title: Camptothecin inhibits both the cleavage and religation reactions of eukaryotic DNA topoisomerase I.
abstract: We investigated the mode of action of the antitumor drug, camptothecin, by use of a partly double-stranded suicide DNA substrate which enables uncoupling of the cleavage and religation half-reactions of topoisomerase I. The suicide DNA substrate contains a single topoisomerase I site at which SDS cleavage is strongly enhanced by camptothecin on normal double-stranded DNA. The results show that the religation reaction of topoisomerase I per se is strongly inhibited at this site compared to site that is only marginally affected by camptothecin on double-stranded DNA. This study hereby directly demonstrates that camptothecin-mediated stability of a topoisomerase I-DNA complex is sequence-dependent. The influence of camptothecin on the suicide cleavage reaction of topoisomerase I was also investigated. Surprisingly, the cleavage reaction per se is strongly inhibited by the drug. However, reformation of a cleavable suicide DNA substrate, which is fully double-stranded downstream from the cleavage position except for a nick, completely reverses the inhibitory effect of the drug on the cleavage reaction. The results suggest that the inhibitory effect of camptothecin on cleavage is due to a general decrease in the noncovalent interaction of topoisomerase I with partly double-stranded suicide DNA substrates. Based on the findings, a plausible model for camptothecin action is discussed.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Kjeldsen
authors.forename: J Q
authors.initials: JQ
authors.lastname: Svejstrup
authors.forename: I I
authors.initials: II
authors.lastname: Gromova
authors.forename: J
authors.initials: J
authors.lastname: Alsner
authors.forename: O
authors.initials: O
authors.lastname: Westergaard
mesh_terms: D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000187:drug effects*; D005057:Eukaryotic Cells / Q000201:enzymology; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data
chemical_list: D046911:Macromolecular Substances; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin