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• W2055602768 abstract "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 ∼9-fold at 5 μm. 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. 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 ∼9-fold at 5 μm. 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. Quinolone antibacterials were first synthesized over 30 years ago (1Goss W.A. Deitz W.H. Cook T.M. J. Bacteriol. 1965; 89: 1068-1074Crossref PubMed Google Scholar, 2Hooper D.C. Wolfson J.S. N. Engl. J. Med. 1991; 324: 384-394Crossref PubMed Scopus (492) Google Scholar, 3****Google Scholar, 4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar). Although the founding member of this drug class, nalidixic acid, had limited medical applications, its discovery spawned tremendous interest in the clinical potential of these compounds. Since that initial breakthrough, the use of quinolone-based drugs to treat bacterial infections in humans has grown considerably (2Hooper D.C. Wolfson J.S. N. Engl. J. Med. 1991; 324: 384-394Crossref PubMed Scopus (492) Google Scholar, 3****Google Scholar, 4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar, 6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar). In fact, this class is now the most active and broad spectrum family of oral antibacterial agents in clinical use (2Hooper D.C. Wolfson J.S. N. Engl. J. Med. 1991; 324: 384-394Crossref PubMed Scopus (492) Google Scholar, 3****Google Scholar, 4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar). Quinolones are targeted to the prokaryotic type II topoisomerases, DNA gyrase and topoisomerase IV (6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar, 7Sugino A. Peebles C.L. Kreuzer K.N. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4767-4771Crossref PubMed Scopus (556) Google Scholar, 8Gellert M. Mizuuchi K. O'Dea M.H. Itoh T. Tomizawa J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4772-4776Crossref PubMed Scopus (595) Google Scholar, 9Kato J. Nishimura Y. Imamura R. Niki H. Hiraga S. Suzuki H. Cell. 1990; 63: 393-404Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 10Peng H. Marians K.J. J. Biol. Chem. 1993; 268: 24481-24490Abstract Full Text PDF PubMed Google Scholar, 11Ferrero L. Cameron B. Manse B. Lagneaux D. Crouzet J. Famechon A. Mol. Microbiol. 1994; 13: 641-653Crossref PubMed Scopus (338) Google Scholar, 12Khodursky A.B. Zechiedrich E.L. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11801-11805Crossref PubMed Scopus (299) Google Scholar, 13Froelich-Ammon S.J. Osheroff N. J. Biol. Chem. 1995; 270: 21429-21432Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 14Ng E.Y. Antimicrob. Agents Chemother. 1996; 40: 1881-1888Crossref PubMed Google Scholar, 15Munoz R. De la Campa A.G. Antimicrob. Agents Chemother. 1996; 40: 2252-2257Crossref PubMed Google Scholar, 16Pan X.-S. Ambler J. Mehtar S. Fisher L.M. Antimicrob. Agents Chemother. 1996; 40: 2321-2326Crossref PubMed Google Scholar, 17Gootz T.D. Zaniewski R. Haskell S. Schmieder B. Tankovic J. Girard D. Courvalin P. Polzer R.J. Antimicrob. Agents Chemother. 1996; 40: 2691-2697Crossref PubMed Google Scholar, 18Maxwell A. Trends Microbiol. 1997; 5: 102-109Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 19Drlica K. Zhao X. Microbiol. Mol. Biol. Rev. 1997; 61: 377-392Crossref PubMed Scopus (1156) Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar). These enzymes are essential to all bacterial species and play fundamental roles in most DNA processes (20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar, 21Luttinger A. Mol. Microbiol. 1995; 15: 601-606Crossref PubMed Scopus (74) Google Scholar, 22Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2054) Google Scholar). DNA gyrase, the only known topoisomerase that can actively underwind nucleic acids (23Gellert M. Mizuuchi K. O'Dea M.H. Nash H.