Project Summary/Abstract Type IIA topoisomerases (topo II) are ubiquitous molecular machines that manage DNA superhelical structure and decatenate DNA entanglements to support critical processes such as transcription, DNA replication, and chromosome segregation. Topo II relies on ATP to capture and pass one DNA segment through a reversible, topo-II mediated, double-strand break in a second DNA segment. With numerous clinically proven antibiotics and anti-cancer drugs targeting the essential, yet risky, activities of these enzymes, the ATP-dependence of the topo II strand passage reaction is well-established; however, how topo IIs use ATP and local DNA interactions to favor unidirectional strand passage activity (ensuring genomic knots are removed, not formed) is unknown. In sharp contrast to other topo IIs, the mitochondrial-specific type IIA topoisomerase from trypanosomes (TxTopoIImt) is reported to possess an unexpected ATP-independent strand passage activity. In addition, TxTopoIImt appears to switch between canonical topo II activities (e.g., decatenating DNA) and the antithetical activity of catenating DNA molecules. Switching of strand passage directionality is believed to be how TxTopoIImt perpetuates networks of mitochondrial DNA, known as kinetoplast or kDNA, which comprise thousands of DNA circles interlocked into a giant structure akin to medieval chain mail. The unique kDNA structure is a hallmark of trypanosomatids, recondite parasites that cause three neglected tropical diseases: African sleeping sickness, Chagas disease, and leishmaniasis. The goal of this project is to develop TxTopoIImt as a therapeutic target by understanding and exploiting the fundamental mechanisms underlying the enzyme’s deviant activities. Strategies outlined in Aim 1 will mechanistically define the strange activities of TxTopoIImt in vitro, which are now possible due to a recent breakthrough in producing soluble TxTopoIImt purified from recombinant sources. Cofactor requirements for TxTopoIImt will be assessed (with in vitro topoisomerase decatenation and supercoil relaxation assays) and the molecular determinants of DNA strand passage directionality will be explored (using singly-catenated DNA substrates of various compositional topologies). The objectives of Aim 2 are to characterize the mechanisms by which small-molecule inhibitors of purified TxTopoIImt (identified with in vitro screens of both clinically known topo II inhibitors and the Johns Hopkins FDA-Approved Drug Library) affect enzymatic activities, and then validate the anti-parasitic therapeutic potential of these inhibitors with killing assays of bloodstream-form African trypanosome cultures. Together, these aims have the potential to offer novel mechanistic insights into general topo II function, establish a molecular understanding of parasitic trypanosomes’ peculiar biology, and support future research efforts to develop novel treatments for trypanosome infections. As such, this project dra...