Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality.
Eukaryotic 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 Top1-DNA complex necessitates some DNA distortion to allow rotation. To investigate the effects of Top1-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and Ser-534, to crosslink 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, Top1-Clamp was catalytically active. 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 Top1-Clamp was cytotoxic, even in the reducing environment of yeast cells. Remarkably, a shift in redox potential in glr1Delta 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 Top1-DNA complexes, and it suggests that the development of novel therapeutics that promote Top1-clamp closure is possible.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.
Michael H
MH
Woo
Carmen
C
Losasso
Hong
H
Guo
Luca
L
Pattarello
Piero
P
Benedetti
Mary-Ann
MA
Bjornsti
D002166:Camptothecin / Q000494:pharmacology; D020134:Catalytic Domain; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry* / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012399:Rotation; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D004220:Disulfides; D046911:Macromolecular Substances; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin