BULGE-SPECIFIC CLEAVAGE IN TRANSACTIVATION RESPONSE REGION RNA AND ITS DNA ANALOG BY NEOCARZINOSTATIN CHROMOPHORE

On the basis of the finding that in the absence of thiol the nonprotei n chromophore of the antitumor drug neocarzinostatin (NCS-chrom) induc es highly efficient site-specific cleavage at a single site on the 3' side of a bulge in single-stranded DNA involving entirely 5' chemistry [Kappen, L. S., and Goldberg, I. H. (1993) Science, 261, 1319-1321], transactivation response region (TAR) RNA (29-mer) and its DNA analogu e which presumably contain bulge structures were tested as potential s ubstrates for NCS-chrom. In TAR RNA NCS-chrom generates a distinct but weak band due to cleavage at U-24 in the bulge. Cleavage at U-24 has a pH dependence and time course similar to those for previously studie d DNA bulges. This band is not produced in drug reactions containing g lutathione, by the protein component of native NCS, or by inactivated NCS-chrom. Cleavage at U-24, albeit weak, occurs in an RNA substrate m ade up of two linear RNA oligomers which presumably can form a bulge a kin to that in TAR RNA. In the DNA analogue of TAR RNA, as well as in a DNA duplex made of two linear oligomers that can form a similar bulg e, NCS-chrom causes strand cleavage at the T residues in the bulge and at the bases flanking the bulge. Cleavage at T-25 in the bulge involv es, in addition to 5' chemistry, 4' attack which results in a fragment with mobility characteristic of 3'-phosphoglycolate-ended fragments. Experiments using DNA substrate having deuterium selectively at the 4' or 5' positions of T-25 confirm 4' attack and show kinetic shuttling between the two positions. Sequence changes in TAR DNA show that the s pecificity and extent of cleavage is sequence-dependent. While TAR DNA differs from previously studied DNA bulge substrates in having multip le attack sites and 4' chemistry at one site, it is only about 10% as good a substrate as the latter.