Effects of mutations in the F361 to R364 region of topoisomerase I (Topo I), in the presence and absence of 9-aminocamptothecin, on the Topo I-DNA interaction.
Steady-state levels and rates of DNA binding and release of wild-type and mutant topoisomerase I (Topo I) proteins were quantified by surface plasmon resonance analysis. The proteins were constructed and expressed as GST fusion proteins. The Topo I mutations analyzed were F361S, R362L and R364G, all altering a highly conserved region of wild-type eukaryotic Topo I. The R362L and R364G mutations resulted in much lower steady-state levels of DNA binding than wild-type. This was due to a large increase in the k(d). The F361S mutation increased the steady-state levels of the protein-DNA interaction by increasing the k(a) 2-fold, while having little effect on the k(d). The F361S mutation has been shown to confer resistance to camptothecin and its analogs. The camptothecin analog 9-aminocamptothecin decreased greatly the overall k(d) of the wild-type Topo I, but had little effect on the F361S mutant. Both the wild-type and the F361S mutant exhibited decreased steady-state levels in the presence of the drug, and this was attributable to decreased association.
Pond
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Department of Pharmacology and Toxicology, University of Utah, Salt Lake City 84112, USA.
Li
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XG
Rubin
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EH
Barrows
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LR
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D013329:Structure-Activity Relationship
D000970:Antineoplastic Agents; C080125:9-aminocamptothecin; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin