Covalent Poisons and Catenation: The Effects of Natural Products and DNA Topology on Reactions Catalyzed by Type II Topoisomerases
Dalvie, Esha Deepak
Type II topoisomerases are necessary enzymes that regulate the topological state of DNA by creating transient double-stranded breaks. Topoisomerase II poisons act by stabilizing the covalent enzyme-cleaved DNA complex, eventually overwhelming the cell. Many natural products are topoisomerase II poisons, and natural products and their derivatives are often used as anticancer and antibacterial agents. In addition, type II topoisomerases have shown important interactions with different DNA substrates that affect their actions within cells. Therefore, it is important to learn more about how natural products and DNA topology affect the activities of type II topoisomerases. The first part of this dissertation characterizes the effects of 6’6”-dihydroxythiobinupharidine (DTBN), a natural product of the yellow water lily Nuphar lutes, known for its medicinal effects, on human topoisomerase IIalpha. N. lutea was previously identified in a screen of 341 different natural products for their effects on type II topoisomerase activity. I discovered that DTBN increased the amount of DNA cleavage produced by topoisomerase IIalpha by covalently binding to the enzyme and elucidated several properties that identified it as a covalent poison of human type II topoisomerases. The second part of this dissertation describes the relationship between the catenation reaction of type II topoisomerases and the handedness of DNA supercoils, which form during many cellular processes. Some type II topoisomerases recognize supercoil geometry during DNA relaxation. Another important reaction of these enzymes is catenation, or the tangling of two DNA molecules, as it helps stabilize the cohesion of sister chromatids prior to separation during mitosis. For this project, I tracked the rates of catenation by type II enzymes from different species with negatively supercoiled, positively supercoiled, and relaxed DNA substrates. I determined that human topoisomerase IIalpha and several bacterial topoisomerase IV enzymes showed supercoil handedness preferences during catenation, while human topoisomerase IIbeta showed no preference between the DNA substrates. I also found that type II topoisomerases maintained lower levels of cleavage complexes on catenated DNA than on monomeric substrates.