Correlation of Free Radical Yields with Strand Break Yields Produced in Plasmid DNA by the Direct Effect of Ionizing Radiation

Abstract

The purpose of this study was to determine how free radical formation (fr) correlates with single strand break (ssb) and double strand break (dsb) formation in DNA exposed to the direct effects of ionizing radiation. Chemical yields have been determined of (i) total radicals trapped on DNA at 4 K, G(∑fr), (ii) radicals trapped on the DNA sugar, G_sugar(fr), (iii) prompt single strand breaks, G_prompt(ssb), (iv) total single strand breaks, G_total(ssb), and (v) double strand breaks, G(dsb). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(∑fr) were measured by EPR applied to films of pEC (10 810 bp) and pUC18 (2686 bp) plasmids hydrated to Γ = 22 mol of water/nucleotide and X-irradiated at 4 K. Using these same samples warmed to room temperature, strand breaks were measured by gel electrophoresis. The respective values for pEC and pUC18 were G(∑fr) = 0.71 ± 0.02 and 0.61 ± 0.01 μmol/J, G_total(ssb) = 0.09 ± 0.01 and 0.14 ± 0.01 μmol/J, G(dsb) = 0.010 ± 0.001 and 0.006 ± 0.001 μmol/J, and G_total(ssb)/G(dsb) ∼9 and ∼20. Surprisingly, G_sugar(fr) ≈ 0.06 μmol/J for pUC18 films, less than half of G_total(ssb). This indicates that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. To explain this disparity, various mechanisms were considered, including one that entails two one-electron oxidations of a single deoxyribose carbon.