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Eukaryofic DNA topoisomerase I (Top1) is a monomeric protein clamp that functions in DNA replication, transcription, and recombination. Opposable "lip" domains form a salt bridge to complete Top1 protein clamping of duplex DNA. Changes in DNA topology are catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723 and a single DNA strand. Substantial protein domain movements are required for DNA binding, whereas the tight packing of DNA within the covalent Topl-DNA complex necessitates some DNA distortion to allow rotation. To investigate the effects of Topi-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and 5er-534, to crossiink protein loops more proximal to the active-site tyrosine than the protein loops held by the Lys-369-Glu-497 salt bridge. In reducing environments, Topi-Clamp was catalytically acfive. However, contrary to crosslinking the salt-bridge loops [Carey, J. F., Schultz, S. J., Sission, L., Fazzio, T. G. & Champoux, J. J. (2003) Proc. Natl. Acad. Sci. USA 100, 5640-5645], crosslinking the active-site proximal loops inhibited DNA rotation. Apparently, subtle alterations in Top1 clamp flexibility impact enzyme catalysis in vitro. Yet, the catalytically active Topi-Clamp was cytotoxic, even in the reducing environment of yeast cells. Remarkably, a shift in redox potential in glrlA cells converted the catalytically inactive Top1Y723F mutant clamp into a cellular toxin, which failed to induce an S-phase terminal phenotype. This cytotoxic mechanism is distinct from that of camptothecin chemotherapeutics, which stabilize covalent Topl-DNA complexes, and it suggests that the development of novel therapeutics that promote Topi-clamp closure is possible. [ABSTRACT FROM AUTHOR]