Yuriy Razskazovskiy

The Release of 5-Methylene-2-Furanone from Irradiated DNA Catalyzed by Cationic Polyamines and Divalent Metal Cations

Abstract Roginskaya, M., Bernhard, W. A., Marion, R. T. and Razskazovskiy, Y. The Release of 5-Methylene-2-Furanone from Irradiated DNA Catalyzed by Cationic Polyamines and Divalent Metal Cations. Radiat. Res. 163, 85–89 (2005). Release of 5-methylene-2-furanone (5-MF), a characteristic marker of DNA deoxyribose oxidative damage at the C1′ position, was observed in significant quantities from X-irradiated DNA. This observation, which held for DNA irradiated either in aqueous solution or as a film, requires postirradiation treatment at 90°C in the presence of polyamines and divalent metal cations at biological pH. The 5-MF product was quantified by using reverse-phase HPLC. The radiation chemical yield of 5-MF comprised more than 30% of the yield of total unaltered base release. Polylysine, spermine and Be(II) showed the strongest catalytic effect on 5-MF release, while Zn(II), Cu(II), Ni(II), putrescine and Mg(II) were substantially less efficient. We have hypothesized that the 5-MF release from irradiated DNA occurs through catalytic decomposition of the 2′-deoxyribonolactone (dL) precursor through two consecutive β- and δ-phosphate elimination reactions. A stepwise character of the process was indicated by the S-shaped time course of 5-MF accumulation. If dL proves to be the precursor to 5-MF formation, it would then follow that dL is a very important lesion generated in DNA by ionizing radiation.

Direct Radiation Damage to Crystalline DNA: What is the Source of Unaltered Base Release?

Abstract Razskazovskiy, Y., Debije, M. G. and Bernhard, W. A. Direct Radiation Damage to Crystalline DNA: What is the Source of Unaltered Base Release? The radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 μmol J–1. The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient.

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.

Strand Breaks Produced in X-irradiated Crystalline DNA: Influence of Base Sequence

Abstract Razskazovskiy, Y., Debije, M. G. and Bernhard, W. A. Strand Breaks Produced in X-Irradiated Crystalline DNA: Influence of Base Sequence. Radiat. Res. 159, 663–669 (2003). This study reports the radiation-chemical yields for DNA single-strand breaks (SSBs) in crystals of CGCACG:CGTGCG (I) and CACGCG:CGCGTG (II) duplexes induced by direct ionization using X rays. The DNA fragmentation products, consisting of 3′- and 5′-phosphate-terminated fragments, were quantified by ion-exchange chromatography using a set of reference compounds. The yields of single-strand breaks in I and II are 0.16 ± 0.03 μmol/J and 0.07 ± 0.02 μmol/J, respectively. The probability of cleavage at a given site is relatively independent of which of the four bases is at that site. For the very small sample of base sequences studied to date, there is no obvious dependence on base sequence. However, there appears to be an increased frequency of strand breaks at the non-phosphorylated termini of the oligodeoxynucleotides. These results show that direct ionization is efficient at producing single-strand breaks in DNA and that its action is relatively indiscriminate with respect to base sequence.

Strand breaks in x-irradiated crystalline DNA : alternating CG-oligomers

Abstract Razskazovskiy, Y., Debije, M. G., Howerton, S. B., Williams, L. D. and Bernhard, W. A. Strand Breaks in X-Irradiated Crystalline DNA: Alternating CG Oligomers. Radiat. Res. 160, 334–339 (2003). Direct ionization of crystalline d(CGCGCGCG) and d(CGCGCGCGCG) oligomers produces 3′- and 5′-phosphate-terminated fragments as the main strand breakage products detectable by ion-exchange chromatography. The nature of the base has no effect on the probability of strand breakage at the given site. The yields of 3′-phosphates are systematically lower than the yields of the 5′-phosphates originating from the same cleavage site, pointing to the possible presence of unidentified products with sugar remnants attached to the 3′-end. These results show that direct ionization is efficient at producing single-strand breaks in DNA and its action is relatively indiscriminate with respect to base sequence.

