Topoisomerase II-targeted agents: Poisons, pathways, and drug mimics

Topoisomerase II controls the topology of DNA by passing an intact double helix through a transient double-stranded DNA break that it generates in a separate segment of DNA. In order to maintain the integrity of the genetic material throughout this process, topoisomerase II forms covalent bonds between active site tyrosyl residues and the newly generated termini of the cleaved DNA. This covalent enzyme-cleaved DNA complex is referred to as the cleavage complex. Normally, cleavage complexes are fleeting intermediates in the catalytic cycle of topoisomerase II and are tolerated by cells. However, increases in the concentration of cleavage complexes can initiate a number of mutagenic events.; Despite the significance of topoisomerase II in the life and death of a cell, the pathways by which topoisomerase II-generated DNA breaks are processed and repaired are not fully understood. The work in this dissertation has investigated the mechanistic basis of topoisomerase II poisoning and cellular pathways that repair topoisomerase II-induced DNA damage.; First, the ability of divalent cations to act as topoisomerase II poisons was analyzed. Cobalt(II) was found to increase levels of topoisomerase II-mediated DNA cleavage in vitro and in cultured cells.; Second, budding yeast was used as a model genetic system to dissect the downstream pathways by which cells repair the damage induced by topoisomerase II poisons. A Saccharomyces cerevisiae haploid deletion library was tested for sensitivity to the topoisomerase II poison etoposide. Each component of the single-strand invasion homologous recombination pathway was identified in the screen. In addition, strains deficient in MMS22 displayed an ∼10-fold hypersensitivity to topoisomerase II poisons. Further studies indicated that Mms22p is a nuclear protein that localizes at discrete foci.; Lastly, a screen designed to isolate a mutant human topoisomerase IIalpha that mimicked the effects of topoisomerase II-targeted drugs identified an enzyme with a Glu87 → Lys mutation. Expression of this mutant enzyme reduced viability of recombination-deficient yeast cells as compared to cells expressing the wild-type type II enzyme. In addition, this mutant enzyme mimicked the characteristics of a type II topoisomerase treated with the mixed-function inhibitor ICRF-193.