title: Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin.
abstract: DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.
authors.affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Woo
authors.forename: John R
authors.initials: JR
authors.lastname: Vance
authors.forename: Ana R Otero
authors.initials: AR
authors.lastname: Marcos
authors.forename: Christian
authors.initials: C
authors.lastname: Bailly
authors.forename: Mary-Ann
authors.initials: MA
authors.lastname: Bjornsti
mesh_terms: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D002227:Carbazoles; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007211:Indoles; D016296:Mutagenesis; D009154:Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D059004:Topoisomerase I Inhibitors
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D002227:Carbazoles; D017931:DNA Primers; D004791:Enzyme Inhibitors; D007211:Indoles; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; C053339:rebeccamycin; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin