Determinants of Etoposide Hypersensitivity Span All of the Top2 Alpha Catalytic Domains.

Abstract

Type II topoisomerases are found in all kingdoms of life and perform key biological functions during replication, transcription, and other DNA metabolic events. These enzymes are also the targets of important classes of anti-bacterial and anti-cancer drugs. Clinically active drugs targeting topoisomerase II primarily act as poisons rather than as enzyme inhibitors. During the normal enzymatic reaction, topoisomerase poisons stabilize the normally reversible covalent enzyme/DNA adduct leading to DNA damage. More recently, Top2 mutants have been identified that exert the same effect as these small molecules, and expression of these mutants in cells leads to genome instability. Mutations that increase the potency of topoisomerase targeting drugs have the potential to mimic drug action and expand insight into the drug mechanism. Here we carried out an extensive screen of mutations in human Top2α to identify mutations that result in elevated levels of sensitivity to the anti-cancer drug etoposide. This screen was conducted by expressing a library of mutagenized human Top2α that complemented a yeast temperature sensitive Top2 allele in rad52- yeast strains. Etoposide hypersensitive mutations were identified throughout the catalytic domains of Top2α. Mutants were typically hypersensitive to other Top2 inhibitors, although some mutants had hypersensitivity that was specific for etoposide. Several examples of mutant proteins identified in the screen were purified and characterized biochemically. These proteins exhibited a broad range of alterations in enzyme activity including altered requirements for divalent cations, altered relaxation and decatenation activity, altered utilization of ATP, and enhanced DNA cleavage in the absence of inhibitors. Our results suggest that diverse mechanisms influence the ability of small molecules to convert type II topoisomerases to DNA damaging agents, and the etoposide hypersensitivity mutations found provide landmarks for the development of small molecules with improved potency.