultured human cells with 2-chloroacetaldehyde, a reactive metabolite of vinyl chloride that generates etheno adducts, increased cellular levels of DNA cleavage by topoisomerase IIalpha. This finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.     

Effect of antineoplastic agents on the DNA cleavage/religation reaction of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide

Beyond its essential physiological functions, topoisomerase II is the primary cellular target for a number of clinically relevant antineoplastic drugs. Although the chemotherapeutic efficacies of these drugs correlate with their abilities to stabilize the covalent topoisomerase II-DNA cleavage complex, their molecular mechanism of action has yet to be described. In order to characterize the drug-induced stabilization of this enzyme-DNA complex, the effect of etoposide on the DNA cleavage/religation reaction of Drosophila melanogaster topoisomerase II was studied. Under the conditions employed, etoposide increased levels of enzyme-mediated double-stranded DNA cleavage 5-6-fold and single-stranded cleavage approximately 4-fold. Maximal stimulation was observed at 80-100 microM etoposide with 50% of the maximal effect at approximately 15 microM drug. By employing a topoisomerase II mediated DNA religation assay [Osheroff, N. & Zechiedrich, E.L. (1987) Biochemistry 26, 4303-4309], etoposide was found to stabilize the enzyme-DNA cleavage complex (at least in part) by inhibiting the enzyme's ability to religate cleaved DNA. Moreover, in order for the drug to affect religation, it has to be present at the time of DNA cleavage.     

Double-stranded DNA cleavage/religation reaction of eukaryotic topoisomerase II: evidence for a nicked DNA intermediate

The DNA cleavage reaction of eukaryotic topoisomerase II produces nicked DNA along with linear nucleic acid products. Therefore, relationships between the enzyme's DNA nicking and double-stranded cleavage reactions were determined. This was accomplished by altering the pH at which assays were performed. At pH 5.0 Drosophila melanogaster topoisomerase II generated predominantly (greater than 90%) single-stranded breaks in duplex DNA. With increasing pH, less single-stranded and more double-stranded cleavage was observed, regardless of the buffer or the divalent cation employed. As has been shown for double-stranded DNA cleavage, topoisomerase II was covalently bound to nicked DNA products, and enzyme-mediated single-stranded cleavage was salt reversible. Moreover, sites of single-stranded DNA breaks were identical with those mapped for double-stranded breaks. To further characterize the enzyme's cleavage mechanism, electron microscopy studies were performed. These experiments revealed that separate polypeptide chains were complexed with both ends of linear DNA molecules generated during cleavage reactions. Finally, by use of a novel religation assay [Osheroff, N., & Zechiedrich, E. L. (1987) Biochemistry 26, 4303-4309], it was shown that nicked DNA is an obligatory kinetic intermediate in the topoisomerase II mediated reunion of double-stranded breaks. Under the conditions employed, the apparent first-order rate constant for the religation of the first break was approximately 6-fold faster than that for the religation of the second break. The above results indicate that topoisomerase II carries out double-stranded DNA cleavage/religation by making two sequential single-stranded breaks in the nucleic acid backbone, each of which is mediated by a separate subunit of the homodimeric enzyme.     

ATP-bound conformation of topoisomerase IV: a possible target for quinolones in Streptococcus pneumoniae

Topoisomerase IV, a C(2)E(2) tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.     

Yeast topoisomerase II is inhibited by etoposide after hydrolyzing the first ATP and before releasing the second ADP.

Topoisomerase II-catalyzed DNA transport requires coordination between two distinct reactions: ATP hydrolysis and DNA cleavage/religation. To further understand how these reactions are coupled, inhibition by the clinically used anticancer drug etoposide was studied. The IC(50) for perturbing the DNA cleavage/religation equilibrium is nucleotide-dependent; its value is 6 microM in the presence of ATP, 25 microM in the presence of a nonhydrolyzable ATP analog, and 45 microM in the presence of ADP or no nucleotide. This inhibition was further characterized using steady-state and pre-steady-state ATPase and decatenation assays. Etoposide is a hyperbolic noncompetitive inhibitor of the ATPase activity with a K(i)(app) of 5.6 microM no inhibition of ATP hydrolysis is seen in the absence of DNA cleavage. In order to determine which steps of the ATPase mechanism etoposide inhibits, pre-steady-state analysis was performed. These results showed that etoposide does not reduce the rate of binding two ATP, hydrolyzing the first ATP, or releasing the second ADP. Inhibition is therefore associated with the first product release step or hydrolysis of the second ATP, suggesting that DNA religation normally occurs at one of these two steps. Multiple turnover decatenation is inhibited when etoposide is present; however, single turnover decatenation occurs normally. The implications of these results are discussed in terms of their contribution to our current model for the topoisomerase II mechanism.     

In vitro photochemical clastogenicity of quinolone antibacterial agents studied by a chromosomal aberration test with light irradiation

The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Exposure to all test compounds, except for DK-507k, increased the incidence of cells with structural aberrations excluding gap (TA) following light irradiation. Test compounds used in the present study under light irradiation were divided into three groups based on their ED(50) values, doses inducing chromosomal aberrations in 50% of cells. The first group with ED(50) values below 30 microg/ml includes sparfloxacin (SPFX), clinafloxacin (CLFX), gemifloxacin (GMFX), lomefloxacin (LFLX), sitafloxacin (STFX), grepafloxacin (GPFX) and fleroxacin (FLRX); the second group with ED(50) values of 100 microg/ml, enoxacin (ENX) and levofloxacin (LVFX); the third group with little or no potency, moxifloxacin (MFLX), trovafloxacin (TVFX) and DK-507k. The photochemical clastogenicity of these compounds correlates well with their reported in vivo phototoxic potentials. In the chemical structure and clastogenicity relationships, substitution of a methoxy group at the C-8 position in the quinolone nucleus was confirmed to reduce not only photochemical clastogenicity, but also the clastogenic potential of quinolone antibacterial agents.