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3872-3876Crossref PubMed Scopus (848) Google Scholar), is required for the maintenance of superhelical density in the bacterial chromosome and relieves torsional stress that accumulates in front of replication forks (20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar, 22Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2054) Google Scholar, 24Kreuzer K.N. Cozzarelli N.R. J. Bacteriol. 1979; 140: 424-435Crossref PubMed Google Scholar, 25Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 26Reece R.J. Maxwell A. Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (543) Google Scholar, 27Menzel R. Gellert M. Adv. Pharmacol. 1994; 29: 39-69Crossref Scopus (33) Google Scholar). Topoisomerase IV is responsible for unlinking newly replicated daughter chromosomes and resolving knots that result from recombination events (9Kato J. Nishimura Y. Imamura R. Niki H. Hiraga S. Suzuki H. Cell. 1990; 63: 393-404Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar, 28Hirota Y. Ryter A. Jacob F. Cold Spring Harbor Symp. Quant. Biol. 1968; 33: 677-694Crossref PubMed Scopus (309) Google Scholar, 29Kato J.-I. Nishimura Y. Yamada M. Suzuki H. Hirota Y. J. Bacteriol. 1988; 170: 3967-3977Crossref PubMed Google Scholar). Although these enzymes play different roles in the cell, they both alter the topological state of nucleic acids by passing a double helix through a transient break that they generate in a separate DNA segment (19Drlica K. Zhao X. Microbiol. Mol. Biol. Rev. 1997; 61: 377-392Crossref PubMed Scopus (1156) Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar, 22Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2054) Google Scholar, 26Reece R.J. Maxwell A. Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (543) Google Scholar, 27Menzel R. Gellert M. Adv. Pharmacol. 1994; 29: 39-69Crossref Scopus (33) Google Scholar). Quinolones do not kill bacterial cells by blocking the essential functions of type II topoisomerases. Rather, they increase the cellular concentration of covalent topoisomerase-cleaved DNA complexes that are intermediates in the DNA strand passage reactions of these enzymes (4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar,5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar, 10Peng H. Marians K.J. J. Biol. Chem. 1993; 268: 24481-24490Abstract Full Text PDF PubMed Google Scholar, 19Drlica K. Zhao X. Microbiol. Mol. Biol. Rev. 1997; 61: 377-392Crossref PubMed Scopus (1156) Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar, 24Kreuzer K.N. Cozzarelli N.R. J. Bacteriol. 1979; 140: 424-435Crossref PubMed Google Scholar, 26Reece R.J. Maxwell A. Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (543) Google Scholar, 30Maxwell A. J. Antimicrob. Chemother. 1992; 30: 409-414Crossref PubMed Scopus (157) Google Scholar, 31Anderson V.E. Gootz T.D. Osheroff N. J. Biol. Chem. 1998; 273: 17879-17885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 32Khodursky A.B. Cozzarelli N.R. J. Biol. Chem. 1998; 273: 27668-27677Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). This action generates high levels of double-stranded breaks in the chromosomes of treated bacteria, triggers the SOS response, and ultimately induces cell death (4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar, 26Reece R.J. Maxwell A. Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (543) Google Scholar, 30Maxwell A. J. Antimicrob. Chemother. 1992; 30: 409-414Crossref PubMed Scopus (157) Google Scholar,32Khodursky A.B. Cozzarelli N.R. J. Biol. Chem. 1998; 273: 27668-27677Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Before topoisomerase IV was discovered in 1990, DNA gyrase was believed to be the only significant target for quinolones in bacterial cells. To a great extent, this has proven true for Gram-negative species (4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar, 6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar, 7Sugino A. Peebles C.L. Kreuzer K.N. