INDUCTION OF TOPOISOMERASE-I CLEAVAGE COMPLEXES BY THE VINYL-CHLORIDEADDUCT 1,N-6-ETHENOADENINE

We used purified mammalian topoisomerases I (top1) and oligonucleotide s to study top1-mediated cleavage and religation in the presence of a potent carcinogenic adduct, 1,N-6-ethenoadenosine (EA) incorporated im mediately downstream of a unique top1 cleavage site. We found tha epsi lon A markedly enhanced top1 cleavage complexes when it was incorporat ed at the +1 position of the top1 cleavage. This enhancement was due t o a reduction of the religation step of the top1 reaction. In addition , epsilon A reduced the top1-mediated cleavage and decreased binding o f the enzyme to DNA. We also studied the effects of the epsilon A addu ct on top1 trapping by camptothecin (CPT), a web known top1 inhibitor. CPT was inactive when epsilon A was present at the +1 position. Alkyl ation of the top1 cleavage complex by 7-chloromethyl-10,11-methylenedi oxycamptothecin (7-ClMe-MDO-CPT) was also blocked by the epsilon A add uct. Altogether, these results demonstrate that the epsilon A carcinog enic ad duct can efficiently trap human top1 and mimic CPT effects. No rmal hydrogen bonding of the base pairs immediately downstream from th e top1 cleavage site is probably essential for efficient DNA religatio n and binding of camptothecins in the top1 cleavage complex.