Correlation of free radical yields with strand break yields produced in plasmid DNA by the direct effect of ionizing radiation

Abstract

The purpose of this study was to determine how free radical formation (fr) correlates with single strand break (ssb) and double strand break (dsb) formation in DNA exposed to the direct effects of ionizing radiation. Chemical yields have been determined of (i) total radicals trapped on DNA at 4 K, G(Sigmafr), (ii) radicals trapped on the DNA sugar, Gsugar(fr), (iii) prompt single strand breaks, Gprompt(ssb), (iv) total single strand breaks, Gtotal(ssb), and (v) double strand breaks, G(dsb). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(fr) were measured by EPR applied to films of pEC (10,810 bp) and pUC18 (2686 bp) plasmids hydrated to Gamma = 22 mol of water/nucleotide and X-irradiated at 4 K. Using these same samples warmed to room temperature, strand breaks were measured by gel electrophoresis. The respective values for pEC and pUC18 were G(fr) = 0.71 +/- 0.02 and 0.61 +/- 0.01 micromol/J, Gtotal(ssb) = 0.09 +/- 0.01 and 0.14 +/- 0.01 micromol/J, G(dsb) = 0.010 +/- 0.001 and 0.006 +/- 0.001 micromol/J, and Gtota)(ssb)/G(dsb) approximately 9 and approximately 20. Surprisingly, Gsugar(fr) approximately 0.06 mumol/J for pUC18 films, less than half of Gtotal(ssb). This indicates that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. To explain this disparity, various mechanisms were considered, including one that entails two one-electron oxidations of a single deoxyribose carbon.

Figure 1

First-derivative, Q-band, EPR spectrum recorded at 4 K of pUC18 plasmid X-irradiated to 2400 Gy at 4 K. The scan is paused at the dashed line, the field sweep center increased by 20 mT, and the signal gain adjusted to record the singlet of the ruby reference at high field. The vertical solid line marks g = 2.0022.

Figure 2

Free radical dose-response curve for X-irradiated pUC18 plasmid films hydrated to Γ = 22 mol water/mol nucleotide. X-irradiation and EPR measurements were done at 4 K. G(∑fr) = 0.61± 0.01 μmol/J.

Figure 3

Loss of supercoiled DNA in films of pUC18, X-irradiated at 4 K, warmed to room temperature, and dissolved in 1X TE buffer. The data set is fitted with a straight line of the form y = c e^−mx. From the slope m, G_prompt(ssb) = 0.12 ± 0.01 μmol/J.

Figure 4

Formation of linearized plasmid in pUC18 plasmid DNA, corresponding to the loss of supercoiled plasmid plotted in Figure 3. The data set is fitted with a least mean squares polynomial of the form y = ax² + bx + c. From the value b, G(dsb) = 0.007 ± 0.001 μmol/J.

Figure 5

Corrected dose-response curve for free radical trapping by pUC18 plasmid film hydrated to Γ = 22 mol of water/nucleotide, fit to the two component model given in eq 1. X-irradiation and EPR were done at 4 K. R² is the goodness of fit, and χ² is the reduced χ²-value calculated by the nonlinear least-squares fitting routine used by ORIGIN. Inset: G(∑fr) = 0.62 ± 0.001 μmol/J was calculated from a fit to the initial linear response.