Strand breakage in poly(C), poly(A), single- and double-stranded DNA induced by nanosecond laser excitation at 193 nm.

Single- and double-stranded calf thymus DNA and two polynucleotides (0.4 mM) were studied in aqueous solution at pH approximately 7 using pulsed, 20 ns laser excitation at 193 nm. Monophotonic ionization of the nucleic acids is suggested from the linear dependences of the concentration of ejected electrons and the number of single- and double-strand breaks (ssb, dsb, respectively) on laser intensity (IL) in the range (0.2-3) x 10(6) W cm-2. The quantum yields of formation of hydrated electrons (phi e-) and ssb and dsb (phi ssb and phi dsb) are therefore independent of IL. In contrast, under 248 nm excitation these quantum yields increase linearly with IL under otherwise comparable conditions. Nevertheless, several effects and mechanistic implications are analogous using lambda exc = 193 and 248 nm. For polycytidylic acid, poly(C), in Ar-saturated solution for example, the efficiency of ssb per radical cation (eta RC = phi ssb/phi e-) is similar to the efficiency of ssb per OH radical (eta OH). For polyadenylic acid, poly(A), and single- and double-stranded DNA eta RC (lambda exc = 193 nm) is significantly smaller than eta OH. The ratio phi ssb (N2O)/phi ssb (Ar) is approximately 2 for poly(C), approximately 4 for poly(A) approximately 10 for DNA; the conversion of hydrated electrons into OH radicals in N2O-saturated solution and smaller eta RC than eta OH values in the case of DNA account for these results. For double-stranded DNA phi dsb does not depend on IL but increases linearly with the dose, indicating an accumulative effect of two ssb to generate one dsb. The critical distance for this event is 60-85 phosphoric acid diester bonds.     

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

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.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

An investigation into the mechanisms of DNA strand breakage by direct ionization of variably hydrated plasmid DNA

The mechanisms by which ionizing radiation directly causes strand breaks in DNA were investigated by comparing the chemical yield of DNA-trapped free radicals to the chemical yield of DNA single strand break (ssb) and double strand break (dsb), as a function of hydration (Gamma). Solid-state films of plasmid pUC18, hydrated to 2.5 < Gamma < 22.5 mol, were X-irradiated at 4 K, warmed to room temperature, and dissolved in water. Free radical yields were determined by EPR at 4 K. With use of the same samples, Gel electrophoresis was used to measure the chemical yield of total strand breaks, which includes prompt plus heat labile ssb; G'total(ssb) decreased from 0.092 +/- 0.016 micromol/J at Gamma= 2.5 to 0.066 +/- 0.008 micromol/J at Gamma= 22.5. Most provocative is that at Gamma= 2.5 the yield of total ssb exceeds the yield of trapped deoxyribose radicals: G'total(ssb) - G'sugar(fr) = 0.06 +/- 0.02 micromol/J. Nearly 2/3 of the strand breaks are derived from precursors other than radicals trapped on the deoxyribose moiety. To account for these nonradical precursors, we hypothesize that strand breaks are produced by two one-electron oxidations at a single deoxyribose residue within an ionization cluster.     

