J. R. Milligan

DNA strand-break yields after post-irradiation incubation with base excision repair endonucleases implicate hydroxyl radical pairs in double-strand break formation

PURPOSE To determine the increases in SSB and DSB yields after post gamma-irradiation incubation of plasmid DNA with the Escherichia coli base excision repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) and endonuclease III (endo III). MATERIALS AND METHODS Aqueous solutions of plasmid DNA were irradiated with 137Cs gamma-rays in the presence of 10(-4) - 10(-1) mol dm(-3) formate. After irradiation, aliquots were treated with FPG and/or endo III. SSB and DSB yields were then determined using gel electrophoresis. RESULTS Both SSB and DSB yields were found to increase after enzyme incubation, with the increase in the DSB yield being approximately equal to the square of the increase in the SSB yield. The correlation between the increases in the SSB and DSB yields was unaffected by the scavenger concentration during irradiation. CONCLUSION Under the conditions used, the majority of DSB appear to be formed from two hydroxyl radical attacks.Purpose : To determine the increases in SSB and DSB yields after post gamma-irradiation incubation of plasmid DNA with the Escherichia coli base excision repair endonucleases formamidopyrimidineDNA N -glycosylase (FPG) and endonuclease III (endo III). Materials and methods : Aqueous solutions of plasmid DNA were irradiated with 137 Cs gamma-rays in the presence of 10 -4 -10 -1 mol dm -3 formate. After irradiation, aliquots were treated with FPG and/or endo III. SSB and DSB yields were then determined using gel electrophoresis. Results : Both SSB and DSB yields were found to increase after enzyme incubation, with the increase in the DSB yield being approximately equal to the square of the increase in the SSB yield. The correlation between the increases in the SSB and DSB yields was unaffected by the scavenger concentration during irradiation. Conclusion : Under the conditions used, the majority of DSB appear to be formed from two hydroxyl radical attacks.

Evaluation of lesion clustering in irradiated plasmid DNA.

Purpose: To measure the yield of DNA strand breaks and clustered lesions in plasmid DNA irradiated with protons, helium nuclei, and γ-rays. Materials and methods: Plasmid DNA was irradiated with 1.03, 19.3 and 249 MeV protons (linear energy transfer = 25.5, 2.7, and 0.39 keV μm – 1 respectively), 26 MeV helium nuclei (25.5 keV μm) and γ-rays (137Cs or 60Co) in phosphate buffer containing 2 mM or 200 mM glycerol. Single-and double-strand breaks (SSB and DSB) were measured by gel electrophoresis, and clustered lesions containing base lesions were quantified by converting them into irreparable DSB in transformed bacteria. Results: For protons, SSB yield decreased with increasing LET (linear energy transfer). The yield of DSB and all clustered lesions seemed to reach a minimum around 3 keV μm – 1. There was a higher yield of SSB, DSB and total clustered lesions for protons compared to helium nuclei at 25.5 keV μm – 1. A difference in the yields between 137Cs and 60Co γ-rays was also observed, especially for SSB. Conclusion: In this work we have demonstrated the complex LET dependence of clustered-lesion yields, governed by interplay of the radical recombination and change in track structure. As expected, there was also a significant difference in clustered lesion yields between various radiation fields, having the same or similar LET values, but differing in nanometric track structure.

A nanodosimetric model of radiation-induced clustered DNA damage yields

We present a nanodosimetric model for predicting the yield of double strand breaks (DSBs) and non-DSB clustered damages induced in irradiated DNA. The model uses experimental ionization cluster size distributions measured in a gas model by an ion counting nanodosimeter or, alternatively, distributions simulated by a Monte Carlo track structure code developed to simulate the nanodosimeter. The model is based on a straightforward combinatorial approach translating ionizations, as measured or simulated in a sensitive gas volume, to lesions in a DNA segment of one-two helical turns considered equivalent to the sensitive volume of the nanodosimeter. The two model parameters, corresponding to the probability that a single ion detected by the nanodosimeter corresponds to a single strand break or a single lesion (strand break or base damage) in the equivalent DNA segment, were tuned by fitting the model-predicted yields to previously measured double-strand break and double-strand lesion yields in plasmid DNA irradiated with protons and helium nuclei. Model predictions were also compared to both yield data simulated by the PARTRAC code for protons of a wide range of different energies and experimental DSB and non-DSB clustered DNA damage yield data from the literature. The applicability and limitations of this model in predicting the LET dependence of clustered DNA damage yields are discussed.

