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 rad9Δ 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.