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A study was conducted to examine adenosine triphosphate (ATP) hydrolysis by Saccharomyces cerevisiae yeast topoisomerase II. The enzyme was expressed and purified to high plasmid levels in the yeast strain BCY123 while thin-layer chromatography was utilized to determine the amount of ATP hydrolyzed at each reaction time points. Results showed that yeast topoisomerase II bound to DNA supports burst kinetics in hydrolyzing ATP.

A study was conducted to examine the kinetic mechanism of the sequential hydrolysis of two adenosine triphosphate (ATP) by Saccharomyces cerevisiae yeast topoisomerase II. A pulse-chase experiment was carried out to test whether the two ATP were hydrolyzed sequentially or simultaneously. Results showed that topoisomerase II hydrolyzes only one of its two bound ATP. Findings also suggested that the study's rate constants were composite values that supported conformation step changes.

A covalently cross-linked dimer of yeast DNA topoisomerase II was created by fusing the enzyme with the GCN4 leucine zipper followed by two glycines and a cysteine. Upon oxidation of the chimeric protein, a disulfide bond forms between the two carboxyl termini, covalently and intradimerically cross-linking the two protomers. In addition, all nine of the cysteines naturally occurring in topoisomerase II have been changed to alanines in this construct. This cross-linked, cysteine-less topoisomerase II is catalytically active in DNA duplex passage as indicated by ATP-dependent DNA supercoil relaxation and kinetoplast DNA decatenation assays. However, these experiments do not directly distinguish between a 'one-gate' and a 'two-gate' mechanism for the enzyme.

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 nonhydroiyzable 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.

ATP binding to a single protomer of type II DNA topoisomerase can induce an allosteric change throughout the enzyme. A topoisomerase with a subunit defective in ATP binding which was immunotagged and one wild-type subunit were used to demonstrate that binding at a single subunit can have conformational effects in all subunits. The techniques used should prove useful in the study of allosteric communication among components of multimeric complexes.