Yields of damage to C4′ deoxyribose and to pyrimidines in pUC18 by the direct effect of ionizing radiation

Abstract

Our mechanistic understanding of damage formation in DNA by the direct effect relies heavily on what is known of free radical intermediates studied by EPR spectroscopy. Bridging this information to stable product formation requires methods with comparable sensitivities, a criterion met by the 32P-post-labeling assay developed by Weinfeld and Soderlind, [Weinfeld,M. and Soderlind,K.-J.M. (1991) 32P-Postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry, 30, 1091–1097] which when applied to the indirect effect, detected phosphoglycolate (pg) and thymine glycol (Tg). Here we applied this assay to the direct effect, measuring product yields in pUC18 films with hydration levels (Γ) of 2.5, 16 or 23 waters per nucleotide and X-irradiated at either 4 K or room temperature (RT). The yields of pg [G(pg)] for Γ ∼ 2.5 were 2.8 ± 0.2 nmol/J (RT) and 0.2 ± 0.3 nmol/J (4 K), which is evidence that the C4′ radical contributes little to the total deoxyribose damage via the direct effect. The yield of detectable base damage [G(B*)] at Γ ∼ 2.5 was found to be 30.2 ± 1.0 nmol/J (RT) and 12.9 ± 0.7 nmol/J (4 K). While the base damage called B*, could be due to either oxidation or reduction, we argue that two reduction products, 5,6-dihydrouracil and 5,6-dihydrothymine, are the most likely candidates.