Regulation of DNA Cleavage by the ATPase Domain of DNA Topoisomerase II
thesisposted on 2019-08-01, 00:00 authored by Matthew Allen Gilbertson
The human type II DNA topoisomerases, topoisomerase II alpha and beta (Top2α and Top2β) are targeted by a variety of small molecules in anti-cancer therapies. Small molecules like etoposide and doxorubicin prolong a normally short-lived protein-DNA covalent intermediate, leading to DNA damage and cell death. Therefore, this class of inhibitors is referred to as Top2 poisons. Targeting Top2 is therapeutically effective but the molecular details of drug action are poorly understood. Our laboratory performed a yeast-based screen to isolate Top2α alleles that confer hypersensitivity to etoposide. We identified mutations throughout the Top2α enzyme, many of which were located far from the etoposide-binding site, and these alleles frequently conferred cross-sensitivity to other Top2 targeting agents. I purified and biochemically characterized several of the Top2α mutant proteins, located in the ATPase domain. I found that these mutant proteins exhibited altered ATP utilization and hydrolysis. These results suggested that ATPase domain affects the regulation of Top2 mediated strand breaks. We also characterized a unique class of Top2 mutant alleles that we term self-poisoning Top2, because they mimic the action of Top2 poisons in the absence of drug. We characterized a de novo mutation, also located in the ATPase domain, which was identified in a human patient with developmental delay and autism spectrum disorder. We found that the purified mutant protein exhibited high levels of drug independent DNA cleavage. We demonstrated that expression of self-poisoning mutant alleles can lead to genotoxicity and cytotoxicity in yeast. This evidence suggests a mechanism by which Top2 alterations could lead to pathogenic changes in genome stability.