Abstract
The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. These ternary complexes, called cleaved complexes because the DNA moiety is broken, block replication, transcription, and bacterial growth. Cleaved complexes readily form in vitro when gyrase, plasmid DNA, and quinolone are combined and incubated; complexes are detected by the linearization of plasmid DNA, generally assayed by gel electrophoresis. The stability of the complexes can be assessed by treatment with EDTA, high temperature, or dilution to dissociate the complexes and reseal the DNA moiety. Properties of the complexes are sensitive to quinolone structure and to topoisomerase amino acid substitutions associated with quinolone resistance. Consequently, studies of cleaved complexes can be used to identify improvements in quinolone structure and to understand the biochemical basis of target-based resistance. Cleaved complexes can also be detected in quinolone-treated bacterial cells by their ability to rapidly block DNA replication and to cause chromosome fragmentation; they can even be recovered from lysed cells following CsCl density-gradient centrifugation. Thus, in vivo and cell-fractionation tests are available for assessing the biological relevance of work with purified components.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Suda K, Hicks L, Roberts R, RJHunkler DL (2013) A national evaluation of antibiotic expenditures by healthcare setting in the United States, 2009. J Antimicrob Chemother 68:715–718
Gellert M, Mizuuchi K, O'Dea MH, Itoh T, Tomizawa JI (1977) Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci U S A 74:4772–4776
Sugino A, Peebles C, Kruezer K, Cozzarelli N (1977) Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc Natl Acad Sci U S A 74:4767–4771
Snyder M, Drlica K (1979) DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid. J Mol Biol 131:287–302
Kato J, Nishimura Y, Imamura R, Niki H, Hiraga S, Suzuki H (1990) New topoisomerase essential for chromosome segregation in E. coli. Cell 63:393–404
Hiasa H, Yousef D, Marians K (1996) DNA strand cleavage is required for replication fork arrest by a frozen topoisomerase-quinolone-DNA ternary complex. J Biol Chem 271:26424–26429
Wentzell L, Maxwell A (2000) The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex. J Mol Biol 304:779–791
Willmott CJR, Critchlow SE, Eperon IC, Maxwell A (1994) The complex of DNA gyrase and quinolone drugs with DNA forms a barrier to transcription by RNA polymerase. J Mol Biol 242:351–363
Manes SH, Pruss GJ, Drlica K (1983) Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling. J Bacteriol 155:420–423
Chow R, Dougherty T, Fraimow H, Bellin E, Miller M (1988) Association between early inhibition of DNA synthesis and the MICs and MBCs of carboxyquinolone antimicrobial agents for wild-type and mutant [gyrA nfxB(ompF) acrA] Escherichia coli K-12. Antimicrob Agents Chemother 32(8):1113
Drlica K, Malik M, Kerns RJ, Zhao X (2008) Quinolone-mediated bacterial death. Antimicrob Agents Chemother 52:385–392
Zhao X, Drlica K (2014) Reactive oxygen species and the bacterial response to lethal stress. Curr Opin Microbiol 21:1–6
Dwyer D, Collins J, Walker G (2015) Unraveling the physiological complexities of antibiotic lethality. Annu Rev Pharmacol Toxicol 55:9.1–9.20
Pan XS, Dias M, Palumbo M, Fisher LM (2008) Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage. Nucleic Acids Res 36:5516–5529
Drlica K, Mustaev A, Towle T, Luan G, Kerns R, Berger J (2014) Bypassing fluoroquinolone resistance with quinazolinediones: studies of drug-gyrase-DNA complexes having implications for drug design. ACS Chem Biol 9:2895–2904
Aldred K, McPherson S, Wang P, Kerns R, Graves D, Turnbough C et al (2012) Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance. Biochemistry 51:370–381
Mustaev A, Malik M, Zhao X, Kurepina N, Luan G, Oppegard L et al (2014) Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding. J Biol Chem 289:12300–12312
Aldred K, McPherson S, Turnbough C, Kerns R, Osheroff N (2013) Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance. Nucleic Acids Res 41:4628–4639
Blower TR, Williamson BH, Kerns RJ, Berger JM (2016) Crystal structure and stability of gyrase-fluoroquinolone cleaved complexes from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 113:1706–1713
Laponogov I, Pan X, Veselkov D, McAuley K, Fisher L, Sanderson M (2010) Structural basis of gate-DNA breakage and resealing by type II topoisomerases. PLoS One 5:e11338
Laponogov I, Sohi M, Veselkov D, Pan X, Sawhney R, Thompson A et al (2009) Structural insight into the quinolone-DNA cleavage complex of type IIA topoisomerases. Nat Struct Mol Biol 16:667–669
Bax B, Chan P, Eggleston D, Fosberry A, Gentry D, Gorrec F et al (2010) Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature 466:935–940
Wohlkonig A, Chan P, Fosberry A, Homes P, Huang J, Kranz M et al (2010) Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance. Nat Struct Mol Biol 17:1152–1153
Schoeffler A, May A, Berger J (2010) A domain insertion in Escherichia coli GyrB adopts a novel fold that plays a critical role in gyrase function. Nucleic Acids Res 38:7830–7844
Cohen S, Chang A, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A 69:2110–2114
Oppegard LM, Streck K, Rosen J, Shwanz HA, Drlica K, Kerns RJ et al (2010) Comparison of in vitro activities of fluoroquinolone-like 2,4- and 1.3-diones. Antimicrob Agents Chemother 54:3011–3014
Goss W, Deitz W, Cook T (1965) Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J Bacteriol 89:1068–1074
Acknowledgments
We thank the following for critical comments on the manuscript: Arkady Mustaev and Marila Gennaro.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Luan, G., Drlica, K. (2018). Fluoroquinolone-Gyrase-DNA Cleaved Complexes. In: Drolet, M. (eds) DNA Topoisomerases. Methods in Molecular Biology, vol 1703. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7459-7_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7459-7_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7458-0
Online ISBN: 978-1-4939-7459-7
eBook Packages: Springer Protocols