Worth L, Frank B L, Christner D F, Absalon M J, Stubbe J, Kozarich J W
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Biochemistry. 1993 Mar 16;32(10):2601-9. doi: 10.1021/bi00061a018.
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'-2H]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.
当将[4'-2H]胸苷掺入DNA中时，已观察到活化的铁-博来霉素（Fe.BLM）对胸苷残基处DNA切割的序列特异性同位素效应。可以通过用磷-32对DNA进行末端标记、通过凝胶电泳生成序列梯以及通过放射自显影或磷成像技术测量切割位点处的相对损伤来对这些效应进行定量。在脱氧核糖环的其他位置（如5'-或2'-碳）氘代的DNA的结果未显示同位素效应，这表明BLM介导的碳氢键切割具有高度的区域特异性。Fe.BLM的4'-同位素效应的大小范围为2至7（±0.3），并且对两种提出的切割途径的分配比变化不敏感。然而，同位素效应对BLM的结构变化（如 tallysomycin）以及金属和切割机制的变化（如Co.BLM）敏感。根据导致观察到V/K同位素效应的因素讨论了效应的变异性。研究结果表明，Fe.BLM切割碳氢键的动力学、热力学和几何结构允许在这一化学步骤中观察到较大的同位素效应。因此，Fe.BLM可被视为一个特殊情况。
