DNA Damage Emanating From a Neutral Purine Radical Reveals the Sequence Dependent Convergence of the Direct and Indirect Effects of γ-Radiolysis

Abstract

Nucleobase radicals are the major intermediates generated by the direct (e.g., dA^•+) and indirect (e.g., dA•) effects of γ-radiolysis. dA• was independently generated in DNA for the first time. The dA^•+/dA• equilibrium, and consequently the reactivity in DNA, is significantly shifted toward the radical cation by a flanking dA. Tandem lesions emanating from dA• are the major products when the reactive intermediate is flanked by a 5'-dGT. In contrast, when dA• is flanked by dA, the increased dA^•+ pK_a results in DNA damage arising from hole transfer. This is the first demonstration that sequence effects lead to the intersection of the direct and indirect effects of ionizing radiation.

Figure 1

dA• induced strand damage in 5′-³²P-2. (a.) Denaturing PAGE analysis (b.) Proposed mechanism for tandem lesion formation.

Figure 2

dA^•+ induced hole transfer in 5′-³²P-6. (a.) Denaturing PAGE analysis (b.) Proposed mechanism for sequence dependent dA^•+ formation, and subsequent hole transfer.

Figure 3

Sequence and pH influence on strand damage. (a) pH 7.2 (b) pH 5.1. G5′, GM, and G3′ refer to the dGs in the dGGG triplet of each respective duplex. Values are the average ± std. dev. of 3 replicates.

Figure 4

pH Dependent strand damage in 5′-³²P-11. (a.) Denaturing PAGE analysis. (b.) Proposed mechanism of pH dependent switching of strand damage induced by dA• and dA^•+.

Scheme 1

Reactive intermediate formation from dA by γ-radiolysis.

Scheme 2

Proposed reaction pathways of dA• in DNA.

Chart 1

DNA duplexes lacking A-stacks.

Chart 2

DNA duplexes containing A-stacks.