Position-specific trapping of topoisomerase I-DNA cleavage complexes by intercalated benzo[a]pyrene diol epoxide adducts at the 6-amino group of adenine

DNA topoisomerase I (top1) is the target of potent anticancer agents, inclu ding camptothecins and DNA intercalators, which reversibly stabilize (trap) top1 catalytic intermediates (cleavage complexes). The aim of the present study was to define the structural relationship between the site(s) of cova lently bound intercalating agents, whose solution conformations in DNA are known, and the site(s) of top1 cleavage. Two diastereomeric pairs of oligon ucleotide 22-mers, derived from a sequence used to determine the crystal st ructure of top1-DNA complexes, were synthesized. One pair contained either a trans-opened 10R- or 10S-benzo[a]pyrene 7,8-diol 9,10-epoxide adduct at t he N-6-amino group of a central 2'-deoxyadenosine residue in the scissile s trand, and the other pair contained the same two adducts in the non-scissil e strand. These adducts were derived from the (+)-(7R,8S,9S,10R)- and (-)-( 7S,8R,9R,10S)-7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. On the basis of analogy with known solut ion conformations of duplex oligonucleotides containing these adducts, we c onclude that top1 cleavage complexes are trapped when the hydrocarbon adduc t is intercalated between the base pairs flanking a preexisting top1 cleava ge site, or between the base pairs immediately downstream (3' relative to t he scissile strand) from this site. We propose a model with the +1 base rot ated out of the duplex, and in which the intercalated adduct prevents relig ation of the corresponding nucleotide at the 5' end of the cleaved DNA. The se results suggest mechanisms whereby intercalating agents interfere with t he normal function of human top1.