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 pare nt 31-mer. In order to characterize this product, it was stabilized by reduction with borane/pyridine, labeled with P-32 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 T-22 at the bulge. Cleaved fragments inclusive of T-22 from the 5' or the 3' end had retarded and anomalou s mobilities and appeared as a smear of bands closer to the starting m aterial, presumably due to the presence of the covalently bound drug. Pyrimidine-specific agents such as hydrazine and potassium permanganat e, but not the DNA sugar-specific probe thiol-activated NCS-Chrom, ind uced strand cleavage at T-22 Mass spectral analysis of the presumed ad duct isolated from anaerobic reactions containing NCS-Chrom and a bulg e duplex substrate made up of a 10-mer and an 8-mer showed that the ad duct contains one molecule of the drug and one molecule of the 10-mer. Taken together, the results show that (i) drug adduction is at T-22 O n the full-length substrate; (ii) the pyrimidine ring is accessible to base-specific chemical modifications, hence, presumably free of the d rug; (iii) it is most likely that drug adduction is via its C6 positio n to the 5' carbon of T-22, 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 bas es by way of inter- or intrastrand cross-linking; and (v) the product is a monoadduct.