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‘ 32P-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‘ 32P-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 μM) and the 10-mer (K d = 1.11 μM), much more strongly than to the 12-mer (K d = 20 μM) and the 9-mer (K d = 41 μM), 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.