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 selectively alkylate a guanine at the topoisomerase II cleavage site to trap the topoisomerase 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 topoisomerase II-induced DNA cleavage. First, we demonstrate that the topoisomerase II-induced alkylation of DNA by psorospermin occurs at a time preceding the topoisomerase II-mediated strand cleavage event, because it occurs in the absence of Mg2+. We confirm that the alkylation of DNA by psorospermin takes place at N-7 of guanine in the presence of topoisomerase II, because substitution of the target guanine by 7-deazaguanine prevents alkylation. Because the stimulation of the topoisomerase II-induced DNA cleavage by psorospermin can be slowly reversed by the addition of excess salt, this indicates 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 psorospermin are more enhanced at acidic pH values, in accordance with the increased stability of the topoisomerase II-DNA complex at acidic pH values. Finally, our results suggest that it is the psorospermin-DNA adducts, not the abasic sites resulting from depurination, that are responsible for the stimulation of the topoisomerase II-mediated cleavage. Because the precise location of the psorospermin within the topoisomerase II cleavage site is known, together with the covalent DNA linkage chemistry and the conformation of the psorospermin-DNA adduct, this structural insight provides an excellent opportunity for the design and synthesis of new, more effective topoisomerase II poisons.
Kwok
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Drug Dynamics Institute, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Hurley
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000478:Alkylation; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D018736:DNA Adducts; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004330:Drosophila / Q000201:enzymology; D006147:Guanine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity; D014966:Xanthenes / Q000494:pharmacology*; D044004:Xanthones
D000972:Antineoplastic Agents, Phytogenic; D018736:DNA Adducts; D014966:Xanthenes; D044004:Xanthones; D000266:Adenylyl Imidodiphosphate; D006147:Guanine; C025543:psorospermin; D000255:Adenosine Triphosphate; D004247:DNA; D004250:DNA Topoisomerases, Type II; D008274:Magnesium