The effect of camptothecin on topoisomerase I catalysis.
The existence of a covalent intermediate in topoisomerase I catalysis allows uncoupling of the cleavage and ligation half-reactions on partially single-stranded DNA substrates containing a highly preferred interaction site. Using this model DNA substrate system we have demonstrated that the cleavage reaction requires bipartite interaction with two distinct DNA duplex regions; One located around the cleavage site (region A) and another located on the side holding the 5'-OH end generated by cleavage (region B). The postcleavage complexes containing the enzyme covalently attached at an internal position are capable of ligating DNA strands matching the noncleaved strand. Previously, we have characterized the effect of the antitumor agent camptothecin on the two half-reactions of topoisomerase I catalysis on DNA substrates allowing bipartite DNA interaction. The obtained results demonstrated that the drug only inhibited the ligation reaction leaving the cleavage reaction unaffected at the studied site. Here, we report that camptothecin also impairs ligation of the cleaved strand to a dinucleotide within region A in the absence of additional DNA contacts. When these results are taken together with the observation that camptothecin-trapped topoisomerase I-DNA complexes preferentially are generated at sites containing guanine immediately 3' to the cleavage position, it suggests that camptothecin inhibits the ligation reaction by forming a reversible ternary complex with the enzyme and DNA at the cleavage site within region A.
Christiansen
K
K
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Westergaard
O
O
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism; D018900:Dinucleotide Repeats; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae; D059004:Topoisomerase I Inhibitors
D000972:Antineoplastic Agents, Phytogenic; D002166:Camptothecin; D004247:DNA; D004264:DNA Topoisomerases, Type I; D015226:Dinucleoside Phosphates; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; C015772:CpG dinucleotide