Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of the antitumor drug camptothecin (Cpt). Mutation of several conserved residues in yeast top1 mutants is sufficient to induce cell lethality in the absence of camptothecin. Despite tremendous differences in catalytic activity, the mutant proteins Top1T722Ap and Top1R517Gp cause cell death via a mechanism similar to that of Cpt, i.e. stabilization of the covalent enzyme-DNA intermediate. To establish the interdomainal interactions required for the catalytic activity of Top1p and how alterations in enzyme structure contribute to the cytotoxic activity of Cpt or specific DNA topoisomerase I mutants, we initiated a genetic screen for intragenic suppressors of the top1T722A-lethal phenotype. Nine single amino acid substitutions were defined that map to the conserved central and C-terminal domains of Top1p as well as the nonconserved linker domain of the protein. All reduced the catalytic activity of the enzyme over 100-fold. However, detailed biochemical analyses of three suppressors, top1C273Y, T722A, top1G295V, T722A, and top1G369D, T722A, revealed this was accomplished via a mechanism of reduced affinity for the DNA substrate. The mechanistic implications of these results are discussed in the context of the known structures of yeast and human DNA topoisomerase I.