The long-term objective of the work in this laboratory is to understand the detailed mechanisms of enzymes that use the chemical energy of nucleotide triphosphates to effect structural changes or movements of enzyme-bound DNA. This proposal addresses the mechanism of the type II DNA topoisomerase. More specifically, these studies are designed to understand how this enzyme utilizes ATP binding/hydrolysis to promote the transport of one DNA duplex through a transient, enzyme-mediated break in another. Apart from being mechanistically challenging and fascinating enzymes, type II topoisomerases are biologically and clinically important. These enzymes are essential in all known organisms and are thought to be required for chromatin condensation and the separation of intertwined chromosomes during both mitosis and meiosis. Through regulating the supercoiling of chromosomal DNA, their activity also affects cell growth and gene expression. They are the targets of a long and growing list of antitumor, antibiotic and anti-fungal drugs, and yet their mechanism of action is largely unknown. The proposed approach is to use a combination of rapid kinetic techniques, mutagenesis and protein chemistry. The single-turnover rates of ATP binding and hydrolysis, as well as DNA transport will be measured under a number of reaction conditions by rapid quench-flow. The mechanism by which phosphorylation of the enzyme increases the steady-state reaction rates will be determined. The comformational changes in the protein and protein-DNA complex that accompany ATP binding, ATP hydrolysis and product dissociation will be followed by stopped-flow fluorescence. Two different types of protein cross-linking experiments will be used to determine whether DNA is transported all the way through the enzyme. Studies on mutant/wild type topoisomerase heterodimers will indicate how the protein conformational changes drive DNA transport. Results from these studies will increase our knowledge of how proteins can couple chemical energy to mechanical work, how topoisomerases may be regulated in the cell, and how to better design topoisomerase II-targeting drugs.
Showing the most recent 10 out of 13 publications
