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

Abstract

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.