Probing DNA single strands for single-base bulges with neocarzinostatin chromophore.
Neocarzinostatin chromophore (NCS-Chrom) induces strong cleavage at a single site (C3) in the single-stranded and 5' (32)P-end-labeled 13-mer GCCAGATTTGAGC in a reaction dependent on a thiol. By contrast, in the duplex form of the same 13-mer, strand cleavage occurs only at the T and A residues, and C3 is not cleaved. To determine the minimal structural requirement(s) for C3 cleavage in the single-stranded oligomer, several deletions and mutations were made in the 13-mer. A 10-mer (GCCAGAGAGC) derived from the 13-mer by deletion of the three T residues was also cleaved exclusively at C3 by NCS-Chrom, generating fragments having 5' phosphate ends. That the cleavage at C3 is initiated by abstraction of its 5' hydrogen is confirmed in experiments using 3' (32)P-end-labeled 10-mer. The competent 13-mer and 10-mer were assigned hairpin structures with a stem loop and a single bulged out A base, placing C3 across from and 3' to the bulge. Removal of the bulged A base from the 13-mer and the 10-mer resulted in complete loss of cutting activity, proving that it is the essential determinant in competent substrates. Studies of thiol post-activated NCS-Chrom binding to the DNA oligomers show that the drug binds to the bulge-containing 13-mer (K(d) = 0.78 microM) and the 10-mer (K(d) = 1.11 microM), much more strongly than to the 12-mer (K(d) = 20 microM) and the 9-mer (K(d) = 41 microM), lacking the single-base bulge. A mutually induced-fit between NCS-Chrom and the oligomer resulting in optimal stabilization of the drug-DNA complex is proposed to account for the site-specific cleavage at C3. These studies establish the usefulness of NCS-Chrom as a probe for single-base bulges in DNA.
Kappen
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Goldberg
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D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000187:drug effects*; D053281:Enediynes; D066298:In Vitro Techniques; D015335:Molecular Probes; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D009353:Zinostatin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D004277:DNA, Single-Stranded; D053281:Enediynes; D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides; D013438:Sulfhydryl Compounds; C032372:neocarzinostatin chromophore; D009353:Zinostatin