Isolation and characterization of suppressors of camptothecin toxicity in Saccharomyces cerevisiae
DNA topoisomerase I catalyzes changes in DNA topology through the transient breakage and religation of one strand of the DNA duplex. The reaction occurs through the formation of a transient enzyme-DNA intermediate, in which the enzyme is covalently bound to one end of the cleaved DNA by a phosphotyrosine linkage. Since the enzyme is able to affect DNA topology, it has been proposed to participate in several processes, including replication, transcription and recombination. The gene encoding DNA topoisomerase I, TOPI, has been shown to be non-essential in yeast. The plant alkaloid anti-tumor drug, camptothecin, is toxic to cells expressing DNA topoisomerase I and acts by stabilizing the covalent enzyme-DNA complex. This represents both a highly specific and reversible interaction. Although levels of DNA topoisomerase I and drug stabilized enzyme-DNA complexes have been shown to remain constant throughout the cell cycle, cell death occurs preferentially during S-phase. These data suggest the involvement of cellular processes, such as DNA replication, in mediating camptothecin cytotoxicity. To identify genes other than TOPI that participate in camptothecin mediated cell death, EMS mutagenized $topI\Delta$ yeast expressing plasmid borne TOPI were screened for mutations which restore cell viability in the presence of drug. Resistant colonies were subsequently rescreened with a second plasmid to eliminate both TOPI and promoter specific mutations. Six dominant mutants were recovered and assigned to one complementation group, SCT1. Fluorescence microscopy of camptothecin treated SCT mutants shows a largely unaffected proliferating cell population, in contrast to the G$\sb2$ arrested wild-type cells. In addition, results obtained from western blot analysis and in vitro catalytic assays, demonstrate that DNA topoisomerase I protein recovered from the mutants is indistinguishable from the wild-type enzyme. Thus, neither post-translational modifications nor altered catalytic activity of the enzyme appear to account for their drug resistance. Experiments with several other DNA damaging agents and topI mutants suggest that resistance is limited to camptothecin induced DNA damage or similar types of damage in the SCT1 mutants. Screening with a genomic library prepared from SCT1-1 DNA has resulted in the isolation of a single clone, which was able to reiterate the camptothecin resistant phenotype. Further characterization of this clone is currently being pursued.
Kauh, Eunkyung Ann, "Isolation and characterization of suppressors of camptothecin toxicity in Saccharomyces cerevisiae" (1996). ETD Collection for Thomas Jefferson University. AAI9633539.