Topoisomerase II is an essential enzyme that controls DNA topology. This enzyme generates a protein-linked DNA break as a catalytic intermediate. This intermediate, known as the cleavage complex, is present in very low levels under normal conditions. Topoisomerase II poisons increase the levels of cleavage complexes and generate double-stranded DNA breaks that are mutagenic and can cause cell death. Abasic sites poison topoisomerase II-alpha. In this dissertation, the effect of exocyclic DNA adducts and abasic sites on enzyme-mediated DNA cleavage were examined. Endogenous DNA lesions such as etheno-dC, etheno-dG, and M1dG were strong topoisomerase II-alpha poisons. Studies of the mechanism by which these lesions poison the enzyme revealed that these lesions did not inhibit enzyme-mediated religation, and that the enzyme did not posses higher affinity for these adducted DNA substrates. These findings suggest that the presence of DNA lesions accelerated the forward rate of topoisomerase II-DNA cleavage complex formation at the chemical scission step. A fluorescence resonance energy transfer system showed a correlation between DNA bending induced by different lesions and the ability of topoisomerase II-alpha to cleave the DNA substrate. Moreover, human cells treated with the alkylating agent 2-chloroacetaldehyde, which induces etheno adducts, displayed increased levels of topoisomerase II-alpha-associated DNA breaks. Furthermore, cells treated with the methylating agent methyl methanesulfonate (MMS) displayed increased levels of enzyme-associated DNA breaks with a parallel increase in the amount of abasic sites. Methylated bases are poor topoisomerase II poisons, but are readily converted to abasic sites by N-methyl purine glycosylase (MPG). In addition, cells overexpressing MPG displayed higher baseline levels of topoisomerase II-alpha-associated DNA breaks. These data suggest that topoisomerase II-alpha interacts with intermediates of the base excision repair pathway. Finally, cells with decreased levels of topoisomerase II-alpha displayed a 40% reduction in the amount of double-stranded DNA breaks generated upon MMS treatment, and displayed a slight resistance to MMS cytotoxicity. These findings demonstrate that topoisomerase II-alpha is able to interact with DNA lesions in a physiological system and that the enzyme mediates a portion of the clastogenic and cytotoxic effects of MMS.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-10112005-183529 |
| Date | 02 November 2005 |
| Creators | Velez-Cruz, Renier |
| Contributors | Michael P. Stone, Neil Osheroff, Walter J. Chazin, Lawrence J. Marnett |
| 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-10112005-183529/ |
| 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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