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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]

Human DNA Topoisomerase I is a 765aa monomeric enzyme composed of four domains: the N-terminal domain, highly charged and responsible for several protein-protein interactions, the core domain that embraces the DNA during catalysis, the highly charged linker domain and the C-teminal domain containing the active site. The enzyme promotes the relaxation of supercoiled DNA by nicking and rejoining one of the strands of the DNA. Its activity is critical for many biological processes including DNA replication, transcription, and recombination. The aim of this review is to analyze the enzyme activity in terms of structure-function relationship.