ISOTOPE EFFECTS ON THE CLEAVAGE OF DNA BY BLEOMYCIN - MECHANISM AND MODULATION

Sequence-specific isotope effects on the cleavage of DNA at thymidine residues by activated iron.bleomycin (Fe.BLM) have been observed upon incorporation of [4'-H-2]thymidine into the DNA. The effects may be quantitated by end-labeling the DNA with phosphorus-32, generating a sequence ladder by gel electrophoresis, and measuring the relative damage at cleavage sites by autoradiography or phosphorimager technology. Results with DNA deuteriated at other positions of the deoxyribose ring, such as, the 5'- or 2'-carbon, afford no isotope effect suggesting high regiospecificity for the BLM-mediated carbon-hydrogen bond cleavage. The magnitude of the 4'-isotope effects for Fe.BLM ranges from 2 to 7 (+/-0.3) and is not sensitive to changes in the partition ratio of the two proposed cleavage pathways. However, the isotope effect is sensitive to structural changes in BLM, such as with tallysomycin, and to changes in metal and cleavage mechanism, such as with Co.BLM. The variability of the effects are discussed in light of the factors responsible for the observation of V/K isotope effects. The findings suggest that the kinetics, thermodynamics, and geometry of carbon-hydrogen bond cleavage by Fe.BLM permit the observation of large isotope effects on this chemical step. Fe.BLM may be viewed, therefore, as a special case.