DNA degradation by bleomycin: evidence for 2'R-proton abstraction and for C-O bond cleavage accompanying base propenal formation.

Reaction of poly(dA-[2'S-3H]dU) with activated bleomycin yields [3H]uracil propenal that completely retains the tritium label. In contrast, we have previously shown that reaction of poly(dA-[2'R-3H]dU) with activated bleomycin affords unlabeled uracil propenal [Wu, J. C., Kozarich, J. W., & Stubbe, J. (1983) J. Biol. Chem. 258, 4694-4697]. We have also prepared both cis- and trans-thymine propenals by chemical synthesis and have observed that the trans isomer is the exclusive product of the bleomycin reaction. Moreover, the cis isomer was found to be stable to the conditions of bleomycin-induced DNA degradation. Taken together, these results establish that the formation of trans-uracil propenal occurs via an anti-elimination mechanism with the stereospecific abstraction of the 2'R proton. The question of phosphodiester bond cleavage during base propenal formation has also been addressed by the analysis of the fate of oxygen-18 in poly(dA-[3'-18O]dT) upon reaction with activated bleomycin. The 5'-monophosphate oligonucleotide ends produced from thymine propenal formation have been converted to inorganic phosphate by the action of alkaline phosphatase, and the phosphate has been analyzed for 18O content by 31P NMR spectroscopy. The oxygen-18 is retained in the inorganic phosphate, establishing that the formation of thymine propenal by activated bleomycin proceeds with C-O bond cleavage at the 3'-position.