A Camptothecin-resistant DNA Topoisomerase I Mutant Exhibits Altered Sensitivities to Other DNA Topoisomerase Poisons

Abstract

The cytotoxic plant alkaloid camptothecin promotes DNA topoisomerase I-linked nicks in DNA by stabilizing a covalently bound enzyme-DNA complex. In the yeast Saccharomyces cerevisiae, substitution of Arg and Ala for the amino acid residues immediately N-terminal to the active site tyrosine in the yeast and human DNA topoisomerase I mutants, top1 vac, results in camptothecin resistance. To examine the mechanism of drug resistance, we assessed the sensitivity of these enzymes to several classes of DNA topoisomerase poisons. Yeast cells expressing the camptothecin-resistant top1 vac mutants were resistant to all of the camptothecin derivatives cytotoxic to wild-type TOP1-expressing cells. This correlated with a significant reduction in drug-induced DNA cleavage in vitro. However, the yeast and human mutant enzymes differed in their responses to the minor groove binding ligand netropsin and to saintopin, a DNA intercalator that targets both DNA topoisomerase I and II. The yeast mutant enzyme demonstrated enhanced sensitivity to the action of saintopin but was resistant to the inhibitory effects of netropsin. In contrast, the human Top1 vac enzyme was resistant to saintopin and indistinguishable from the wild-type enzyme in its response to the netropsin. These results are discussed in terms of enzyme function and the different modes of action of these DNA topoisomerase poisons.

Increased Camptothecin Toxicity Induced in Mammalian Cells Expressing Saccharomyces cerevisiae DNA Topoisomerase I

“…eScTOP1 and eScTop1p). Complementary oligos encoding the sequence MDYKDDDDKAI were cloned into an NcoI site, previously engineered into the sequences encoding the initiating methionine residue in the eScTOP1 plasmid YCpGAL1-TOP1 (17), to yield YCpGAL1-eScTOP1. The epitope tag (underlined in the sequence above) was immediately amino-terminal to the original methionine in the fusion protein eScTop1p.…”

“…* says included wild-type yeast TOP1 (eScTOP1), the camptothecin-resistant mutant eSctop1vac (17,21), a catalytically inactive mutant (eSctop1Y727F) (25), and the lethal mutant eSctop1T722A, which mimics that action of camptothecin in stabilizing the covalent enzyme DNA intermediate (20). Given the difficulties inherent in the selection of cytotoxic phenotypes in mammalian cells, the effects of such lethal mutations on mammalian cell viability have not previously been described.…”

“…These results demonstrate the specificity of camptothecin for eukaryotic DNA topoisomerase I and the utility of using yeast as a model system for the analysis of drug-enzyme interactions. In fact, mutations in yeast and human TOP1 that render the enzyme resistant to camptothecin (17,18) or render the enzyme cytotoxic even in the absence of camptothecin (19,20) 1,2 have been defined using this yeast system.…”

“…These results demonstrate the specificity of camptothecin for eukaryotic DNA topoisomerase I and the utility of using yeast as a model system for the analysis of drug-enzyme interactions. In fact, mutations in yeast and human TOP1 that render the enzyme resistant to camptothecin (17,18) or render the enzyme cytotoxic even in the absence of camptothecin (19, 20) 1,2 have been defined using this yeast system.These results indicate a significant conservation of function between the yeast and human enzymes, consistent with extensive similarities in TOP1 sequences. Nevertheless, differences between these proteins do exist.…”

“…For example, expression of a camptothecin-resistant yeast or human DNA topoisomerase I mutant (top1vac) has different effects on the viability of yeast strains defective in the repair of double strand DNA breaks (17). In vitro, these mutant enzymes exhibit different sensitivities to other DNA topoisomerase I poisons, including saintopin, a DNA intercalator that targets both DNA topoisomerase I and II, and the minor groove binding ligand netropsin (21). The human enzyme plays a direct role in transcriptional activation in vitro (22)(23)(24) and suppresses the basal level of transcription (22).…”