PhD

dissertationDNA topoisomerase II is a molecular machine that mediates the passage of one DNA duplex through a transient break in another duplex, an essential event for chromosome segregation and cell viability. This process requires coordination between two distinct catalytic reactions: ATP hydrolysis and DNA cleavage/religation. The details linking these two processes are not currently well-understood. Additional characterization of the ATPase mechanism along with how it is correlated with DNA transport are reported here. Topoisomerase II has been found to have a surprisingly complex mechanism for ATP hydrolysis; rapid quench and pulse chase experiments suggest the enzyme binds two ATP and hydrolyzes them sequentially. Two key elements in the mechanism, Pi release and ATP resynthesis were investigated using stopped flow fluorescence and 18O exchange experiments, respectively. Pi release is shown to occur with a lag. Since hydrolysis of one ATP occurs with a burst, the rate-limiting step in the reaction cycle must occur after hydrolysis and before or concurrent with Pi release. The very limited 18O exchange seen is consistent with a slow rate of ATP resynthesis. Together, these data support the sequential hydrolysis mechanism. To gain a better understanding of how the individual ATP active sites participate in the DNA transport process, heterodimeric topoisomerase II mutants were created. The heterodimers contained one wild-type protomer and one protomer mutant in ATP binding or hydrolysis. Using these enzymes the role of steps in the first half of the ATPase mechanism. Steady-state ATPase and DNA relaxation results suggest a mechanism in which two ATP need to bind the enzyme but only one needs to be hydrolyzed for efficient DNA transport. Studies of DNA decatenation in the pre-steady-state support this model and indicate that hydrolysis of the first ATP precedes and accelerates DNA transport. A model for the mechanism for topoisomerase II is presented to illustrate how ATP could be used to drive the transport of one DNA duplex through another