Effect of ribonucleotide substitution on nucleic acid bulge recognition by neocarzinostatin.
Bulged RNA structures are not as good substrates for cleavage by the enediyne antibiotic neocarzinostatin chromophore in the general base-catalyzed reaction as are DNA bulges. In an effort to determine why this is so, we have systematically substituted ribonucleotide residues in a DNA bulged structure (CCGATGCG.CGCAGTTCGG) (cleaved residue is underlined) known to be an excellent substrate. It was found that ribonucleotide substitution at the bulge target site, as well as at other regions involving duplex formation had a small effect on the cleavage reaction, unless either of the two strands was entirely of the ribo form. By contrast, changing the A.T base pair on the 5' side of the target nucleotide (T residue) to the ribo A.U resulted in an 87% decrease in cleavage; in fact, conversion of the A alone to the ribo form caused a 68% loss in cleavage. This result can be understood from the recent solution structure of the complex formed between an analogue of the drug radical species and a bulged DNA (Stassinopoulos, A.; Ji, J.; Gao, X.; Goldberg, I.H. Science 1996, 272, 1943), since the 2' hydroxyl group of the ribo A would be expected to clash sterically with the 7"-O-methyl moiety of the drug. Additional studies on substrate bulge-dependent drug product formation and protection against spontaneous drug degradation support the cleavage experiments, and imply that bulge-specific drug binding is required for efficient cleavage.
Kappen
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
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Goldberg
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D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D053281:Enediynes; D009690:Nucleic Acid Conformation; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D012313:RNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D012265:Ribonucleotides / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D009353:Zinostatin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D000903:Antibiotics, Antineoplastic; D053281:Enediynes; D019384:Nucleic Acid Synthesis Inhibitors; D012265:Ribonucleotides; D012313:RNA; C032372:neocarzinostatin chromophore; D004247:DNA; D009353:Zinostatin