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

Abstract

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 G sugar (fr) = 0.04-0.07 μmol/J. We found that (1) guanine release exceeded cytosine release by more than twofold, (2) G sugar (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.

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

“…The films were prepared so as to strongly favor the duplex form, (d(CG) n ) 2 and the hydration level for all samples was Γ= 2.5 mol H 2 O per mol nucleotide. In each case, the HPLC profiles were comparable to those published previously for d((CG) 3 ) 2 15. Typical dose response curves are shown in Figure 1 for d(CG) 5 .…”

X-ray induced damage in DNA monitored by X-ray photoelectron spectroscopy

“…25 Moreover, base release under ionizing radiation is known to be correlated with DNA strand breaks; however the mechanism of possible pathways to form strand break is still a subject of many studies. 26 N loss is also observed at low binding energy attributed to the imino site; however this signal begins to decrease only after 4 h of continuous irradiation. After this time, the peak area for the imino group drops about 15% within 1 h ͓see Fig.…”

“…1,2 The first type is due to the attack of DNA by highly reactive radicals that are products of ionizations in the surrounding medium. However, the mobility of formed radicals is not significant and, additionally, the presence of radical scavengers in cells often protects the DNA molecule.…”

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

“…Upon warming of the plasmid film to room temperature and after dissolution in water, reactions (5) and (6) are proposed to account for SSB formation. Reaction (4) raises the possibility that base damage influences deoxyribose damage (19), which increases the importance of understanding the evolution of base damage in the same plasmid samples. The initial radicals generated by reactions (2) and (3) are unstable at higher temperatures, and thus, when samples irradiated at low temperatures are warmed or samples are irradiated at room temperature, these radicals give rise to a reaction sequence that results in radicals that are more stable and eventually in stable end products.…”