Yield of DNA Strand Breaks after Base Oxidation of Plasmid DNA

We have irradiated aerobic aqueous solutions of plasmid DNA with 137 Cs γ rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (· OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [$({\rm SCN})_{2}{}^{\cdot -}$ and ${\rm Br}_{2}{}^{\cdot -}$, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the · OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for $({\rm SCN})_{2}{}^{\cdot -}$ with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of $({\rm SCN})_{2}{}^{\cdot -}$ with DNA as $1.6\times 10^{4}\ {\rm dm}^{3}\ {\rm mol}^{-1}\ {\rm s}^{-1}$, and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to · OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.