Topoisomerase II drives DNA transport by hydrolyzing one ATP

DNA topoisomerase II is a homodimeric molecular machine that couples ATP us age to the transport of one DNA segment through a transient break in anothe r segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is k nown 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. Usi ng rapid-quench techniques, topoisomerase II recently was shown to hydrolyz e its two bound ATPs in a strictly sequential manner. This result is incong ruous 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 featu res of this enzyme's mechanism previously have been overlooked because of t he reliance on nonhydrolyzable analogs for studying a single reaction turno ver. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.