SITE-SPECIFIC CLEAVAGE AT A DNA BULGE BY NEOCARZINOSTATIN CHROMOPHOREVIA A NOVEL MECHANISM

The chromophore of the anticancer drug neocarzinostatin (NCS-Chrom) ox idatively cleaves single-stranded or duplex DNA site-specifically in t he absence of activating thiol provided that the DNA contains a bulged structure. Point mutations, deletions, and insertions in the DNA anal ogue and its complement of the 3'-terminus of yeast tRNA(Phe) show tha t for a single-stranded DNA to be cleaved by NCS-Chrom the DNA must ge nerate a hairpin structure with an apical loop and at least a two-base -pair stem hinged to a region of duplex structure via a bulge containi ng a target nucleotide at its 3' side. The size of the loop is not cri tical so long as it contains at least three nucleotides; the bulge req uires a minimum of two nucleotides but must have fewer than five. With a notable exception involving base-pair changes immediately 3' to the bulge, base changes in the bulge and base-pair changes immediately 5' to the bulge retain substrate activity for NCS-Chrom. Maintenance of the bulged structure requires stable duplex regions on each side of th e bulge. A similar bulged structure, but lacking a loop, formed by the annealing of a linear 8-mer and a 6-mer is an excellent target for cl eavage in the thiol-independent reaction. Drugs such as netropsin, whi ch sequester the DNA into nonbulge containing structures inhibit the r eaction. In the absence of O2 strand cleavage is blocked and quantitat ively replaced by a presumed drug-DNA covalent adduct. The cleavage re action has a pH optimum of about 9 and is much slower than the thiol-d ependent reaction. The site-specificity of the cleavage, however, is m uch greater than in the thiol-dependent reaction. Thus, NCS-Chrom clea ves DNA containing a bulge via a novel mechanism not involving thiol. These studies extend the usefulness of this enediyne as a probe for un usual DNA structures.