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: Michael H Woo, John R Vance, Ana R Otero Marcos, Christian Bailly, Mary-Ann Bjornsti
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 277 Issue 6 Pg. 3813-22 (Feb 08 2002) ISSN: 0021-9258 [Print] United States
• PMID: 11733535 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Aminoglycosides
• Anti-Bacterial Agents
• Carbazoles
• DNA Primers
• Enzyme Inhibitors
• Indoles
• Recombinant Proteins
• Topoisomerase I Inhibitors
• rebeccamycin
• DNA Topoisomerases, Type I
• Camptothecin

Topics

• Aminoglycosides
• Anti-Bacterial Agents (pharmacology)
• Base Sequence
• Binding Sites
• Camptothecin (pharmacology)
• Carbazoles
• DNA Primers
• DNA Topoisomerases, Type I (genetics)
• Enzyme Inhibitors (pharmacology)
• Humans
• Indoles
• Mutagenesis
• Mutation
• Recombinant Proteins (antagonists & inhibitors, genetics)
• Saccharomyces cerevisiae (genetics)
• Topoisomerase I Inhibitors