Investigating the Direct and Indirect Effects of Ionizing Radiation on DNA Via Independent Generation of Nucleobase Radicals and Investigating DNA Damage by Enediyne C-1027
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Date
2014-03-19
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Johns Hopkins University
Abstract
Ionizing radiation damages DNA indirectly via the formation of •OH, which reacts with DNA to form nucleobase and deoxyribose radicals; or directly to generate nucleobase radical cations. These nucleobase reactive intermediates have been suggested to result in direct strand scission in DNA. Pyrimidine peroxyl radicals are also proposed to initiate electron transfer. The reactivity of these intermediates must be examined more thoroughly in order to fully understand the chemical effects of ionizing radiation on DNA.
We synthesized photochemical precursors for the reactive intermediates produced by the effects of ionizing radiation on thymidine, specifically, 5,6-dihydro-5-hydroxy-thymidin-6-yl (2) and its analog (5,6-dihydrouridin-6-yl, 23), thymidine radical cation (1), and 5,6-dihydro-thymidin-5-yl radical (22). These were studied as monomers in solution or within chemically synthesized oligonucleotides.
Herein, we report that radicals 2 and 23, along with their respective peroxyl radicals, did not produce direct strand breaks or base-labile lesions in DNA. We also did not find evidence that the pyrimidine peroxyl radicals initiate electron transfer. The pyrimidine peroxyl radicals, however, add to the 5'-adjacent nucleotide (with a preference for dG) to form biochemically deleterious tandem and clustered lesions.
The majority of the reactivity (93%) of thymidine radical cation (1) involves hydration at the C5 position, while a minor amount (~7%) of products results from the deprotonation pathway. In DNA, 1 does not generate direct strand scission. However, 1 abstracts hydrogen atom(s) from the 5'-adjacent deoxyribose sugar, leading to NaOH-labile sugar lesions. Electron transfer to 1 occurs from the 5'-adjacent nucleotide, forming oxidized nucleobases. We also show that 2 does not dehydrate to 1, in contrast to earlier proposals.
C-1027 is a potent antitumor agent that damages DNA by forming oxidized sugar lesions resulting from hydrogen atom abstraction from the C1', C4', and C5' positions. It also has the unique ability to generate interstrand cross-links (ICLs) under anaerobic conditions. We find that the damage produced by C-1027 on different DNA sequences is more diverse than previously thought. We also propose that the abasic site (AP) results from a covalent modification of the nucleobase by the drug. Analysis of the chemical stability of the ICLs suggests that in some of the ICLs, direct addition of C-1027 to the nucleobase occurs.
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DNA damage, oxidized lesions, clustered lesions, nucleobase radicals, thymidine radical cation, hydroxyl radical adducts of thymidine, oxidized abasic sites