DOUBLE-STRANDED DAMAGE OF DNA-CENTER-DOT-RNA HYBRIDS BY NEOCARZINOSTATIN CHROMOPHORE - SELECTIVE C-1' CHEMISTRY ON THE RNA STRAND

Glutathione-activated neocarzinostatin chromophore generates bistrande d lesions in the hybrid formed by yeast tRNA(phe) and DNA complementar y to its 31-mer 3' terminus. To elucidate the chemistry of the RNA cle avage reaction and to show that the lesions are double-stranded (ds), a series of shorter oligoribonucleotides containing the target sequenc e r(AGAAUUC).(GAATTCT) (underlining indicates major attack site) was s tudied as substrates. In addition to cleavage at both U residues, majo r damage was produced in the form of an abasic site at the U residues. Evidence for abasic site formation on the RNA strand was obtained fro m sequencing-gel analysis and measurement of uracil base release. Init ial evidence for the ds nature of the damage came from experiments in which 2'-O-methyluridine was substituted for uridine in the RNA at one or both of the target sites. The site containing the substitution was not a target for cleavage or abasic site formation, and the particula r T residue, staggered two nucleotides in the 3' direction on the comp lementary DNA strand, was cleaved significantly less. These studies we re valuable in identifying the DNA ds partner of the RNA attack site. Direct evidence for ds lesions came from analysis of the products from a hairpin oligonucleotide construct in which the RNA and DNA strands were linked by four T residues and contained an internal P-32 label at the 3' end of the RNA strand. Substitution of deuterium for hydrogen at the C-1' position of the SI residues led to a substantial isotope e ffect (k(1H)/k(2H) = 3) upon the formation of the RNA abasic lesion an d the RNA cleavage products, providing conclusive evidence for selecti ve 1' chemistry. On the other hand, cleavage at the T residues on the complementary DNA strand involved C-5' hydrogen abstraction, as was al so true for the T residue in an oligodeoxynucleotide analogue of the R NA strand. Chemical mechanisms to account for the RNA cleavage and aba sic site formation via C-1' hydrogen abstraction are proposed.