DNA strand break yields after post-high LET irradiation incubation with endonuclease-III and evidence for hydroxyl radical clustering

Purpose : To determine the increase in single- (SSB) and double-strand break (DSB) yields after post-high LET irradiation incubation of plasmid DNA with the endonuclease-III (endo-III) of Escherichia coli. Materials and methods : Plasmid DNA in aerobic aqueous solution was irradiated with one of five radiation types: 137 Cs γ-rays (LET ~0.3keV μ m -1) , 244 Cm α -particles (140-190 keV μ m -1) , 4 He ions (97 keV μ m -1) , 56 Fe ions (143keV μ m -1) or 197 Au ions (1440keV μ m -1) . The irradiated samples were then incubated with endo-III. SSB and DSB yields were quantified by agarose gel electrophoresis. Results : Endo-III incubation produced an increase in the SSB and DSB yields. The increases were in general lower after the high LET irradiation than after γ-irradiation. This may reflect inhibition of the activity of endo-III by the nearby DNA damage expected from high LET radiation. It can be shown that even if the activity of endo remains unchanged, significantly lower increases in SSB and DSB yields would still be expected. Conclusion : The results provide evidence for clustered DNA damage after high LET irradiation.

Mechanism of DNA damage by thiocyanate radicals

Purpose : It was previously shown that γ-irradiation of aqueous solutions of plasmid DNA in the presence of millimolar concentrations of thiocyanate ions leads to the formation in very high yields of sites recognized by the base excision repair endonuclease formamido-pyrimidine-DNA N -glycosylase (FPG). The authors wished to characterize the mechanism responsible for the production of these FPG-sensitive sites. Materials and methods : An aqueous solution of plasmid DNA containing thiocyanate ions was irradiated with 137 Cs γ-rays. After irradiation, aliquots were treated with FPG. Break yields were determined using neutral agarose gel electrophoresis. Results : The yield of FPG-sensitive sites decreased with decreasing enzyme activity, increasing thiocyanate concentration, increasing dose-rate, increasing ionic strength, increasing nitrite or iodide concentration, and decreasing oxygen concentration. Conclusions: The observations suggest that the monomeric thiocyanate radical SCN •is an intermediate in the reaction, and that the yields of FPG-sensitive sites are determined by competition between the disproportionation of the dimeric radical anion (SCN) 2 •- and the fate of a one-electron oxidized guanine species in DNA. The latter can react with oxygen to produce an FPGsensitive site or can be reduced without producing an FPGsensitive site. The results help to clarify the mechanisms responsible for DNA damage by the direct eÚect of ionizing radiation.PURPOSE It was previously shown that gamma-irradiation of aqueous solutions of plasmid DNA in the presence of millimolar concentrations of thiocyanate ions leads to the formation in very high yields of sites recognized by the base excision repair endonuclease formamido-pyrimidine-DNA N-glycosylase (FPG). The authors wished to characterize the mechanism responsible for the production of these FPG-sensitive sites. MATERIALS AND METHODS An aqueous solution of plasmid DNA containing thiocyanate ions was irradiated with 137Cs gamma-rays. After irradiation, aliquots were treated with FPG. Break yields were determined using neutral agarose gel electrophoresis. RESULTS The yield of FPG-sensitive sites decreased with decreasing enzyme activity, increasing thiocyanate concentration, increasing dose-rate, increasing ionic strength, increasing nitrite or iodide concentration, and decreasing oxygen concentration. CONCLUSION The observations suggest that the monomeric thiocyanate radical SCN* is an intermediate in the reaction, and that the yields of FPG-sensitive sites are determined by competition between the disproportionation of the dimeric radical anion (SCN)*2- and the fate of a one-electron oxidized guanine species in DNA. The latter can react with oxygen to produce an FPG-sensitive site or can be reduced without producing an FPG-sensitive site. The results help to clarify the mechanisms responsible for DNA damage by the direct effect of ionizing radiation.

