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.
Highlights
DNA topoisomerases catalyze changes in DNA topology through cycles of transient DNA strand breakage and religation
Since DNA synthesis inhibitors abolish the cytotoxic activity of camptothecin [10, 11], the drugstabilized enzyme-DNA adducts are presumably converted into lethal lesions following their interaction with DNA replication forks [12]
The yeast Saccharomyces cerevisiae has been exploited to examine the mechanism of action of camptothecin (13-15, 2931), and specific mutations have been identified in the yeast and human DNA topoisomerase I genes (ScTOP1 and hTOP1, respectively) that affect drug sensitivity [29,30,31]
Summary
DNA topoisomerases catalyze changes in DNA topology through cycles of transient DNA strand breakage and religation (reviewed in Refs. 1-4). The active site tyrosine in eukaryotic DNA topoisomerase I becomes covalently linked to the 3'-end of a single-stranded nick in the DNA duplex, while DNA topoisomerase II becomes linked to staggered 5 '-ends of the cleaved DNA duplex These enzymes play a critical role in DNA replication and transcription and have been shown to be the targets of an ever increasing number of anti-tumor agents [1,2,3,4,5]. Additional antitumor and anti-fungal agents have been identified that act as DNA topoisomerase I poisons [23,24,25,26,27,28] These include indolocarbazole derivatives (KT6006 and KT6528), minor groove binding ligands (bulgarein and netropsin), and bisbenzimide dyes (Hoechst 33258 and Hoechst 33342) [23,24,25,26]. Hoechst 33258, like DNA intercalators, causes an unwinding of DNA at high
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