Human topoisomerase I and topoisomerase IIα and IIβ play critical roles in regulating the topological state of DNA and are targets for some of the most widely prescribed anticancer drugs currently in clinical use. These drugs act by stabilizing covalent-topoisomerase cleavage complexes that are requisite intermediates in the enzyme catalytic cycles. It is the accumulation of these cleavage complexes ahead of DNA tracking systems, on overwound DNA, that ultimately leads to cell death. Therefore, work in this dissertation more fully characterizes the effects that DNA interactions have on the activities of topoisomerase I and topoisomerase II and drugs that target these enzymes. Results show that all clusters of positively charged residues located in the C-terminal domain of topoisomerases IIα are necessary for the preferential relaxation of overwound substrates. Experiments also show that topoisomerase I preferentially relaxes and cleaves overwound substrates in the absence or presence of anticancer drugs. Additionally, DNA intercalating compounds were found to act as topological poisons of topoisomerase I, enhancing cleavage activity on underwound substrates and in cells by changing the apparent topological state of the DNA. Finally, F14512, an etoposide derivative that contains a spermine moiety in place of the C4 carbohydrate, was shown to be a more potent poison of human topoisomerase II than the parent compound. The drug shows similar contacts as etoposide in the enzyme-drug binary complex but, unlike etoposide, interacts with DNA in the absence of enzyme. The enhanced activity of the drug correlates with a stronger interaction of the compound in the ternary enzyme-drug-DNA complex, and the covalent linkage of the spermine moiety to the drug core is necessary for this enhanced activity. Together, data presented in this dissertation increase our understanding of the role that topoisomerase-DNA interactions play in enzyme regulation of DNA topology and highlight the importance of enzyme-DNA-drug interactions when designing new topoisomerase-targeted compounds with potentially enhanced therapeutic potential.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03152011-094529 |
| Date | 18 March 2011 |
| Creators | Gentry, Amanda Cormine |
| Contributors | Dr. Daniel Kaplan, Dr. Martin Egli, Dr. Zu-Wen Sun, Dr. Michael Stone, Dr. Neil Osheroff |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03152011-094529/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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