ULTRAVIOLET LIGHT INDUCES DOUBLE-STRAND BREAKS IN DNA OF CULTURED HUMAN P3 CELLS AS MEASURED BY NEUTRAL FILTER ELUTION

Neutral filter elution at pH 7.2 and 9.6 was used to measure the induction of DNA lesions in human P3 teratocarcinoma cells by monochromatic 254-, 270-, 313-, 334-, 365-, and 405-nm radiation and by 60 gamma rays. In this assay DNA double-strand breaks (dsb) increase the rate of elution of DNA from cell lysates on a filter. Yields of dsb as measured by this procedure were determined by using a calibration of the assay that correlates elution parameters with number of dsb caused by disintegration of 125I incorporated into the DNA. Analysis of fluence responses obtained by using the calibrated assay indicated that the number of dsb induced per dalton of DNA as measured by this assay is proportional to the square of the fluence at all the energies of radiation studied, implying that the induction of these lesions may be a two-hit event. Analysis of the relative efficiencies for the induction of dsb by ultraviolet radiation, corrected for quantum efficiency, revealed a spectrum that coincided closely with that for the induction of single-strand breaks (ssb) in the same cells, having a close fit with the spectrum of nucleic acid in the UVC and UVB region below 313 nm, and a shoulder in the UVA region. It was calculated, however, that there may be too few ssb for dsb to result from randomly distributed closely opposed ssb.     

Biological responses to the simulated Martian UV radiation of bacteriophages and isolated DNA

Mars is considered as a main target for astrobiologically relevant exploration programmes. In this work the effect of simulated Martian solar UV radiation was examined on bacteriophage T7 and on isolated T7 DNA. A decrease of the biological activity of phages, characteristic changes in the absorption spectrum and in the electrophoretic pattern of isolated DNA/phage and the decrease of the amount of PCR products were detected indicating damage of isolated and intraphage T7 DNA by UV radiation. Further mechanistic insights into the UV-induced formation of intraphage/isolated T7 DNA photoproducts were gained from the application of appropriate enzymatic digestion and neutral/alkaline agarose gel electrophoresis. Our results showed that intraphage DNA was about ten times more sensitive to simulated Martian UV radiation than isolated T7 DNA indicating the role of phage proteins in the DNA damage. Compared to solar UV radiation the total amount of DNA damage determined by QPCR was about ten times larger in isolated DNA and phage T7 as well, and the types of the DNA photoproducts were different, besides cyclobutane pyrimidine dimers (CPD), double-strand breaks (dsb), and single-strand breaks (ssb), DNA-protein cross-links were produced as well. Surprisingly, energy deposition as low as 4-6eV corresponding to 200-400nm range could induce significant amount of ssb and dsb in phage/isolated DNA (in phage the ratio of ssb/dsb was approximately 23%/12% and approximately 32%/19% in isolated DNA). 5-8% of the CPD, 3-5% of the AP (apurinic/apyrimidinic) sites were located in clusters in DNA/phage, suggesting that clustering of damage occur in the form of multiple damaged sites and these can have a high probability to produce strand breaks. The amount of total DNA damage in samples which were irradiated in Tris buffer was reduced by a factor approximately 2, compared to samples in phosphate buffer, suggesting that some of the photoproducts were produced via radicals.     

TRANSIENTS OF URACIL and THYMINE DERIVATIVES and THE QUANTUM YIELDS OF ELECTRON EJECTION and INTERSYSTEM CROSSING UPON 20 ns PHOTOLYSIS AT 248 nm

Transients of uracil and a series of 17 correlated pyrimidines, e.g. methylated bases, nucleosides, nucleotides, and polyuridylic acid [poly(U)] were studied after 248 nm excitation by 20 ns laser pulses. The transient absorption spectra in aqueous solution at room temperature reveal the triplet state and the hydrated electron (e-aq), while the corresponding radical cation could not be observed at pH 6-7. Fast loss of the chromophore in the 260-290 nm range within 0.1 microsecond was observed in aqueous solution in some cases [e.g. poly(U), 5'-UMP, uridine, uracil] and in others (thymine, thymidine) virtually not. This photobleaching is assigned to formation of the photohydrate. The concentration of e-aq shows a quadratic dependence on the laser pulse intensity (IL) in the range (0.2-2) x 10(7) W cm-2 and the quantum yield of electron ejection (phi c-) thus depends linearly on IL. This behaviour, suggesting that the photoionization involves a two-step absorption process, was found for poly(U) and all pyrimidine monomers examined. At a constant IL value of 2 x 10(7) W cm-2, phi c- ranges from 3 x 10(-3) for 1,3-dimethylthymine to 4 x 10(-2) for poly(U). The triplet state shows a much larger transient absorbance (delta A, typically in a broad range, e.g. 290-500 nm) than that of the neutral radical resulting from the radical cation. The triplet state in organic solvents (acetonitrile and ethanol) shows generally a significantly larger delta A value than in aqueous solution. The estimated quantum yields of intersystem crossing at room temperature are compared with those of phosphorescence at -196 degrees C.     

