Topoisomerase inhibitors block topoisomerases II and I by stabilizing cleavage complexes that probably correspond to natural intermediates in the DNA stand passage reactions of these enzymes. Drugs of different chemical class stabilize different patterns of cleavage sites. The basis for selectivity of drug action was investigated by sequencing a large number of sites induced by inhibitors of topoisomerase I (camptothecin) and topoisomerase II (anthracyclines, m-AMSA, VP-16, VM-26, mitoxantrone and ellipticines). DNA sequencing was performed using purified mouse leukemia L1210 topoisomerases and 32P-end labeled DNA fragments from SV40 DNA and the human MYC gene. A computer program was developed in order to analyze the statistical probability of a random occurrence at each position between positions [-20] and [+20] from the cleavage sites. For topoisomerase I in the absence of drug the strongest preference was T[-1]. Camptothecin stabilized a subset of those sites with a strong preference for those having G[+1]. For topoisomerase II in the absence of drug a core region of strong base preference was found between positions [-3] and [+7] but no single base was strongly preferred at any of these positions. For anthracyclines, A[-1] at one of the cleavage sites of a DNA double-strand break was always present. For acridines (m-AMSA), it was A[+1], and for epipodophyllotoxins (VP-16 and VM-26), it was C[-1]. We now propose a model in which topoisomerase inhibitors bind inside a cavity formed by the base pairs flanking the cleavage site (-1 and +1 bases) and the enzyme. We have isolated a Chinese hamster cell line resistant to camptothecin. Since purified topoisomerase I from these cells is camptothecin-resistant, we are planning to clone the topoisomerase I gene(s) in order to determine the mutation sites. Mutation sites may correspond to amino acids that interact with camptothecin.
