In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites

Gmünder, Hans; Kuratli, Karin; Keck, Wolfgang

doi:10.1093/nar/25.3.604

Cited by 21 publications

(18 citation statements)

References 33 publications

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“…This is in agreement with the results of Cove et al (30) and Gmü nder et al (31) who found that gyrase can cleave DNA fragments as small as 20 bp in the presence of quinolones. DNA fragments smaller than 100 bp were shown to be unable to stimulate efficiently the ATPase of drug-free gyrase and when they did so, the need for DNA binding at two sites on the enzyme was evident (23).…”

Section: Discussionsupporting

confidence: 93%

The DNA Gyrase-Quinolone Complex

Kampranis¹,

Maxwell

1998

Journal of Biological Chemistry

109

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Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis which now proceeds through an alternative pathway involving two different conformations of the enzyme (Kampranis, S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606 -22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence of DNA fragments as small as 20 base pairs. By observing the conversion of the ATPase rate to the quinolone characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone-induced DNA cleavage proceeds via a mechanism consisting of two cleavage events that is modulated in the presence of a nucleotide cofactor. We demonstrate that quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase.DNA gyrase is the intracellular target of the quinolone group of antibacterial agents. The addition of quinolone drugs to an in vitro reaction containing DNA gyrase, relaxed closed-circular DNA, and ATP leads to the inhibition of supercoiling (2). If this reaction is terminated by the addition of a protein denaturant, like SDS, the DNA is found to be cleaved in both strands with a gyrase A subunit (GyrA) 1 covalently attached to each 5Ј-phosphoryl terminus (3). Quinolones also inhibit the relaxation, catenation, and decatenation reactions of gyrase (2, 4).The molecular details of the interaction of the drugs with the gyrase-DNA complex are unknown. When the binding of the drugs to the enzyme was investigated, insignificant binding of quinolones to the A or B subunit (GyrB) or to the gyrase holoenzyme (A 2 B 2 ) was detected (5-7). Binding of the drugs to the DNA is weak, and more efficient in the case of singlecompared with double-stranded DNA (8,9). Quinolones were found to bind strongly to the complex formed by gyrase and DNA, and ATP appeared to assist this interaction (5). These results led to the proposal that the drugs bind to the singlestranded DNA region that is revealed when gyrase cleaves DNA during turnover (10). This proposal was addressed by site-directed mutagenesis of the gyrase active site. Mutation of tyrosine 122 to serine or phenylalanine abolished supercoiling and quinolone-induced DNA cleavage but the mutants could still bind the drugs equally well as wild-type (11). Clearly DNA cleavage is not required for drug binding. Similar results have also been obtained with topoisomerase IV (12).Using limited proteolysis we found that quinolones stabilize a conformational ...

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Section: Discussionsupporting

confidence: 93%

The DNA Gyrase-Quinolone Complex

Kampranis¹,

Maxwell

1998

Journal of Biological Chemistry

109

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“…8Aii); to do this, the cytosine would have to be "flipped out." Hydrogen bonding of quinolones to guanine has been suggested previously (33), and DNA cleavage assays have indicated a preference for G's around the cleavage site (7,15). To explain the role of DNA gyrase, particularly GyrB, in the interaction with quinolones, we propose that, during a normal catalytic cycle, gyrase undergoes a large conformational change involving the 47-kDa subunit of GyrB.…”

