Mechanisms of strand break formation in DNA due to the direct effect of ionizing radiation: the dependency of free base release on the length of alternating CG oligodeoxynucleotides

Abstract

The question of how NA base sequence influences the yield of DNA strand breaks produced by the direct effect of ionizing radiation was investigated in a series of oligodeoxynucleotides of the form (d(CG)(n))(2) and (d(GC)(n))(2). The yields of free base release from X-irradiated DNA films containing 2.5 waters/nucleotide were measured by HPLC as a function of oligomer length. For (d(CG)(n))(2), the ratio of the Gua yield to Cyt yield, R, was relatively constant at 2.4-2.5 for n = 2-4 and it decreased to 1.2 as n increased from 5 to 10. When Gua was moved to the 5' end, for example going from d(CG)(5) to d(GC)(5), R dropped from 1.9 +/- 0.1 to 1.1 +/- 0.1. These effects are poorly described if the chemistry at the oligomer ends is assumed to be independent of the remainder of the oligomer. A mathematical model incorporating charge transfer through the base stack was derived to explain these effects. In addition, EPR was used to measure the yield of trapped-deoxyribose radicals at 4 K following X-irradiation at 4 K. The yield of free base release was substantially greater, by 50-100 nmol/J, than the yield of trapped-deoxyribose radicals. Therefore, a large fraction of free base release stems from a nonradical intermediate. For this intermediate, a deoxyribose carbocation formed by two one-electron oxidations is proposed. This reaction pathway requires that the hole (electron loss site) transfers through the base stack and, upon encountering a deoxyribose hole, oxidizes that site to form a deoxyribose carbocation. This reaction mechanism provides a consistent way of explaining both the absence of trapped radical intermediates and the unusual dependence of free base release on oligomer length.

Figure 1

Free base release in ((CG)₅)₂ films at a hydration level of 2.5 waters per nucleotide: Cyt (●), Gua (○), and Cyt + Gua (▴). Straight line fits to points over the entire dose range are dashed lines and the fit to dose points <60 kGy are solid lines.

Figure 2

Cyt and Gau release in (CG)_n and (GC)_n. The data point representing large N was arbitrarily set at N = 200 and these values of G(Gua) and G(Cyt) were calculated from the M. luteus data by extrapolating the yields measured for the actual GC content of 72% to a content of 100% (Table 1). The nonlinear-least-squares fit is shown as dashed lines for the one-part model. For the two-part model, the fit is shown as solid lines for the (CG)_n data and as dotted lines for the (GC)_n data.

Figure 3

Q-band EPR spectra of ((CG)₅)₂ films X-irradiated and observed at 4 K. The small vertical bar marks g = 2.002. At low dose, 9 kGy, the signal is due primarily to base-centered radicals. At high dose, 525 kGy, the broad signal due to deoxyribose radicals is more intense than that of the base-centered radicals. A signal due to radicals in the quartz sample tube also grows in at high dose.

Figure 4

Free-radical dose response for ((CG)₅)₂ films, at Γ 2.5, X-irradiated, and measured at 4 K. The signal due to quartz becomes apparent above 400 kGy and it was subtracted out to track the signal due to DNA alone, shown as solid circles. The inset shows the linearity of response at low dose. The solid curve is the least-squares fit to C = (G_b/k_b)[1 − exp(−k_bD)] + (G_d/k_d)[1 − exp(−k_dD)], where C is radical concentration, G_b and G_d are the respective yields of base and deoxyribose radicals, k_b and k_d are the respective destruction cross sections for the base and deoxyribose radicals, and D is the dose. The values of G_dRib(fr) = G_d and GΣ(fr) = G_b + G_d are given in Table 2.

SCHEME 1

Reaction Pathways Initiated by Direct Ionization of the Deoxyribose Moiety of DNA

SCHEME 2

Proposed Mechanism by which the 3′-End Sensitizes Deoxyribose Damage in Oligodeoxynucleotides by the Direct Effect of Ionizing Radiation

SCHEME 3

Diffusion-Dependent Double Oxidation of the Deoxyribose Requires Two Oxidation Events on the Oligomer: A Carbon-Centered Radical Trapped on Deoxyribose Due to Deprotonation of a Radical Cation and a Radical Cation Formed on the Base Stack that Diffuses through the Base Stack^{a a}Illustrated here is diffusion-dependent double oxidation resulting in carbocation formation at the 3'-end of DNA decamer.