Sensitivity of topoisomerase II to anticancer drugs and DNA damage

Type II topoisomerases are essential enzymes that alter DNA topology by creating a transient double-stranded break in one helix and passing a second intact helix through the break. While the fleeting covalent enzyme-DNA intermediate (called the cleavage complex) is tolerated by the cell, the DNA tracking activities of replication or transcription machineries may collide with the cleavage complex and create a permanent double-stranded break in the genome. This potentially lethal consequence of having requisite enzymes that make breaks in the genome is exploited in cancer chemotherapy. Several anticancer agents kill cells by stabilizing the cleavage complex, thereby increasing the likelihood that this protein-linked and reversible DNA break will be converted into a lethal break. Spontaneous forms of DNA damage also have been found to stabilize the cleavage complex, and researchers have proposed that DNA lesions may be endogenous counterparts to these anticancer agents.; To further our understanding of the effects of DNA damage and anticancer drugs on topoisomerase II, the work in my dissertation describes the effects of additional DNA lesions on the enzyme, and mechanisms underlying altered cellular sensitivity to drugs. Several lesions, including 1,N6-ethenoadenine (an adduct formed following exposure vinyl chloride), were identified that also can significantly stabilize topoisomerase II-DNA cleavage complexes. These lesions did not affect the ability of topoisomerase II to religate its substrate. As such, the lesions stabilize the cleavage complex by increasing either DNA binding, the forward rate of cleavage, or both.; Alterations in either topoisomerase II or the DNA repair capacity of a cell can significantly affect topoisomerase II-targeted drug cytotoxicity. Top2G436S, a mutant yeast topoisomerase II, confers resistance to yeast expressing it because the protein is inherently unstable and therefore reduces the level of drug target in the cell. On the other hand, deletion of RAD50, RAD52, or RAD54 greatly increases sensitivity of yeast to killing by these cytotoxic agents, which implicates the single-strand invasion pathway of homologous recombination as being critical to the repair and survival of topoisomerase II-mediated DNA breaks.