DNA TOPOISOMERASE I-MEDIATED FORMATION OF STRUCTURALLY MODIFIED DNA DUPLEXES - EFFECTS OF METAL-IONS AND TOPOISOMERASE INHIBITORS

The ability of DNA topoisomerase I to mediate the formation of structu rally modified DNA duplexes was studied utilizing suicide substrates c ontaining high-efficiency cleavage sites and acceptor oligonucleotides in which the 5'-terminal nucleotides were varied, When the substrates were nicked duplexes, the divalent cations Mg2+ and Ca2+ were found t o facilitate the topoisomerase I-mediated formation of ligation produc ts containing 3-nucleotide deletions on the scissile strand, but to su ppress the formation of l-nucleotide deletions. The presence of a comp lementary nucleotide at the 5'-end of the acceptor strand was not requ ired for the ligation reaction to proceed, but duplex formation to pro duce duplexes containing a mismatch proceeded more slowly than formati on of the fully complementary duplex. Topoisomerase I-mediated mismatc h formation in the ligation reaction was inhibited more readily by cam ptothecin than the corresponding ligation reaction to form a fully com plementary duplex; the extent of inhibition was comparable for all thr ee mismatches studied. In comparison, the topoisomerase I inhibitors n itidine and coralyne exhibited quite different effects on the same lig ation reactions.