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4767-4771Crossref PubMed Scopus (556) Google Scholar, 8Gellert M. Mizuuchi K. O'Dea M.H. Itoh T. Tomizawa J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4772-4776Crossref PubMed Scopus (595) Google Scholar,20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar). Although topoisomerase IV is a secondary target for these compounds in Escherichia coli, the cellular consequences of its interaction with quinolones are revealed only in the presence of drug-resistant DNA gyrase mutants (4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar, 12Khodursky A.B. Zechiedrich E.L. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11801-11805Crossref PubMed Scopus (299) Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar). However, in Gram-positive species, topoisomerase IV appears to be the primary cytotoxic target for most quinolones (4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar, 6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar, 11Ferrero L. Cameron B. Manse B. Lagneaux D. Crouzet J. Famechon A. Mol. Microbiol. 1994; 13: 641-653Crossref PubMed Scopus (338) Google Scholar, 14Ng E.Y. Antimicrob. Agents Chemother. 1996; 40: 1881-1888Crossref PubMed Google Scholar, 15Munoz R. De la Campa A.G. Antimicrob. Agents Chemother. 1996; 40: 2252-2257Crossref PubMed Google Scholar, 16Pan X.-S. Ambler J. Mehtar S. Fisher L.M. Antimicrob. Agents Chemother. 1996; 40: 2321-2326Crossref PubMed Google Scholar, 17Gootz T.D. Zaniewski R. Haskell S. Schmieder B. Tankovic J. Girard D. Courvalin P. Polzer R.J. Antimicrob. Agents Chemother. 1996; 40: 2691-2697Crossref PubMed Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar, 33Gootz T.D. Zaniewski R. Haskell S. Schmieder B. Tankovic J. Girard D. Courvalin P. Polzer R.J. Antimicrob. Agents Chemother. 1996; 40: 2691-2697Crossref PubMed Google Scholar). This change in target reflects the increased sensitivity of Gram-positive topoisomerase IV to quinolone-based compounds coupled with the naturally drug-resistant form of gyrase found in these bacteria (4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 20Levine C. Hiasa H. Marians K.J. Biochim. Biophys. Acta. 1998; 1998: 29-44Crossref Scopus (311) Google Scholar,34Hopewell R. Oram M. Briesewitz R. Fisher L.M. J. Bacteriol. 1990; 172: 3481-3484Crossref PubMed Scopus (43) Google Scholar). Quinolones targeted to topoisomerase IV, for the first time, have opened many Gram-positive species to this family of potent antibacterial drugs. This has allowed improved treatment of many infections (especially those of the respiratory tract) that were resistant to other antibiotics (4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar, 6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar, 35Pechère J.-C. Gootz T.D. Eur. J. Clin. Microbiol. Infect. Dis. 1998; 17: 405-412Crossref PubMed Google Scholar). Although Gram-positive topoisomerase IV has become an important new target for antibacterial drug discovery, its interactions with quinolones are poorly understood. Therefore, the present study characterized the effects of quinolones on the DNA cleavage/religation activity of topoisomerase IV fromStaphylococcus aureus. In marked contrast to the actions of antineoplastic quinolones against eukaryotic type II topoisomerases (which increase levels of DNA breakage by stimulating the forward scission reaction), quinolones act primarily by inhibiting the ability of topoisomerase IV to religate cleaved DNA molecules. Even though the functional drug interaction domain on type II topoisomerases has been maintained from bacterial to eukaryotic species, it appears that the mechanistic basis for quinolone action has not been conserved across evolutionary boundaries. Topoisomerase IV was cloned from S. aureus 4220, overexpressed as the separate subunits (GrlA and GrlB) in E. coli, and purified by a modification of the procedure of Hallettet al. (36Hallett P. Grimshaw A.J. Wigley D.B. Maxwell A. Gene (Amst.). 1990; 93: 139-142Crossref PubMed Scopus (81) Google Scholar). Briefly, cells from log-phase cultures were pelleted, resuspended in TED buffer (50 mm Tris-HCl, pH 7.6, 1 mm EDTA, and 5 mm dithiothreitol), lysed by sonication, and repelleted by centrifugation at 18,000 ×g for 30 min. Topoisomerase IV subunits were extracted from both the soluble fraction and the cell pellet. The two extracts were combined and dialyzed against TEDG buffer (TED plus 10% glycerol) and the subunits were purified by the method of Hallett et al. (36Hallett P. Grimshaw A.J. Wigley D.B. Maxwell A. Gene (Amst.). 