Determination of Decatenation Activity of Topoisomerase II Mutant Alleles
Gillentine, Madelyn A.
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Topoisomerases are the magicians of the DNA world, functioning as enzymes to decatenate, or unlink, DNA. Topoisomerase II is the enzyme responsible for unlinking double stranded DNA, which is crucial for segregation of sister chromatids in mitosis across all organisms. Recently, a fourth cell cycle checkpoint, the topoisomerase II/decatenation checkpoint, thought to monitor topoisomerase II activity, has been uncovered with implications in cancer research. For decades, topoisomerase II has been a target of anticancer drugs, and activation of its checkpoint may result in protected tumor cells. Therefore, it is important to understand how topoisomerase II and its checkpoint function in order to understand the molecular nature of cancer treatment. This study hypothesized that mutant topoisomerase II alleles would decrease the decatenation activity of topoisomerase II. An assay to measure the decatenation activity of topoisomerase II in S. cerevisiae was developed, as yeast are ideal model organisms due to their charactized cell cycle and genetic malleability. Ideal conditions to quantify wild-type topoisomerase II activity were achieved. Two mutant allele strains known to activate the topoisomerase II/decatenation checkpoint were assayed: top2-B44, containing a point mutation, and the degron strain, which targets endogenous topoisomerase II for degradation. top2-B44 cells were found to have no detectable activity in the assay, raising questions about the recruitment of topoisomerase II to catenated genomic DNA. Conditions to assay the degron allele activity were not achieved due to the leaky GAL-UBR1 promoter. Further manipulation of this system may result in conditions to further explore the topoisomerase II/decatenation checkpoint.