Effects of ionizing radiation on deoxyribonucleic acid. Part 7. Electron capture at cytosine and thymine

Exposure of a range of DNA samples in various media to 60Co γ-rays at 77 K gives electron-capture centres characterized by an EPR doublet with A(1H) of ca. 16 G. Since the electron-adducts of C and T give very similar doublet EPR spectra in irradiated DNA, it is difficult to judge the proportions of C˙– and T˙– formation by inspection. The possibility, suggested by others, that computer fits can be used to give a quantitative measure of these species is discussed. However, in view of the variability of the features directly assignable to C˙– and T˙– units in different environments, we suggest that this approach has only qualitative significance.The alternative method involves annealing to convert T˙– into TH˙ radicals in which a hydrogen atom is added to C6, the resulting radical having a completely characteristic octet EPR spectrum. It is argued that the ejected electrons move through the stacked DNA bases, becoming trapped at C or T depending upon the relative rates at which C˙– and T˙– are protonated to give C˙–(H+)(protonated at N3) and T˙–(H+)(protonated on oxygen). If this is correct, interconversion between C˙– and T˙– on annealing is unlikely, and only T˙– can lead to TH˙ formation.This is also not accurate, since TH˙ decay sets in within the same temperature range as it is being formed. By generating TH˙ from frozen aqueous DNA with ultraviolet light, the pure decay annealing curve has been obtained, and using this we have been able to extrapolate the data for TH˙ from the γ-irradiated samples to give the real yields of TH˙. The results show that ca. 36% of the doublet must be due to T˙– centres, the remainder (64%) being assigned to C˙– centres.The ratio of C˙– to T˙– varies with the DNA source, and with the environment. We suggest that it is largely governed by the relative rates of protonation to give C˙–(H+) and T˙–(H+), and the factors controlling these rates are discussed. The use of lithium chloride glasses completely suppresses the formation of G˙+ centres, leaving well-defined radical-anion spectra, but on annealing, conversion to TH˙ is negligible despite the rapid, and complete, loss of the doublet species. This result is discussed in terms of reaction with Cl2˙– radicals formed in abundance in these glasses.Studies designed to detect any site-specificity in the DNA damage leading to strand breaks suggest that all possible sites are damaged. These results strongly support the postulate that yields of C˙– and T˙– are comparable. The possibility that some A˙+ cations are formed in addition to G˙+ cations is also considered in the light of these results.