1990; 93: 139-142Crossref PubMed Scopus (81) Google Scholar) with the following changes. In the first step, the crude fraction was applied to a heparin-Sepharose column, washed with TEDG plus 0.25m NaCl, followed by a linear gradient of 0.25 to 1.0m NaCl in TEDG. Active fractions were combined, concentrated, brought to a final concentration of 1 m(NH4)2SO4 and loaded on a phenyl-Superose FPLC column. Samples were eluted with a linear gradient starting at 1 m(NH4)2SO4. Individual subunits were assayed for catalytic activity using an excess of the complementing subunit. The specific activity of S. aureus topoisomerase IV was 3.1 × 104 decatenation units/mg of protein (decatenating 200 ng of catenated kinetoplast DNA in 30 min at 37 °C). Human topoisomerase IIα was expressed inSaccharomyces cerevisiae (37Wasserman R.A. Austin C.A. Fisher L.M. Wang J.C. Cancer Res. 1993; 53: 3591-3596PubMed Google Scholar) and purified by the protocol of Kingma et al. (38Kingma P.S. Greider C.A. Osheroff N. Biochemistry. 1997; 36: 5934-5939Crossref PubMed Scopus (123) Google Scholar). Etoposide and ciprofloxacin were obtained from Sigma. The quinolone CP-115,953 was synthesized at Pfizer Central Research. Etoposide was stored as a 10 mm stock in dimethyl sulfoxide at 4 °C. Ciprofloxacin and CP-115,953 were stored as 40 and 30 mmstock solutions, respectively, in 0.1 n NaOH at −20 °C, then diluted one-fifth with 10 mm Tris-HCl, pH 7.9, immediately prior to use. Tris and ethidium bromide were obtained from Sigma; SDS and proteinase K were from Merck; restriction endonucleases, calf intestine alkaline phosphatase, and T4 polynucleotide kinase were from New England BioLabs; ATP and [γ-32P]ATP (6000 Ci/mmol) were from Amersham Pharmacia Biotech. All other chemicals were analytical reagent grade. Negatively supercoiled pBR322 DNA was isolated from E. coli as described previously (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A uniquely end-labeled 564-base pair DNA substrate (residues 376–939 in pBR322) was prepared as described by Burden et al.(40Burden D.A. Kingma P.S. Froelich-Ammon S.J. Bjornsti M.-A. Patchan M.W. Thompson R.B. Osheroff N. J. Biol. Chem. 1996; 271: 29238-29244Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). DNA cleavage assays in the absence or presence of drugs were performed as described by Corbett et al. (41Corbett A.H. Zechiedrich E.L. Lloyd R.S. Osheroff N. J. Biol. Chem. 1991; 266: 19666-19671Abstract Full Text PDF PubMed Google Scholar). Briefly, 5 nm negatively supercoiled pBR322 DNA was incubated with 15 nm topoisomerase IV in 20 μl of cleavage buffer (35 mm Tris-HCl, pH 7.9, 10 mmMgCl2, 5 mm dithiothreitol, and 50 μg/ml bovine serum albumin) at 37 °C. Unless stated otherwise, reactions were carried out for 30 min. In drug competition assays, ciprofloxacin and etoposide both were present in the reaction mixture prior to the addition of topoisomerase IV, so that the enzyme was exposed simultaneously to both compounds. DNA cleavage reactions were stopped by the addition of SDS (0.5% final concentration) followed by EDTA (15 mm final concentration). Samples were digested with proteinase K (80 μg/ml final concentration) for 30 min at 45 °C. Following the addition of 60% 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 μg/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. Sites of DNA cleavage were determined in the absence or presence of drugs using the protocol of Burden et al. (40Burden D.A. Kingma P.S. Froelich-Ammon S.J. Bjornsti M.