Degradation of DNA by iron-bleomycin: mechanistic implications of product 18O incorporation

Interaction of d(CGCGCG) with Bleomycin (BLM), activated either with Fe(III) and H2O2 or Fe(II), O2 and one electron, results in production of cytosine and a modified oligonucleotide strand (1). Reduction of 1 with NaBD4 followed by enzymatic digestion, derivatization, and GC-MS permits the identification of 2-deoxypentitols-1,4-d2 as their tetra-trimethylsilyl (TMS) derivatives. Similar products have also been isolated from calf thymus DNA and poly(dG-dC). These results provide unequivocal evidence for the intermediacy of a 4' ketone, 1' aldehyde modified carbohydrate. An alternate mode of DNA degradation requires additional O2 and leads to formation of 3' phosphoglycolate termini and base propenals. Glycolate (GA), released from calf thymus DNA, poly(dA-dT) or d(CGCGCG) by enzymatic digestion, can be isolated by chromatography on DEAE Sephadex, silylated and analyzed by GC-MS. This analysis, after incubation with Fe(II) x 18O2 x BLM or Fe(III) x H216O2 x BLM plus 18O2, reveals the incorporation of a single atom of 18O at the C-1 position. Pulse-chase experiments demonstrate that it is the excess molecular oxygen and not the O2 required for drug activation that is incorporated into the carboxylate group of 3' phosphoglycolate and provide evidence for the proposed addition of O2 to a C4' carbon radical. Isotopic enrichments of the other products of DNA oxidation, formed in the presence of 18O2 are also being determined