BASE RELEASE FROM DNA AND POLYNUCLEOTIDES UPON 193 nm LASER EXCITATION

Release of bases form calf thymus DNA and three polynucleotides, induced by 20 ns excitation at 193 nm in aqueous solution at pH 7, was detected by HPLC. The quantum yields of formation of free bases (phi B) from double-stranded DNA (0.4 mM) are independent of intensity, indicating a one-quantum mechanism of N-glycosidic bond cleavage. The phi B values increase in the order guanine, thymine, adenine, cytosine, the latter being phi C approximately 7 x 10(-4) for double-stranded DNA under Ar and O2. The larger phi B values in N2O-saturated solution, e.g., phi C = 1.2 x 10(-3), are ascribed to additional base release via OH-adduct radicals. The phi B values of homopolynucleotides increase in the order poly(G), poly(A) and poly(C), e.g. phi C = 7 x 10(-3) under Ar, as do the efficiencies for base release per radical cation (eta B). A comparison of the eta B values with the efficiencies of single-strand breakage for poly(C), poly(A) and DNA shows a similar trend; both are markedly larger for pyrimidines than for purines. Pathways to undamaged bases, initiated from base radical cations, are proposed.     

Hydrated Electron Transfer to Nucleobases in Aqueous Solutions Revealed by Ab Initio Molecular Dynamics Simulations

We present an ab initio molecular dynamics (AIMD) simulation study into the transfer dynamics of an excess electron from its cavity‐shaped hydrated electron state to a hydrated nucleobase (NB)‐bound state. In contrast to the traditional view that electron localization at NBs (G/A/C/T), which is the first step for electron‐induced DNA damage, is related only to dry or prehydrated electrons, and a fully hydrated electron no longer transfers to NBs, our AIMD simulations indicate that a fully hydrated electron can still transfer to NBs. We monitored the transfer dynamics of fully hydrated electrons towards hydrated NBs in aqueous solutions by using AIMD simulations and found that due to solution‐structure fluctuation and attraction of NBs, a fully hydrated electron can transfer to a NB gradually over time. Concurrently, the hydrated electron cavity gradually reorganizes, distorts, and even breaks. The transfer could be completed in about 120–200 fs in four aqueous NB solutions, depending on the electron‐binding ability of hydrated NBs and the structural fluctuation of the solution. The transferring electron resides in the π*‐type lowest unoccupied molecular orbital of the NB, which leads to a hydrated NB anion. Clearly, the observed transfer of hydrated electrons can be attributed to the strong electron‐binding ability of hydrated NBs over the hydrated electron cavity, which is the driving force, and the transfer dynamics is structure‐fluctuation controlled. This work provides new insights into the evolution dynamics of hydrated electrons and provides some helpful information for understanding the DNA‐damage mechanism in solution.     

193 NM LIGHT INDUCES SINGLE STRAND BREAKAGE OF DNA PREDOMINANTLY AT GUANINE

Irradiation of DNA with 193 nm light results in monophotonic photoionization, with the formation of a base radical cation and a hydrated electron (φ_P1 = 0.048–0.065). Although >50% of the photoionization events initially occur at guanine in DNA, migration of the “hole” from the other bases to guanine occurs to yield predominantly its radical cation or its deprotonated form. From sequence analysis, the data reveal that 193 nm light induces single strand breaks (ssb) in double-stranded DNA preferential 3’ to a guanine residue. However, it has previously been reported that 193 nm light yields very low yields of ssb (<2% of the yield of eaq). The distribution of these ssb at guanine is nonrandom, showing a dependence on the neighboring base moiety. The efficiency of ssb formation at nonguanine sites is estimated to be at least one order of magnitude lower. The preferred cleavage at guanine is consistent with migration and localization of the electron loss center at guanine. It is argued that singlet oxygen and the photoionized phosphate group of the sugar moiety are not major precursors to ssb. At present, the mechanisms of strand breakage are not known although a guanine radical or one of its products remain potential precursors.     