Protection of DNA against Direct Radiation Damage by Complex Formation with Positively Charged Polypeptides

Abstract Roginskaya, M., Bernhard, W. A. and Razskazovskiy, Y. Protection of DNA against Direct Radiation Damage by Complex Formation with Positively Charged Polypeptides. Radiat. Res. 166, 9–18 (2006). Radioprotection of DNA from direct-type radiation damage by histones has been studied in model systems using complexes of positively charged polypeptides (PCPs) with DNA. PCPs bind to DNA via ionic interactions mimicking the mode of DNA-histone binding. Direct radiation damage to DNA in films of DNA-PCP complexes was quantified as unaltered base release, which correlates closely with DNA strand breaks. All types of PCPs tested protected DNA from radiation, with the maximum radioprotection being approximately 2.5-fold compared with non-complexed DNA. Conformational changes of the DNA induced by PCPs or repair of free radical damage on the DNA sugar moiety by PCPs are considered the most feasible mechanisms of radioprotection of DNA. The degree of radioprotection of DNA by polylysine (PL) increased dramatically on going from pure DNA to a molar ratio of PL monomer:DNA nucleotide ∼1:2, while a further increase in the PL:DNA ratio did not offer more radioprotection. This concentration dependence is in agreement with the model of PCP binding to DNA that assumes preferential binding of positively charged side groups to DNA phosphates in the minor groove, so that the maximum occupancy of all minor-groove PCP binding sites is at a molar ratio of PCP:DNA = 1:2.

Identification of the C4'-oxidized abasic site as the most abundant 2-deoxyribose lesion in radiation-damaged DNA using a novel HPLC-based approach.

A novel analytical high-performance liquid chromatography (HPLC)-based method of quantification of the yields of C4′-oxidized abasic sites, 1, in oxidatively damaged DNA has been elaborated. This new approach is based on efficient conversion of 1 into N-substituted 5-methylene-Δ3-pyrrolin-2-ones, 2, upon treatment of damaged DNA with primary amines in neutral or slightly acidic solutions with subsequent quantification of 2 by HPLC. The absolute and relative radiation-chemical yields of 1 in irradiated DNA solutions were re-evaluated using this method. The yields were compared with those of other 2-deoxyribose degradation products including 5-methylene-2(5H)-furanone, malondialdehyde, and furfural resulting from the C1′, C4′ and C5′-oxidations, respectively. The yield of free base release (FBR) determined in the same systems was employed as an internal measure of the total oxidative damage to the 2-deoxyribose moiety. Application of this technique identifies 1 as the most abundant sugar lesion in double-stranded (ds) DNA irradiated under air in solution (36% FBR). In single-stranded (ss) DNA this product is second by abundance (33% FBR) after 2-deoxyribonolactones (C1′-oxidation; 43% FBR). The production of nucleoside-5′-aldehydes (C5′-oxidation; 14% and 5% FBR in dsDNA and ssDNA, respectively) is in the third place. Taken together with the parallel reaction channel that converts C4′-radicals into malondialdehyde and 3′-phosphoglycolates, our results identify the C4′-oxidation as a prevalent pathway of oxidative damage to the sugar-phosphate backbone (50% or more of all 2-deoxyribose damages) in indirectly damaged DNA.

