Topoisomerase II site-directed alkylation of DNA by psorospermin and its effect on topoisomerase II-mediated DNA cleavage

Psorospermin, a plant-derived antitumor agent, has been shown to selectivel y alkylate a guanine at the topoisomerase II cleavage site to trap the topo isomerase II-DNA cleaved complex. The results of this study provide further important insight into the mechanism of the topoisomerase II site-directed alkylation of DNA by psorospermin and its subsequent effects on the topois omerase II-induced DNA cleavage. First, we demonstrate that the topoisomera se II-induced alkylation of DNA by psorospermin occurs at a time preceding the topoisomerase II-mediated strand cleavage event, because it occurs in t he absence of Mg2+. We confirm that the alkylation of DNA by psorospermin t akes place at N-7 of guanine in the presence of topoisomerase II, because s ubstitution of the target guanine by 7-deazaguanine prevents alkylation. Be cause the stimulation of the topoisomerase II-induced DNA cleavage by psoro spermin can be slowly reversed by the addition of excess salt, this indicat es that alkylation of DNA by psorospermin traps a reversible topoisomerase II-DNA complex, Both the DNA alkylation by psorospermin in the presence of topoisomerase II and the enzyme-mediated DNA cleavage elevated by psorosper min are more enhanced at acidic pH values, in accordance with the increased stability of the topoisomerase ZI-DNA complex at acidic pH values. Finally , our results suggest that it is the psorospermin-DNA adducts, not the abas ic sites resulting from depurination, that are responsible for the stimulat ion of the topoisomerase II-mediated cleavage. Because the precise location of the psorospermin within the topoisomerase II cleavage site is known, to gether with the covalent DNA linkage chemistry and the conformation of the psorospermin-DNA adduct, this structural insight provides an excellent oppo rtunity for the design and synthesis of new, more effective topoisomerase I I poisons.