Hydroxyl radical quenching agents protect against DNA breakage caused by both 365-nm UVA and by gamma radiation

The ability of hydroxyl radical (.OH) scavengers to reduce DNA breakage in isolated DNA from Bacillus subtilis by either gamma radiation or monochromatic radiation in the UVA region (365 nm) was examined by comparing dose reduction factors (the ratio of dose required to induce n DNA breaks in the absence to the presence of quencher). Previous data have demonstrated that acetate, formate, azide, and mannitol protect supercoiled DNA against gamma-radiation-induced ssb (single-strand breaks-relaxation of supercoil by first nick) in close agreement with the rate at which their solutions quench .OH. Here we show that these quenchers also protect against 365-nm-induced ssb. The ratios for protection against 365-nm induced DNA ssb in isolated B. subtilis DNA by the four quenchers are also in proportion to their ability to quench .OH. In view of the diverse chemical nature of the quenchers and the wide range of concentrations involved, these findings are evidence that both these radiations may induce ssb in DNA via a common step that might involve .OH.     

Physiological Doses of Ultraviolet Irradiation Induce DNA Strand Breaks in Cultured Human Melanocytes, as Detected by Means of an Immunochemical Assay

Abstract— An immunochemical assay, i.e. sandwich enzyme-linked immunosorbent assay, has been modified to detect UV-induced damage in cellular DNA of monolayer-grown human melanocytes. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The melanocytes derived from human foreskin of skin type II individuals were suspended and exposed to UVA, UVB, solar-simulated light or γ-rays. Following physiological doses of UVA, UVB or solar-simulated light, a dose-related DNA unwinding comprising a considerable number of single-strand breaks (ssb) was observed. No correlation was found between different seeded cell densities or different culturing periods and the UVA sensitivity of the cells. After UVA irradiation, 0.07 ssb/10¹⁰ Da/kJ/m² were detected and after UVB irradiation 1.9 ssb/10¹⁰ Da/kJ/m² were seen. One minimal erythema dose of solar-simulated light induced 2.25 ssb/10¹⁰ Da. Our results from melanocytes expressed in ssb/Da DNA are comparable and have the same sensitivity toward UVA as well as toward UVB as nonpigmented skin cells. As low doses of UVA have already been shown to induce detectable numbers of ssb, this assay is of great interest for further investigations about the photoprotecting and/or photosensitizing effects of melanins in human melanocytes derived from different skin types.     

A comparative pulse radiolysis study on DNA's of various molecular masses under anoxic and salt-free conditions,based on conductivity measurements

OH radical attack on various molecular mass DNA molecules in aqueous solutions in O₂-free, N₂O-saturated solutions at room temperature and with lower doses have been studied. The two phenomena, resulting in a negative and a positive conductivity build-up under a short electron pulse (0.4–1 s), are discussed in the light of their dependence on the pH, dose rate, concentration and temperature. The positive conductivity build-up observed at lower concentrations of DNA and higher doses is attributed to a degradation process resulting in ssb formation and liberation of counter ions, whereas the negative conductivity build-up at the higher concentrations of DNA and relatively lower doses is attributed to an intermolecular reaction, resulting in cross-links and condensation of the counter ions. The dependence of the applied potential (20–100 V) on the rate constant and conductivity build-up for both processes are shown.     

Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.     

