Topoisomerase II drives DNA transport by hydrolyzing one ATP

Abstract

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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Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

“…However, it is unlikely that phosphate release is the overall rate-limiting step for strand passage as single-molecule measurements of Drosophila Topo II indicate that a single tension-dependent, chirality-independent, rate-limiting step governs strand passage (24). Moreover, rapid kinetic measurements showed that ATP hydrolysis is at least 5-fold faster than the strand passage rate at saturating ATP (43). Tension-dependent unlinking measurements of htopo II␣ suggest a more complex relationship between strand passage and tension than was observed with Drosophila Topo II (Fig.…”

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DNA topoisomerase II, genotoxicity, and cancer

“…Second, topoisomerase II uses the energy of adenosine triphosphate (ATP) to drive the overall DNA strand passage reaction [66,69,75,76]. While ATP is not required for either DNA cleavage or ligation, the binding of this nucleoside triphosphate triggers the translocation of DNA through the double-stranded nucleic acid gate [75,77]. ATP hydrolysis is necessary for enzyme recycling [66].…”

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