Section: Discussionmentioning

confidence: 88%

Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Heddle

Maxwell

2002

Antimicrob Agents Chemother

103

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DNA gyrase is a prokaryotic type II topoisomerase and a major target of quinolone antibacterials. The majority of mutations conferring resistance to quinolones arise within the quinolone resistance-determining region of GyrA close to the active site (Tyr 122 ) where DNA is bound and cleaved. However, some quinolone resistance mutations are known to exist in GyrB. Present structural data suggest that these residues lie a considerable distance from the quinolone resistance-determining region, and it is not obvious how they affect quinolone action. We have made and purified two such mutant proteins, GyrB(Asp 426 3Asn) and GyrB(Lys 447 3Glu), and characterized them in vitro. We found that the two proteins behave similarly to GyrA quinolone-resistant proteins. We showed that the mutations exert their effect by decreasing the amount of quinolone bound to a gyrase-DNA complex. We suggest that the GyrB residues form part of a quinolonebinding pocket that includes DNA and the quinolone resistance-determining region in GyrA and that large conformational changes during the catalytic cycle of the enzyme allow these regions to come into close proximity.Quinolone drugs are a large and widely used class of synthetic antibacterial compounds (10,11,19). First-generation (acidic) quinolones include nalidixic acid and oxolinic acid (OXO). Subsequent generations have been modified to increase spectrum and potency. The most significant modification has been the addition of a fluorine atom at position C-6 in drugs such as ciprofloxacin (CFX), which results in a considerable increase in activity (30). The newer drugs also commonly contain a secondary amine in addition to the carboxylic acid group common to most quinolones, making them amphoteric rather than acidic (Fig. 1A). More recent modifications that increase drug potency include the presence of a methoxy group at C-8 (48).In Escherichia coli the major target for the quinolones is DNA gyrase (14,35). DNA gyrase is a type II topoisomerase that is able to alter the topological state of DNA by cleaving both strands, passing a double strand of DNA through the gap and resealing the ends (6). In the presence of ATP, this results in negative supercoiling, a reaction unique to gyrase. The crystal structures of the 43-kDa N-terminal domain of GyrB (responsible for ATP hydrolysis and capturing a DNA strand) and a 59-kDa fragment of the 64-kDa N-terminal domain of GyrA (containing the residues for DNA binding and cleavage and the quinolone resistance-determining region [QRDR; residues 67 to 106]) have been determined (24, 39). The structure of the 47-kDa C-terminal domain of GyrB is not known but can be inferred from the analogous enzyme from Saccharomyces cerevisiae, topoisomerase II, for which the crystal structures of a 92-kDa region have been solved (5, 12).Resistance to quinolones commonly arises in DNA gyrase via mutations in the QRDR (44). Such mutations include GyrA(Ser 83 3Trp), which gives Ϸ20-fold resistance to a wide range of quinolones (9, 46). This region of GyrA is close to t...

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“…This HTH motif and its counterpart in E. coli gyrase are mutational hotspots for resistance to drugs that stabilize the cleaved state of DNA (15,39). DNA footprinting has shown that for top2 and DNA gyrase approximately 15-35 base pairs of DNA are protected by the enzyme (40,41). In addition, a 29-kDa fragment containing the active-site tyrosine and the HTH motif can be cross-linked to DNA (42), and protein footprinting has demonstrated that the presence of DNA protects the HTH motif from chemical modification (43).…”

Section: Reversibility Of the Etoposide-induced Cleavage Complexesmentioning

confidence: 99%

Mutation of a Conserved Serine Residue in a Quinolone-resistant Type II Topoisomerase Alters the Enzyme-DNA and Drug Interactions

Strumberg

Nitiss²,

Rose³

et al. 1999

Journal of Biological Chemistry

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A Ser740 3 Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser 83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2 S740W in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2 S740W induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position ؊2 and G at position ؉6 were observed for the top2 S740W in addition to the previously reported C؊1 and G؉5 for the wildtype top2. The VP-16-induced top2 S740W cleavage complexes were also more stable. The most stable sites had strong preference for C؊1, whereas the most reversible sites showed no base preference at positions ؊1 or ؊2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser 740 3 Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.DNA topoisomerases are enzymes that catalyze changes in the topology of DNA via a mechanism involving the transient breakage and rejoining of phosphodiester bonds in the DNA backbone (1). Studies in both prokaryotic and eukaryotic cells have demonstrated the importance of topoisomerases in transcription, DNA replication, and chromosome segregation. The type II topoisomerases, which make transient double-strand breaks and change the linking number of DNA in steps of two, play key roles in chromosome structure. In eukaryotic cells, these enzymes are essential for chromosome condensation/decondensation and decatenation of chromosome loops during mitosis (2, 3).Topoisomerase II (top2) 1 has also been identified as a major target of chemotherapeutic agents that are specifically active against prokaryotic (4) or cancer cells (5-8). Fluoroquinolones are antibacterial agents that target DNA gyrase (the prokaryotic type II topoisomerase) (9), whereas a variety of DNAintercalating agents such as anthracyclines, amsacrine, and mitoxantrone and nonintercalating agents such as the epipodophyllotoxin etoposide (VP-16) are active against eukaryotic top2 and are clinically important anti-cancer agents (5-8).Azatoxin is also a nonintercalative top2 poison (10).Recently, it has been shown that 6,8-difluoro-7-(4Ј-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid (CP-115, 953), a fluoroquinolone closely related to ciprofloxacin, is highly toxic to mammalian cells in culture (11,12). Studies in yeast demonstrated that top2 is the primary physiological target for this quinolone (13). Unlike etoposide, which stabilizes top2-mediated DNA cleavage primarily by inhibiting the religation reaction of top2, CP-115,953 stabilizes DNA cleavage by enhancing the forward rate o...

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In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites

Cited by 21 publications

References 33 publications

The DNA Gyrase-Quinolone Complex

The DNA Gyrase-Quinolone Complex

Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Mutation of a Conserved Serine Residue in a Quinolone-resistant Type II Topoisomerase Alters the Enzyme-DNA and Drug Interactions

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