Redox reactivity of guanyl radicals in plasmid DNA.

Purpose : It has been previously argued that γ-irradiation of plasmid DNA in the presence of thiocyanate ions produces products recognized by the E. coli base excision-repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG), and there that derive from an intermediate guanyl radical species. The wish was to characterize the reactivity of this intermediate with reducing agents. Materials and methods : Aqueous solutions of plasmid DNA containing either bromide or thiocyanate (10 -3 to 10 -1 mol dm -3) and also one of six other additives (azide, ferrocyanide, iodide, nitrite, promethazine, tryptophan, 10 -7 to 10 -3 mol dm -3) were subjected to 137 Cs γ-irradiation (662 keV). After irradiation, the plasmid was incubated with FPG. Strand break yields before and after incubation were determined by agarose gel electrophoresis under neutral conditions. Results : The very high yields of FPG-sensitive sites in the presence of SCN - or Br - decreased significantly with increasing concentrations of all of the six additives, with promethazine and tryptophan being the most efficient additives, and azide and iodide the least. Conclusions : From the results it is possible to estimate values of the rate constants for the reduction of the DNA guanyl radical (5x10 5, 2x10 5, 10 7 and 10 7 dm 3 mol -1 s -1 for ferrocyanide, nitrite, promethazine and tryptophan respectively).PURPOSE It has been previously argued that gamma-irradiation of plasmid DNA in the presence of thiocyanate ions produces products recognized by the E. coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG), and there that derive from an intermediate guanyl radical species. The wish was to characterize the reactivity of this intermediate with reducing agents. MATERIALS AND METHODS Aqueous solutions of plasmid DNA containing either bromide or thiocyanate (10(-3) to 10(-1) mol dm(-3)) and also one of six other additives (azide, ferrocyanide, iodide, nitrite, promethazine, tryptophan, 10(-7) to 10(-3) mol dm(-3)) were subjected to 137Cs gamma-irradiation (662 keV). After irradiation, the plasmid was incubated with FPG. Strand break yields before and after incubation were determined by agarose gel electrophoresis under neutral conditions. RESULTS The very high yields of FPG-sensitive sites in the presence of SCN- or Br- decreased significantly with increasing concentrations of all of the six additives, with promethazine and tryptophan being the most efficient additives, and azide and iodide the least. CONCLUSIONS From the results it is possible to estimate values of the rate constants for the reduction of the DNA guanyl radical (5 x 10(5), 2 x 10(5), 10(7) and 10(7) dm3 mol(-1) s(-1) for ferrocyanide, nitrite, promethazine and tryptophan respectively).

Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength.

Purpose : Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN) 2 £ -. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN) 2 £ - is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of oneelectron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. Materials and methods : Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137 Cs n -irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. Results : The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). Conclusions : It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN) 2 £ - and DNA-G £ +. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G £ + /DNA of E 7 = +1.39 - 0.01V.

Modification of ionizing radiation clustered damage: estimate of the migration distance of holes through DNA via guanyl radicals under physiological conditions.

