Shubhadeep Purkayastha

On the Chemical Yield of Base Lesions, Strand Breaks, and Clustered Damage Generated in Plasmid DNA by the Direct Effect of X Rays

Abstract Purkayastha, S., Milligan, J. R. and Bernhard, W. A. On the Chemical Yield of Base Lesions, Strand Breaks, and Clustered Damage Generated in Plasmid DNA by the Direct Effect of X Rays. Radiat. Res. 168, 357–366 (2007). The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Γ < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Γ) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Γ was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 ± 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 ± 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.

Mechanisms of Direct Radiation Damage in DNA, Based on a Study of the Yields of Base Damage, Deoxyribose Damage, and Trapped Radicals in d(GCACGCGTGC)2

Abstract Swarts, S. G., Gilbert, D. C., Sharma, K. K., Razskazovskiy, Y., Purkayastha, S., Naumenko, K. A. and Bernhard, W. A. Mechanisms of Direct Radiation Damage in DNA, Based on a Study of the Yields of Base Damage, Deoxyribose Damage, and Trapped Radicals in d(GCACGCGTGC)2. Radiat. Res. 168, 367–381 (2007). Dose–response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)2prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3′ or 5′ terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were Gtotal(fr) = 618 ± 60, G(fbr) = 93 ± 8, G(8-oxoGua) = 111 ± 62, G(8-oxoAde) = 4 ± 3, G(diHUra) = 127 ± 160, and G(diHThy) = 39 ± 60, respectively. The yields were determined and the dose–response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamers GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 ± 16% 8-oxoGua, 3 ± 3% 8-oxoAde, 28 ± 46% diHThy, 23 ± 32% diHUra, and 27 ± 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is ∼3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose–response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.

Unaltered Free Base Release from d(CGCGCG)2 Produced by the Direct Effect of Ionizing Radiation at 4 K and Room Temperature

Abstract Sharma, K. K., Purkayastha, S. and Bernhard, W. A. Unaltered Free Base Release from d(CGCGCG)2 Produced by the Direct Effect of Ionizing Radiation at 4 K and Room Temperature. Radiat. Res. 167, 501–507 (2007). Unaltered free base release in d(CGCGCG)2 exposed to X rays at 4 K or room temperature was measured by HPLC. Samples were prepared either as films hydrated to a level of Γ = 2.5 mol water/mol nucleotide or as polycrystalline with Γ ∼ 7.5 mol water/mol nucleotide. X irradiation of films at 4 K, followed by annealing to room temperature, resulted in yields for cytosine and guanine of G(Cyt) = 0.036 ± 0.001 μmol/J and G(Gua) = 0.090 ± 0.002 μmol/J. Irradiation of films at room temperature gave similar yields. The yields for polycrystalline d(CGCGCG)2 X-irradiated at room temperature were G(Cyt) = 0.035 ± 0.005 μmol/J and G(Gua) = 0.077 ± 0.023 μmol/J. The total free base release yield, G(fbr), was 0.124 ± 0.008 μmol/J for films and 0.112 ± 0.028 μmol/J for polycrystalline samples. G(fbr) is believed to be a good estimate of total strand break yield. The yields of total free radicals trapped [G(Σfr)] by the d(CGCGCG)2 films at 4 K were measured by EPR. The measured value, G(Σfr) = 0.450 ± 0.005 μmol/J, was used to calculate the yield of trappable sugar radicals, giving Gsugar(fr) = 0.04–0.07 μmol/J. We found that (1) guanine release exceeded cytosine release by more than twofold, (2) Gsugar(fr) cannot account for more than half of the free base release, and (3) G(fbr), G(Cyt) and G(Gua) were independent of the sample temperature during irradiation. Finding (1) suggests that base and or sequence influences sugar damage, and finding (2) is consistent with our working hypothesis that an important pathway to strand break formation entails two one-electron oxidations at the same sugar site.

The Role of Hydration in the Distribution of Free Radical Trapping in Directly Ionized DNA

Abstract Purkayastha, S., Milligan, J. R. and Bernhard, W. A. The Role of Hydration in the Distribution of Free Radical Trapping in Directly Ionized DNA. Radiat. Res. 166, 1–8 (2006). The purpose of this study was to elucidate the role of hydration (Γ) in the distribution of free radical trapping in directly ionized DNA. Solid-state films of pUC18 (2686 bp) plasmids were hydrated to Γ in the range 2.5 ≤ Γ ≤ 22.5 mol water/mol nucleotide. Free radical yields, G(Σfr), measured by EPR at 4 K are seen to increase from 0.28 ± 0.01 μmol/J at Γ = 2.5 to 0.63 ± 0.01 μmol/J at Γ= 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 (Γ < 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 (∼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.

One-Electron Oxidation of DNA by Ionizing Radiation: Competition between Base-to-Base Hole-Transfer and Hole-Trapping

The distance of hole migration through DNA determines the degree to which radiation-induced lesions are clustered. It is the degree of clustering that confers to ionizing radiation its high toxicity. The migration distance is governed by a competition between hole transfer and irreversible trapping reactions. An important type of trapping is reactions that lead to the formation of deoxyribose radicals, which are precursors to free base release (fbr). Using HPLC, fbr was measured in X-irradiated films of d(CGCGCGCGCG)(2) and d(CGCGAATTCGCG)(2) as well as three genomic DNAs: M. luteus, calf thymus, and C. perfringens. The level of DNA hydration was varied from Gamma = 2.5 to 22 mol waters/mol nucleotide. The chemical yields of each base, G(base), were measured and used to calculate the modification factor, M(base). This factor compensates for differences in the GC/AT ratio, providing a measure of the degree to which a given base influences its own release. In the DNA oligomers, M(Gua) > M(Cyt), a result ascribed to the previously observed end effect in short oligomers. In the highly polymerized genomic DNA, we found that M(Cyt) > M(Gua) and that M(Thy) is consistently the smallest of the M factors. For these same DNA films, the yields of total DNA trapped radicals, G(tot)(fr), were measured using EPR spectroscopy. The yield of deoxyribose radicals was calculated using G(dRib)(fr) = approximately 0.11 x G(tot)(fr). Comparing G(dRib)(fr) with total fbr, we found that only about half of the fbr is accounted for by deoxyribose radical intermediates. We conclude that for a hole on cytosine, Cyt(*+), base-to-base hole transfer competes with irreversible trapping by the deoxyribose. In the case of a hole on thymine, Thy(*+), base-to-base hole transfer competes with irreversible trapping by methyl deprotonation. Close proximity of Gua protects the deoxyribose of Cyt but sensitizes the deoxyribose of Thy.

WHICH DNA DAMAGE IS LIKELY TO BE RELEVANT IN HORMETIC RESPONSES

Working under the assumption that hormesis is triggered by specific types of DNA damage, this report focuses on the types of damage which form the signature of ionizing radiation. The key attribute of the signature is the clustering of damage, arising from clusters of energy deposition such that more than one site within a 10 base pair segment of DNA has been chemically altered. A brief overview is given on what is currently believed to be the primary components of clustered damage produced by the direct effect. The overview draws primarily on studies that utilize electron paramagnetic resonance to measure free radical intermediates and gel electrophoresis to measure clustered damage in plasmid DNA. Based on this information, the threshold for a radiation induced biological response is calculated.