Factors Affecting the Yields of C1′ and C5′ Oxidation Products in Radiation-Damaged DNA: The Indirect Effect

Abstract This study reports the effects of denaturation and deoxygenation on radiation-induced formation of 2-deoxyribonolactone (2-dL) and 5′-aldehyde (5′-Ald) lesions in highly polymerized DNA. The radiation-chemical yields of 2-dL were determined through quantification of its dephosphorylation product 5-methylenefuranone (5MF). The formation of 5′-Ald was monitored qualitatively through the release of furfural (Fur) under the same conditions. The yields of 2-dL were found to be 7.3 ± 0.3 nmol J−1, or about 18% of the yield of free base release measured in the same samples. Denaturation increased the efficiency of 2-dL formation approximately twofold while deoxygenation resulted in a fourfold decrease. The release of Fur is about twofold lower than that of 5MF in aerated native DNA samples and is further reduced by denaturation of the DNA. Unlike 5MF, the formation of Fur requires the presence of molecular oxygen, which is consistent with peroxyl radical-mediated oxidation of C5′ radicals into 5′-Ald. In contrast, the existence of an oxygen-independent pathway of 2-dL formation suggests that C1′ sugar radicals can also be oxidized by radiation-produced oxidizing intermediates such as electron-loss centers on guanines.

Reductively Activated Cleavage of DNA Mediated by o,o′-Diphenylenehalonium Compounds

Abstract Razskazovskiy, Y., Roginskaya, M., Jacobs, A. and Sevilla, M. D. Reductively Activated Cleavage of DNA Mediated by o,o′-Diphenylenehalonium Compounds. o,o′-Diphenylenehalonium (DPH) cations represent a novel class of DNA-affinic compounds characterized by binding constants within the range of 105–106 M–1. The maximum binding capacity of 2–2.5 base pairs per DPH cation and about 30% hypochromic reduction in the optical absorption of DPH cations upon binding to DNA suggest intercalation as a likely binding mode. In a DNA-bound form, DPH cations induce strand breaks upon reduction by radiation-produced electrons in aqueous solutions. In keeping with this mechanism, the cleavage is strongly inhibited by oxygen and is not affected by OH radical scavengers in the bulk. The yields of DPH-mediated base release significantly exceed the yield of base release caused by hydroxyl radical (in the absence of scavenger) in anoxic solutions. The yields are weakly dependent on DNA loading within the range from 5 to 50 base pairs per intercalator, which indicates the ability of excess electrons in DNA to react with a scavenger separated by tens of base pairs from the electron attachment site. The question regarding the mechanism by which the distant reactants reach each other in DNA remains unanswered, although it most likely involves electron hopping rather than a single-step long-distance tunneling. The latter conclusion is based on our finding that the electron affinity of DPH cations does not affect their properties as electron scavengers in DNA as would be expected if the direct long-distance tunneling is involved.

Radiation-Activated Nuclease Activity of o,o′-Diphenyleneiodonium Cations (DPI): A Reductively Initiated Chain Reaction Involving the C1′ Chemistry

Abstract Razskazovskiy, Y. Radiation-Activated Nuclease Activity of o,o′-Diphenyleneiodonium Cations (DPI): A Reductively Initiated Chain Reaction Involving the C1′ Chemistry. Radiat. Res. 159, 543–549 (2003). o,o′-Diphenyleneiodonium cations (DPI) convert relatively harmless radiation-produced electrons into efficient DNA cleaving agents. The cleavage products are unaltered DNA bases, 5-methylenefuranone (5-MF), and a complete set of 3′ and 5′-phosphorylated DNA fragments. The production of alkali-labile sites is a minor factor in the process. Based on the production of 5-MF, it is concluded that DNA cleavage by DPI cations involves (but may not be limited to) the C1′ chemistry. The loss of 3-aminoDPI (ADPI) cations bound to highly polymerized calf thymus DNA appears to be due to a short-chain reaction with an apparent length of up to 2.1 ADPI cations consumed for each radiation-produced electron. The suggested chain reaction mechanism includes the one-electron oxidation of DNA radicals (including the C1′ sugar radical) by ADPI cations bound to the same duplex. The yields of DNA loss in complexes formed by ADPI with short synthetic duplexes indicate that there is more than a 60% probability of DNA damage after one-electron reduction of ADPI.