REGULATION OF STRAND SCISSION IN DNA TOPOISOMERASE II: BIOCHEMICAL, COMPUTATIONAL, AND STRUCTURAL STUDIES
Johns Hopkins University
Type II topoisomerases are essential enzymes that generate transient breaks in chromosomes to pass one double-stranded DNA segment through another, supporting a number of critical processes including DNA replication, transcription, and chromosome segregation. In addition to their cellular utility, type IIA topoisomerases are targets of clinically validated antibacterial and anti-cancer agents. The most commonly used class of topoisomerase antagonists are compounds known as ‘poisons,’ which stabilize cleaved-DNA intermediates and convert the enzyme into a genotoxic agent. To better understand the action and selection mechanisms of various poison compounds, I performed biochemical analyses with a series of eukaryotic type IIA topoisomerase variants that impart drug resistance. I also determined the first structure of a human topoisomerase II breakage-reunion region in complex with oligonucleotide DNA and a fluoroquinolone compound. This structure revealed a unique mode of binding compared to previously published structures, identified interactions which may account for specificity of individual fluoroquinolone compounds for human topo II, and highlights molecular contexts that may be useful in the development of novel topoisomerase poisons for chemotherapeutic treatments. To better understand mechanisms that underlie poison sensitivity, I characterized the in vitro activities of human topoisomerase IIβ (hTOP2β) variants sensitized to etoposide poisoning and uncovered a mechanistic interplay between elevated sensitivity to poison compounds and the innate cleavage propensity of the enzyme in the absence of drug. Computational analyses helped to delineate some of the mechanisms underlying breakage-reunion dysfunction and were subsequently used to identify cancer cell samples harboring DNA-damaging hTOP2β variants, highlighting the potential of the enzyme to act as a clastogen capable of maintaining or driving cellular transformation. To better understand how topoisomerases are fundamentally regulated to prevent unwanted breaks, I performed biochemical analyses showing that the ATPase elements of type IIA topoisomerases are not required for strand passage activity, but that their presence moderates aberrant cleavage activity by the enzyme’s DNA binding and cleavage elements. These data lend support to a longstanding hypothesis in the field that the ATPase elements of type II topoisomerases may have been acquired in the course of evolution to suppress erroneous DNA damage by the enzyme.
DNA, topoisomerase, computation, chemotherapeutics, crystallography