Alterations in the catalytic activity of yeast DNA topoisomerase I result in cell cycle arrest and cell death.
Eukaryotic DNA topoisomerase I catalyzes the relaxation of supercoiled DNA through a concerted mechanism of DNA strand breakage and religation. The cytotoxic activity of camptothecin results from the reversible stabilization of a covalent enzyme-DNA intermediate. Mutations in two conserved regions of yeast DNA topoisomerase I induced a similar mechanism of cell killing, albeit through different effects on enzyme catalysis. In Top1T722Ap, substituting Ala for Thr722 reduced enzyme specific activity by 3-fold, yet enhanced the stability of the covalent enzyme-DNA complex. In contrast, Top1R517Gp was 1,000-fold less active and camptothecin resistant. Nevertheless, salt-stable DNA-enzyme intermediates were detected. Mutation of the active-site tyrosine abrogated mutant enzyme activity and cytotoxicity, while sublethal levels of top1T722A expression increased rDNA recombination. In checkpoint proficient cells, pGAL1-induced top1 expression coincided with the accumulation of a terminal G2-arrested phenotype. Although the acquisition of this phenotype did not require Rad9p, Top1R517Gp- and Top1T722Ap-induced lethality was enhanced in rad9Delta strains. Thus, despite mechanistic differences between Top1R517Gp and Top1T722Ap, the DNA lesions resulting from the enhanced stability of the covalent enzyme-DNA intermediates were sufficient to cause cell cycle arrest and cell death.
Megonigal
M D
MD
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Fertala
J
J
Bjornsti
M A
MA
D017209:Apoptosis; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D016195:G2 Phase; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D014443:Tyrosine
D014443:Tyrosine; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin