INTERACTION OF AN ALKYLATING CAMPTOTHECIN DERIVATIVE WITH A DNA-BASE AT TOPOISOMERASE I-DNA CLEAVAGE SITES

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme, It is speci fically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of it s derivatives are presently in clinical trial and exhibit remarkable a nticancer activity, The present study is a further investigation of th e molecular interactions between the drug and the enzyme-DNA complex, We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,1 1-methylenedioxycamptothecin (7-CIMe-MDO-CPT), and compared its activi ty against calf thymus top1 in a DNA oligonucleotide containing a sing le top1 cleavage site with the activity of its nonalkylating analog, 7 -ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT), In the presenc e of top1, 7-CIMe-MDO-CPT produced a DNA fragment that migrated more s lowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT, T op1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C, This fragment was resis tant to piperidine treatment and was also formed with an oligonucleoti de containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base +1), It was however cleaved by formic acid treatment followe d by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-CIMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence,that camptothecins inhibit top1 by binding at the enzyme-DNA interface.