Mechanism of DNA topoisomerase I poisoning: The role of DNA bending.

摘要

Human DNA topoisomerase I (TOP1), is a nuclear enzyme that changes the topology of DNA by making a reversible single stranded break on DNA. Its ability to perform cleavage-religation on DNA contributes to its role as an anticancer drug target. The prototypic TOP1-targeting drug (TOP1 poison), camptothecin, is known to function by inhibiting the religation step of the cleavage-religation reaction. In addition, a group of DNA binders have been shown to poison TOP1 as well. Some of them were shown to have their TOP1 poisoning ability attributed to their DNA binding. In this study, a pair of anthracyclines, menogaril and nogalamycin, whose structures only differ by a nogalose sugar, were used to elucidate the mechanism of poisoning of TOP1 by DNA binders. It was shown that, menogaril, which is a DNA intercalator, poisons DNA topoisomerase II (TOP2) but not TOP1. On the other hand, nogalamycin, which has an extra nogalose sugar that binds to the minor groove of DNA, poisons TOP1 but not TOP2. A nogalamycin-induced TOP1-mediated DNA cleavage site was mapped within an AT-rich sequence, containing a few G.C base pairs. DNase I footprinting showed that nogalamycin binds to the distal G.C base pairs rather than the G.C base pair right at the +2 position (relative to the cleavage site). Using systematic mutational analysis, the critical nogalamycin binding site was shown to be at the −6 position. Replacement of the nogalamycin-DNA complex with DNA bend-inducing sequence (A₅), induced the same site-specific cleavage. Since nogalamycin is known to induce DNA bending, it is possible that nogalamycin poisons TOP1 by inducing a DNA bend structure, which is a preferred substrate for TOP1, thus enhancing the cleavage reaction. In other words, nogalamycin may poison TOP1 by enhancing the forward reaction, distinct from camptothecin, which inhibits the religation reaction.