Characterization of a covalent monoadduct of neocarzinostatin chromophore at a DNA bulge

Neocarzinostatin chromophore (NCS-Chrom) induces highly efficient site-specific strand cleavage at the bulge of a folded single-stranded 31-mer DNA in the presence of oxygen [Kappen, L. S., and Goldberg, I. H. (1993) Science 261, 1319-1321]. Under anaerobic conditions, the major product is a material having gel mobility slower than that of the parent 31-mer. In order to characterize this product, it was stabilized by reduction with borane/pyridine, labeled with 32P at its 5' or 3' end, and subjected to chemical cleavage dependent on base elimination or modification, and the cleavage products were analyzed on a sequencing gel. A cleavage pattern comparable to that of the 31-mer was obtained until the bases on either side of T22 at the bulge. Cleaved fragments inclusive of T22 from the 5' or the 3' end had retarded and anomalous mobilities and appeared as a smear of bands closer to the starting material, presumably due to the presence of the covalently bound drug. Pyrimidine-specific agents such as hydrazine and potassium permanganate, but not the DNA sugar-specific probe thiol-activated NCS-Chrom, induced strand cleavage at T22. Mass spectral analysis of the presumed adduct isolated from anaerobic reactions containing NCS-Chrom and a bulge duplex substrate made up of a 10-mer and an 8-mer showed that the adduct contains one molecule of the drug and one molecule of the 10-mer. Taken together, the results show that (i) drug adduction is at T22 on the full-length substrate; (ii) the pyrimidine ring is accessible to base-specific chemical modifications, hence, presumably free of the drug; (iii) it is most likely that drug adduction is via its C6 position to the 5' carbon of T22, based on the current results and the known chemistry of the hydrogen abstraction by the drug in the presence or absence of oxygen; (iv) there is no involvement of the neighboring bases by way of inter- or intrastrand cross-linking; and (v) the product is a monoadduct.