New technique for uncoupling the cleavage and religation reactions of Eukaryotic Topoisomerase I.: The mode of action of camptothecin at a specific recognition site

Abstract

A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 m m, while the religation reaction seemed unaffected by concentrations as high as 3 m-NaCl. The divalent cations Mg 2+, Ca 2+ and Mn 2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu 2+ and Zn 2+ inhibited both reactions. Furthermore, the effect of the antineoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation.