The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. Saccharomyces cerevisiae cells expressing yeast DNA topoisomerase I mutant top1 vac (I725R, N726A) or top1N726L, in which the amino acid residues N terminus to the active site tyrosine Tyr-727 were changed as indicated, were found to be camptothecin-resistant even though the mutant proteins expressed in Escherichia coli were previously shown to be active. Assays of enzyme-catalyzed relaxation of supercoiled DNA in vitro and in vivo in yeast showed that the camptothecin resistance of these mutants arises by entirely different mechanisms. Top1N726L-catalyzed DNA relaxation was not detected in yeast. The Top1 vac protein was catalytically active; however, camptothecin was inefficient in trapping the covalent intermediate formed between the Top1 vac enzyme and DNA. Yeast cells expressing human mutant htop1 vac, with similar substitutions near the active site tyrosine Tyr-723, were also camptothecin-resistant. Surprisingly, in the absence of camptothecin, yeast rad52 mutants defective in the repair of double-stranded DNA breaks were nonviable when top1N726L or top1 vac was overexpressed but viable when htop1 vac was overexpressed. These results suggest differences between yeast and human enzyme function in vivo.