Mechanisms of DNA topoisomerase I-induced cell killing in the yeast Saccharomyces cerevisiae.
DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a mechanism of transient DNA strand cleavage characterized by the formation of a phosphotyrosyl bond between the DNA end and active site tyrosine. Camptothecin reversibly stabilizes the covalent enzyme-DNA intermediate by inhibiting DNA religation. During S-phase, collisions with advancing replication forks convert these complexes into potentially lethal lesions. To define the DNA damage induced by alterations in Top1p catalysis and the cellular processes that mediate the repair of such lesions, the yeast Saccharomyces cerevisiae was used. Substitution of conserved residues N-terminal to the active site tyrosine (Tyr-727) produced alterations in the camptothecin sensitivity or catalytic cycle of DNA Top1. For example, substituting Ala for Thr-722 in Top1T722A increased the stability of the covalent enzyme DNA intermediate. As with camptothecin, Top1T722A-induced cytotoxicity was ascribed to a reduction in DNA religation. By contrast, enhanced covalent complex formation by Top1N726H resulted from a relative increase in the rate of DNA cleavage. Conditional yeast mutants were also selected that exhibit temperature-sensitive growth only in the presence of the self-poisoning Top1T722A enzyme. Subsequent analyses of these tah mutants identified 9 genes whose function suppresses the cytotoxic action of camptothecin and Top1T722A. These include genes encoding essential DNA replication proteins (CDC45 and DPB11) and proteins involved in SUMO- or ubiquitination (UBC9 and DOA4).
Fiorani
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P
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38103, USA.
Bjornsti
M A
MA
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity; D002166:Camptothecin / Q000633:toxicity; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000633:toxicity; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D059004:Topoisomerase I Inhibitors
D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin