Effect of CPT on the DNA cleavage/religation reaction mediated by calf thymus Topoisomerase I: evidence of an inhibition of DNA religation. Inhibition of Topoisomerase I-mediated DNA religation by CPT.
The uncoupling of the calf thymus Topoisomerase I-mediated forward DNA cleavage reaction from the religation event by a rapid shift of cleavage temperature either from 37 degrees C to 0 degrees C or from 37 degrees C to 56 degrees C has been studied and utilized to elucidate the molecular mechanism by which camptothecin, a clinically relevant antineoplastic agent, influences the half reactions of the enzyme. Results of heating and cooling religation-inducing treatments have been compared: both temperature extremes reduce the amount of protein-linked DNA breaks to background levels, thereby affecting cleavage reversal. Camptothecin is found to stabilize the enzyme-DNA intermediate, by inhibition of the Topoisomerase I-mediated rejoining of cleaved DNA, even when the drug is added after formation of the complex. We conclude that: 1. Heating and cooling treatments show a pronounced effect on the DNA cleavage-religation equilibrium. The efficacy of cold is more pronounced than that of heat. 2. Reversal of the enzyme-DNA intermediate favors the DNA resealing versus the closed relaxed form. 3. Camptothecin affects the heat or cold induced religation: in fact in both cases the drug delays the religation step.
Carboni
M C
MC
Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy.
Coderoni
S
S
D000818:Animals; D002166:Camptothecin / Q000494:pharmacology*; D002417:Cattle; D003080:Cold Temperature; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D006358:Hot Temperature; D011485:Protein Binding / Q000187:drug effects; D013950:Thymus Gland / Q000201:enzymology; D059004:Topoisomerase I Inhibitors
D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin