Effects of DNA Geometry and Topoisomerase II Poisons on Human and Bacterial Type II Topoisomerases

Abstract

The topological state of DNA has a dramatic effect on nucleic acid processes in cells. Type II topoisomerases are necessary enzymes that help regulate the topological state of the genome, but their activity requires the creation of transient DNA breaks. This potential to induce DNA damage is exploited by topoisomerase II poisons, which stabilize the covalent enzyme-cleaved DNA complex and eventually overwhelm the cell with DNA breaks. Although several topoisomerase II poisons are highly successful chemotherapeutic drugs or antibacterials, much about the interactions between topoisomerase II poisons, type II topoisomerases, and their DNA substrates has remained elusive. The first part of this dissertation examines interactions between human topoisomerase IIα and covalent poisons. Thymoquinone, a natural product from black seed, acts as a covalent poison even in its complex herbal form. This suggests that its chemopreventive properties may be related to its effects on topoisomerases. Covalent poisons cannot enhance cleavage mediated by the catalytic core of topoisomerase IIα, but can still inhibit the enzyme if incubated with it prior to the addition of DNA. Therefore, covalent poisons appear to have multiple interaction sites within the enzyme, although not every interaction stabilizes cleavage complexes. The second part of this dissertation describes the recognition of DNA geometry by bacterial type II topoisomerases. Bacillus anthracis gyrase relaxes overwound DNA quickly and processively while maintaining low levels of cleavage complexes, indicating that it is well suited to work on overwound DNA ahead of replication forks and tracking systems. Conversely, topoisomerase IV relaxes overwound DNA faster than underwound molecules but is unable to distinguish supercoil geometry during cleavage. Based on results with different constructs of Mycobacterium tuberculosis gyrase, it appears that the ability to distinguish supercoil geometry during cleavage is embedded within the N-terminal domain. Finally, the last part of this dissertation describes the biochemical basis of interactions between quinolone antibacterials and B. anthracis gyrase. Results indicate that the primary interaction occurs through a conserved water-metal ion bridge. However, altering quinolone substituents can allow for new interactions and overcome resistance.