The role of hydration in the distribution of free radical trapping in directly ionized DNA

Abstract

The purpose of this study was to elucidate the role of hydration (Gamma) in the distribution of free radical trapping in directly ionized DNA. Solid-state films of pUC18 (2686 bp) plasmids were hydrated to Gamma in the range 2.5 < or = Gamma < or = 22.5 mol water/mol nucleotide. Free radical yields, G(Sigmafr), measured by EPR at 4 K are seen to increase from 0.28 +/- 0.01 micromol/J at Gamma = 2.5 to 0.63 +/- 0.01 micromol/J at Gamma= 22.5, respectively. Based on a semi-empirical model of the free radical trapping events that follow the initial ionizations of the DNA components, we conclude that two-thirds of the holes formed on the inner solvation shell (Gamma < 10) transfer to the sugar-phosphate backbone. Likewise, of the holes produced by direct ionization of the sugar-phosphate, about one-third are trapped by deprotonation as neutral sugar-phosphate radical species, while the remaining two-thirds are found to transfer to the bases. This analysis provides the best measure to date for the probability of hole transfer (approximately 67%) into the base stack. It can thus be predicted that the distribution of holes formed in fully hydrated DNA at 4 K will be 78% on the bases and 22% on the sugar-phosphate. Adding the radicals due to electron attachment (confined to the pyrimidine bases), the distribution of all trapped radicals will be 89% on the bases and 11% on the sugar-phosphate backbone. This prediction is supported by partitioning results obtained from the high dose-response curves fitted to the two-component model. These results not only add to our understanding of how the holes redistribute after ionization but are also central to predicting the yield and location of strand breaks in DNA exposed to the direct effects of ionizing radiation.

FIG. 1

First-derivative, Q-band, EPR spectra of pUC18 plasmid X-irradiated with a dose of 3 kGy and recorded at 4 K at various Γ. The vertical line corresponds to the position of g = 2.0022. The modulation amplitude was 0.40 mT, and the microwave power attenuation was 50 dB, delivering 0.3 nW to the cavity. The signal from ruby, used as an internal standard to determine the free radical concentration, lies about 40 mT up field from the DNA signal and is not shown here.

FIG. 2

First-derivative, Q-band, EPR spectra of pUC18 plasmid X-irradiated with a dose of 90.0 kGy and recorded at 4 K at various Γ. The spectral width was 40 mT. The scan was paused at the line as shown; the field sweep center was then increased by 20 mT and the signal gain adjusted before continuing the sweep to record the strong singlet of the ruby reference at high field. The vertical line corresponds to the position of g = 2.0022. The vertical arrows indicate the broad wing-line features of the putative sugar radicals.

FIG. 3

Corrected dose–response curve for free radical (FR) trapping by pUC18 plasmid film hydrated to a Γ = 2.5 mol water/mol nucleotide fitted to the two-component model described previously (). X irradiation and EPR were done at 4 K. Note that the slope shown in the inset (G) is the yield obtained from a fit to the initial linear response measured at low dose (~6 kGy). The free radical yield, G(∑fr), of this pUC18 plasmid (2686 bp) sample determined from the fit is found to be 0.285 ± 0.006 μmol/J. R² (~0.998) is the goodness of fit and χ² (~7.04 × 10²⁸) is the reduced χ² value calculated by the nonlinear least-squares fitting routine used by ORIGIN.

FIG. 4

Plot of the free radical (FR) concentration as a function the X-ray dose for irradiated pUC18 plasmid samples at various Γ (mol water/mol nucleotide). The data includes plasmids at (■) Γ = 2.5, (●)Γ = 7.5, (▲) Γ = 11.5, (○) Γ = 15.0, and (♦) Γ = 22.5 mol water/mol nucleotide. The data for Γ = 22.5 have been published previously (). X irradiation and EPR measurements were done at 4 K. A vertical offset of 0.2 units has been introduced between each curve. The linear region of the dose–response data has been fitted to a straight line by least squares. The free radical yield of the pUC18 sample at each Γ is determined from the slope of this line.

FIG. 5

Dependence of the total free radical yield, G(∑fr), on Γ (mol water/mol nucleotide) for X-irradiated pUC18 plasmid samples measured at 4 K.