Type II topoisomerases are a ubiquitous class of proteins that use ATP to actively transport one DNA duplex through another. This reaction is essential for supercoiling homeostasis and resolving cytotoxic chromosome tangles prior to cell division. Type II topoisomerases are also targeted by drugs that serve as frontline clinical therapies for cancer and bacterial infections. The long-term objective of this proposal is to investigate the molecular basis of type II topoisomerase function and drug inhibition. Although rough framework for the catalytic cycle of these enzymes is in place, there remain many critical questions surrounding the mechanisms by which type II topoisomerases discriminate between different topological states of DNA, undergo allosteric transitions to drive DNA transport, and are inhibited by small molecule """"""""poisons"""""""" that stimulate DNA cleavage. Using a combination of structural, biochemical, and biophysical methods we aim to fill these gaps by: 1) Determining the structure of a """"""""poisoned"""""""" type II topoisomerase/DNA complex, 2) Establishing how a novel DNA binding and bending domain in bacterial type II topoisomerases controls substrate selectivity and functional output, and 3) Defining the molecular mechanisms and kinetics of DNA deformations and key structural rearrangements in the topoisomerase catalytic cycle. Our proposed studies will define the physical events by which type II topoisomerases facilitate the passage of one DNA segment through another to globally control DNA topology, and by which anticancer and antibacterial inhibitors subvert enzyme function. Data resulting from such efforts broadly impact a number of important scientific fronts, from understanding the dynamics of ATP-dependent molecular machines and regulation of chromosome superstructure, to defining the physical action of anti-topoisomerase therapies and aiding drug development. PROJECT NARRATIVE: Type II topoisomerases are molecular machines that disentangle DNA, as well as validated targets for frontline antibacterial and anticancer therapies. This proposal aims to understand the biochemistry and mechanics of the topoisomerase reaction, and to determine how some of the most widely-used anti-topoisomerase drugs block enzyme function.
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