-A. Patchan M.W. Thompson R.B. Osheroff N. J. Biol. Chem. 1996; 271: 29238-29244Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Assay mixtures contained 1.4 nm labeled linear 564-mer DNA (25 ng) and 6 nm topoisomerase IV in cleavage buffer, and were incubated at 37 °C for 15 min. Alternatively, assays contained 60 nm human topoisomerase IIα in 10 mm Tris-HCl, pH 7.9, 100 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, and 2.5% glycerol, and were incubated at 37 °C for 15 min. In both cases, DNA cleavage complexes were trapped by the addition of SDS and samples were digested with proteinase K in the presence of EDTA, as above. DNA cleavage products were precipitated twice with ethanol, dried, and resuspended in 40% formamide, 8.4 mm EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol FF. Samples were subjected to electrophoresis in 8% sequencing gels (42Knab A. Fertala J. Bjornsti M.-A. J. Biol. Chem. 1993; 268: 22322-22330Abstract Full Text PDF PubMed Google Scholar), fixed in 10% methanol, 10% acetic acid, and dried. Reaction products were visualized using a Molecular Dynamics PhosphorImager. Reactions were carried out by the procedure of Anderson et al. (31Anderson V.E. Gootz T.D. Osheroff N. J. Biol. Chem. 1998; 273: 17879-17885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). DNA cleavage/religation equilibria were established in the presence or absence of 5 μmquinolone using negatively supercoiled pBR322 substrates as described in the preceding section. Religation was initiated by shifting the temperature from 37 to 65 °C and stopped at various times up to 120 s by the addition of SDS (0.5% 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. Ciprofloxacin is the most widely prescribed quinolone in clinical use and represents one of the most active oral antibacterial agents currently available (2Hooper D.C. Wolfson J.S. N. Engl. J. Med. 1991; 324: 384-394Crossref PubMed Scopus (492) Google Scholar, 3****Google Scholar, 4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar, 6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar). It is cytotoxic to a broad range of bacteria and has well documented activity against topoisomerase IV from Gram-negative species (2Hooper D.C. Wolfson J.S. N. Engl. J. Med. 1991; 324: 384-394Crossref PubMed Scopus (492) Google Scholar, 4Hooper D.C. Biochim. Biophys. Acta. 1998; 1400: 45-61Crossref PubMed Scopus (132) Google Scholar, 5Gootz T.D. Brighty K.E. Med. Res. Rev. 1996; 16: 433-486Crossref PubMed Scopus (170) Google Scholar, 6Hooper D.C. Clin. Infect. Dis. 1998; 27 Suppl. 1: S54-S63Crossref PubMed Scopus (137) Google Scholar, 9Kato J. Nishimura Y. Imamura R. Niki H. Hiraga S. Suzuki H. Cell. 1990; 63: 393-404Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 12Khodursky A.B. Zechiedrich E.L. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11801-11805Crossref PubMed Scopus (299) Google Scholar, 31Anderson V.E. Gootz T.D. Osheroff N. J. Biol. Chem. 1998; 273: 17879-17885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Therefore, it was chosen as the model quinolone for the present study. As previously observed for its Gram-negative counterpart (31Anderson V.E. Gootz T.D. Osheroff N. J. Biol. Chem. 1998; 273: 17879-17885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), topoisomerase IV from S. aureus displayed a robust DNA scission activity with negatively supercoiled substrates. A time course for DNA cleavage in the absence of drugs is shown in Fig. 1. The DNA cleavage/religation equilibrium of the enzyme was established within 30 s of the start of the reaction, and at a 3:1 ratio of topoisomerase IV:plasmid molecule, ∼5% of the initial DNA substrate was cleaved. This is in contrast to human topoisomerase IIα, which cleaves <1% of the DNA molecules at a 30:1 ratio of enzyme:plasmid (31Anderson V.E. Gootz T.D. Osheroff N. J. Biol. Chem. 1998; 273: 17879-17885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The DNA cleavage/religation equilibrium of S. aureustopoisomerase IV was profoundly affected by addition of ciprofloxacin (Fig. 1, inset, and Fig. 2). DNA scission increased as much as 9-fold in the presence of 5 μm drug. The concentration of quinolone required to double levels of cleavage (i.e. the CC2 value) was ∼150 nm. The effect of ciprofloxacin on the Gram-positive enzyme was even