Type II DNA topoisomerases are essential enzymes that unlink intertwined chromosomes. By altering DNA topology they participate in chromosome segregation, condensation, replication, recombination and transcription. These enzymes are the targets of numerous clinically-used and trial-phase anticancer and antibiotic drugs, making the understanding of their mechanism medically relevant. The reaction these enzymes catalyze is the ATP-dependent transport of one segment of DNA through a transient break in a second segment of DNA. Recent pre-steady-state ATPase results indicate that the homodimeric topoisomerase II binds two ATP to initiate the reaction cycle, but hydrolyzes only one rapidly. The second ATP is hydrolyzed later in the reaction cycle, after a rate-determining step in the reaction. This strict sequential hydrolysis of the two ATP was completely unexpected and does not agree with any previous models for the mechanism of this enzyme. The goals of the present proposal are to further elucidate the ATPase mechanism, analyze the protein-DNA complex conformational changes during the reaction cycle, and determine when in this cycle DNA transport actually occurs. By combining all of these pre-steady-state and steady- state results, a more accurate mechanism for DNA topoisomerase II catalyzed reactions will be determined.