Purpose : Guanyl radicals are produced in DNA when it is subjected to oxidation or ionizing radiation. The sites at which stable products can be identified can be located dozens of base pairs away from the initial site of the electron loss. This migration will modify the spatial distribution of damage and tends to mitigate the clustering of initial damage generally associated with ionizing radiation. The migration distance is presumably a function of the lifetime of the intermediate guanyl radical, and we wished to quantify the relationship between them. Materials and methods : Aqueous solutions containing plasmid DNA and thiocyanate ions were treated with γ-irradiation. These conditions result in the very efficient production of guanyl radicals in the plasmid. We quantified the formation of stable guanine oxidation products in the plasmid as strand breaks by using the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). The effect of two additives on the yield of guanine oxidation, nitrite ions and the DNA binding ligand doxorubicin (adriamycin), were examined. Results : The presence during irradiation of the DNA-binding ligand doxorubicin attenuated the yields of stable oxidized guanine products formed. The additional presence of nitrite decreased this effect of doxorubicin. Conclusion : Because doxorubicin binds strongly to DNA, its ability to attenuate guanine oxidation can be interpreted in terms of the migration distance of the intermediate guanyl radical. Because nitrite repairs these intermediate guanyl radicals by electron transfer, its presence during irradiation decreases their lifetime. Therefore, we derived an estimate of the migration distance of guanyl radicals as a function of their lifetime. The presence in cells of antioxidants such as glutathione sets an upper limit to the likely lifetime and, therefore, the migration distance of guanyl radicals. It was concluded that the migration of guanyl radicals may not decrease the clustering of DNA damage in vivo to a great extent.

Reaction of guanyl radicals in plasmid DNA with biological reductants : chemical repair of DNA damage produced by the direct effect of ionizing radiation

Purpose : It has been previously argued that the use of the one-electron oxidants (SCN) 2 •- and Br 2 •- with plasmid DNA leads to the formation of DNA guanyl radicals. These guanyl radical species are intermediates in the DNA damage produced by processes such as photo-ionization and ionizing irradiation. The present paper evaluates the use of thallium(II) ions (Tl II OH +) as the one-electron oxidant, and also determines rate constants for the reduction (repair) of guanyl radicals in plasmid DNA by a variety of reducing agents including the biologically important compounds ascorbate and glutathione. Materials and methods : Aqueous solutions of plasmid DNA containing 10 -3 mol dm -3 thiocyanate or thallous ions and a reducing agent (azide, nitrite, ferrocyanide, hexachloroiridate(III), iodide, ascorbate, glutathione, glutathione disulphide, methionine, tyrosine, 5-hydroxyindole-3-acetic acid, 10 -7 -10 -4 mol dm -3) were irradiated with 137 Cs γ-rays (662 keV). After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). Strand break yields after incubation were quantified by means of agarose gel electrophoresis. Results : High yields of FPG-sensitive sites produced by the oxidants (SCN)2 •- and Tl II OH + were strongly attenuated by the presence of the reducing agents. Conclusions : From the results, it is possible to arrive at estimates of the rate constants for the reduction of the DNA guanyl radical by the reducing agents. Values lie in the range 10 4 -10 7 dm 3 mol -1 s -1. Using the values for ascorbate and glutathione, it is possible to estimate an upper limit on the order of milliseconds for the lifetime of DNA guanyl radicals under cellular conditions. The implication is that there may well be a significant chemical repair of DNA base damage by the direct effect of ionizing radiation.

One-electron oxidation of plasmid DNA by selenium(V) species

Purpose : To employ the γ-radiation-generated selenium(V) one-electron-oxidizing agent SeO 3 •- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species. Materials and methods : Plasmid DNA in aerobic aqueous solution was irradiated with 137 Cs γ-rays (662 keV). The solutions also contained up to 4 ×10 -2 mol dm -3 sodium selenate (Na 2 SeO 4) and/or up to 10 -1 mol dm -3 sodium biselenite (NaHSeO 3) , as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis. Results : In general, γ-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after γ-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after γ-irradiation in the presence of 10 -2 mol dm -3 sodium selenate and 10 -1 mol dm -3 glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO 3 •-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical. Conclusion : By comparing the yields of breaks produced after γ-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.