DNA gyrase as a drug target

Maxwell, Anthony

doi:10.1042/bst0270048

Cited by 130 publications

(82 citation statements)

References 12 publications

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“…These enzymes are the targets of numerous antibiotic and anticancer drugs (1)(2)(3)(4), and their mechanisms have been well studied (for recent reviews, see Refs. 5 and 6).…”

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confidence: 99%

“…The eukaryotic enzymes are homodimers (7-10) whose primary dimer interface is near the COOH terminus (11). The enzyme cleaves one segment of DNA, the gated or G segment, 1 through the transient covalent attachment with a pair of active site tyrosines (12,13). The enzyme traps a second segment of DNA, the transported or T segment, when ATP binding causes the NH 2 -terminal ATPase domains to dimerize (14 -17).…”

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confidence: 99%

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Steady-state and Rapid Kinetic Analysis of Topoisomerase II Trapped as the Closed-clamp Intermediate by ICRF-193

Morris¹,

Baird²,

Lindsley

2000

Journal of Biological Chemistry

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DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme. Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC 50 of 6.5 ؎ 1 M for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP. When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP. By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP. These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.Type II DNA topoisomerases are essential and ubiquitous enzymes that catalyze the ATP-dependent transport of one double-stranded DNA through an enzyme-mediated break in another. These enzymes are the targets of numerous antibiotic and anticancer drugs (1-4), and their mechanisms have been well studied (for recent reviews, see Refs. 5 and 6). The eukaryotic enzymes are homodimers (7-10) whose primary dimer interface is near the COOH terminus (11). The enzyme cleaves one segment of DNA, the gated or G segment, 1 through the transient covalent attachment with a pair of active site tyrosines (12, 13). The enzyme traps a second segment of DNA, the transported or T segment, when ATP binding causes the NH 2 -terminal ATPase domains to dimerize (14 -17). This form of the enzyme has been called the closed-clamp intermediate (16). The enzyme rapidly hydrolyzes one of its two bound ATP molecules (18) and then transports the T segment through the cleaved G segment (19). Only after the first ADP has been released from the enzyme does the topoisomerase hydrolyze the second ATP (20). This unusual type of sequential ATP hydrolysis mechanism is presently only known to be shared with the DNA polymerase clamp-loading enzymes (21).There are two very different types of drugs that inhibit topoisomerase II: the DNA-cleaved complex-stabilizing drugs exemplified by etoposide and the catalytic inhibitors including the bisdioxopiperazines (e.g. (22,23). The in vivo target of ICRF-193 and related compounds was shown to be t...

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“…These enzymes are the targets of numerous antibiotic and anticancer drugs (1)(2)(3)(4), and their mechanisms have been well studied (for recent reviews, see Refs. 5 and 6).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Steady-state and Rapid Kinetic Analysis of Topoisomerase II Trapped as the Closed-clamp Intermediate by ICRF-193

Morris¹,

Baird²,

Lindsley

2000

Journal of Biological Chemistry

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“…The N-terminal half of the enzyme is homologous to Escherichia coli gyrase B and contains the ATPase active site; the C-terminal half is homologous to gyrase A and contains the active site tyrosine for DNA cleavage and the primary dimerization interface. In addition to being mechanistically fascinating, the prokaryotic and eukaryotic members of this enzyme family are the targets of numerous antibiotic and anticancer drugs, respectively (5)(6)(7).…”

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confidence: 99%

Topoisomerase II drives DNA transport by hydrolyzing one ATP

Baird

Harkins

Morris

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

124

128

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DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport. Current models for the enzyme's mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction. Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner. This result is incongruous with the models based on the nonhydrolyzable ATP analog data. Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport. These results indicate that important features of this enzyme's mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.T ype II DNA topoisomerases are ubiquitous enzymes essential for the unlinking of intertwined chromosomes, chromosomal condensation͞decondensation, and manipulation of DNA supercoiling (1, 2). These enzymes catalyze the ATP-dependent transport of one segment of DNA through a transient, enzymemediated break in a second DNA segment (for recent reviews, see refs. 3 and 4). The eukaryotic topoisomerase II enzymes are homodimers, with a monomer molecular mass of Ϸ160-180 kDa, making them relatively simple macromolecular machines for studying the coupling of ATP usage to complex protein movements. The N-terminal half of the enzyme is homologous to Escherichia coli gyrase B and contains the ATPase active site; the C-terminal half is homologous to gyrase A and contains the active site tyrosine for DNA cleavage and the primary dimerization interface. In addition to being mechanistically fascinating, the prokaryotic and eukaryotic members of this enzyme family are the targets of numerous antibiotic and anticancer drugs, respectively (5-7).The mechanism of topoisomerase II is known to involve several steps and associated protein conformational changes (reviewed in ref. 4). The enzyme binds a segment of DNA, the gate or G segment. Both strands of this DNA segment are cleaved and religated by a pair of active site tyrosines, one in each monomer of the dimer. The cleavage reaction results in a four-base staggered break in the DNA, with an enzyme monomer covalently attached to each 5Ј phosphate. Upon binding ATP, the amino-terminal ATPase domains of the enzyme dimerize, capturing a second segment of DNA, the transport or T segment, within the enzyme clamp. The ends of the cleaved G segment are separated, and the T segment is transported through the opening. The gate in the G segment is closed. The T segment is transported out of the topoisomerase, most likely through the C-terminal dimerization interface. The amino terminal dimerization interface re...

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“…Quinolo- nes are capable to inhibit DNA synthesis that results in fragmented DNA and leads to the rapid bacterial cell death [9,10]. For a long time the mechanism of action of fluoroquinolone antibiotics has not been well understood.…”

Section: Issn 2308-8303mentioning

confidence: 99%