greater than that previously observed forE. coli topoisomerase IV (31Anderson V.E. Gootz T.D. Osheroff N. J. Biol. Chem. 1998; 273: 17879-17885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). With this latter enzyme, cleavage levels increased only 4.5-fold, and ∼500 nmquinolone was required to double scission (Fig. 2). Maximal levels of quinolone-induced DNA cleavage are attained within 1–2 min in the presence of eukaryotic type II topoisomerases fromDrosophila and humans (not shown). By comparison, it takes DNA gyrase ∼1 h to re-establish its cleavage/religation equilibrium when ciprofloxacin is included in reaction mixtures (43Cove M.E. Tingey A.P. Maxwell A. Nucleic Acids Res. 1997; 24: 2716-2722Crossref Scopus (20) Google Scholar). S. aureus topoisomerase IV is intermediate to these two enzymes; maximal levels of DNA scission were observed within 10 min following incubation with ciprofloxacin (Fig. 3). As determined by their effects on DNA religation, drugs enhance DNA cleavage mediated by type II topoisomerases by two alternative (but not mutually exclusive) mechanisms (44Corbett A.H. Guerry P. Pflieger P. Osheroff N. Antimicrob. Agents Chemother. 1993; 37: 2599-2605Crossref PubMed Scopus (25) Google Scholar, 45Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (490) Google Scholar). While some drug classes strongly inhibit enzyme-mediated DNA religation, others have little effect on this reaction. Agents in this latter category presumably act by stimulating the forward rate of DNA cleavage. Specific members of the quinolone family (typified by CP-115,953, which is shown with ciprofloxacin in Fig. 4) display antineoplastic activity and are potent enhancers of DNA scission mediated by eukaryotic type II topoisomerases (31Anderson V.E. Gootz T.D. Osheroff N. J. Biol. Chem. 1998; 273: 17879-17885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 46Barrett J.F. Gootz T.D. McGuirk P.R. Farrell C.A. Sokolowski S.A. Antimicrob. Agents Chemother. 1989; 33: 1697-1703Crossref PubMed Scopus (86) Google Scholar, 47Robinson M.J. Martin B.A. Gootz T.D. McGuirk P.R. Moynihan M. Sutcliffe J.A. Osheroff N. J. Biol. Chem. 1991; 266: 14585-14592Abstract Full Text PDF PubMed Google Scholar, 48Robinson M.J. Martin B.A. Gootz T.D. McGuirk P.R. Osheroff N. Antimicrob. Agents Chemother. 1992; 36: 751-756Crossref PubMed Scopus (96) Google Scholar, 49Elsea S.H. Osheroff N. Nitiss J.L. J. Biol. Chem. 1992; 267: 13150-13153Abstract Full Text PDF PubMed Google Scholar, 50Yamashita Y. Ashizawa T. Morimoto M. Hosomi J. Nakano H. Cancer Res. 1992; 52: 2818-2822PubMed Google Scholar, 51Corbett A.H. Osheroff N. Chem. Res. Toxicol. 1993; 6: 585-597Crossref PubMed Scopus (220) Google Scholar, 52Gootz T. Osheroff N. Hooper D.C. Wolfson J.S. Quinolone Antimicrobial Agents. American Society of Microbiology, Washington, D. C.1993: 139-160Google Scholar, 53Elsea S.H. McGuirk P.R. Gootz T.D. Moynihan M. Osheroff N. Antimicrob. Agents Chemother. 1993; 37: 2179-2186Crossref PubMed Scopus (62) Google Scholar, 54Coughlin S.A. Danz D.W. Robinson R.G. Klingbeil K.M. Wentland M.P. Corbett T.H. Waud W.R. Zwelling L.A. Altschuler E. Bales E. Rake J.B. Biochem. Pharmacol. 1995; 50: 111-122Crossref PubMed Scopus (43) Google Scholar, 55Clement J.J. Burres N. Jarvis K. Chu D.T. Swiniarski J. Alder J. Cancer Res. 1995; 55: 830-835PubMed Google Scholar). These drugs show little or no ability to inhibit DNA religation mediated by these enzymes (47Robinson M.J. Martin B.A. Gootz T.D. McGuirk P.R. Moynihan M. Sutcliffe J.A. Osheroff N. J. Biol. Chem. 1991; 266: 14585-14592Abstract Full Text PDF PubMed Google Scholar, 48Robinson M.J. Martin B.A. Gootz T.D. McGuirk P.R. Osheroff N. Antimicrob. Agents Chemother. 1992; 36: 751-756Crossref PubMed Scopus (96) Google Scholar). Thus, it has been suggested that antineopla" @default.
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• W2055602768 title "Quinolones Inhibit DNA Religation Mediated by Staphylococcus aureus Topoisomerase IV" @default.
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