Radiation-induced DNA double-strand breaks in dependence on protein concentration and under aerobic and anaerobic conditions

Double-stranded DNA alone and with serum albumin in eight different weight ratios were irradiated with X-rays in phosphate buffer in air-free and aerated media. Double-strand breaks (dsb) were determined by electrophoresis. The oxygen enhancement ratio for dsb increased with increasing protein concentration from 0.8 to 3.0, presumably caused by the reaction of protein peroxyl radicals. In air-free media, serum albumin protects against radiation-induced double-strand breaking more effectively than in the presence of air, because a part of DNA radicals does not form dsb, but DNA–protein crosslinks.     

MECHANISM OF DNA CLEAVAGE MEDIATED BY PHOTOEXCITED NON-STEROIDAL ANTIINFLAMMATORY DRUGS

DNA damage photoinduced by four nonsteroidal antiinflammatory drugs (NSAID) have been investigated by neutral agarose gel electrophoresis. Upon irradiation at 300 nm, in phosphate buffered solution, benoxaprofen, naproxen, ketoprofen, tiaprofenic acid photosensitized the formation of single-strand breaks (SSB) in double stranded supercoiled phi X174 DNA. The efficiency of the cleavage is higher in argon saturated solutions than in aerated solutions and it is not correlated with the quantum yield of photodegradation of the drugs. Simultaneously with the DNA strand breaks, NSAID promote a weak reduction of the electrophoretic mobility of the supercoiled form that may be attributed to the formation of pyrimidine dimers or other DNA unwinding products. These photodimerization processes suggest the involvement of a triplet-triplet energy transfer between NSAID and DNA. Addition of mannitol and superoxide dismutase decreases the efficiency of the cleavage suggesting that HO. and O2.- are involved in the DNA cleavage. Unexpectedly, addition of sodium azide quenches the cleavage both in aerated or in deaerated solutions. Substituting H2O by D2O does not change the number of SSB thus suggesting that 1O2 does not take an important place in the cleavage of DNA. From our data we tentatively assume that the cleavage occurs through a radical mechanism that may involve in a first step an energy or an electron transfer. Gel sequencing on NSAID-photoinduced DNA breakage exhibits no particular specificity except in the case of benoxaprofen where a slight selectivity for cytosine is observed.     

Analysis of a photosensitive lesion induced by sunlamp UV greater than 315 nm exposure of 254-nm-irradiated human cells

Normal human skin fibroblasts were exposed to 0-10 J m-2 of 254 nm UV, incubated 0-16 h and then treated with 0-150 kJ m-2 of sunlamp UV greater than 315 nm. For each treatment, the cells were subjected to alkaline elution in order to measure the yield of single strand breaks (ssb) produced. It was found that treatment of 254-nm-irradiated cells with sunlamp UV greater than 315 nm resulted in the production of a higher level of ssb than that produced by separate exposures. Hence, lesions are produced by the 254 nm irradiation that are photolyzed through exposure to sunlamp UV greater than 315 nm. Approximately 50% of these lesions are removed following a 2-4 h incubation of the 254-nm-irradiated cells and nearly complete removal is achieved by 16 h. In addition, the profiles for elutions performed at pH 12.8 with cells exposed to the combined treatment were indicative of the presence of alkali labile sites. The repair kinetics of this lesion and alkaline lability of the photolysis product suggest that this photosensitive lesion may represent pyrimidine(6-4)pyrimidone photoproducts. Hence, this approach may represent a relatively simple and sensitive assay for the measurement of this DNA damage.     

SINGLE-STRAND BREAKS INDUCED IN DNA BY VACUUM-ULTRAVIOLET RADIATION

Abstract— The efficiency of vacuum u.v. for producing single-strand breaks in DNA was determined for wavelengths between 58 and 254 nm (corresponding to photon energies of 21·2 and 4·9 eV, respectively) by using the supertwisted RF-DNA of bacteriophage φX174. The cross-section for production of single-strand breaks increases continuously by about 5 orders of magnitude between 5 and 10 eV photon energy, whereas from 11 to 21 eV the number of strand breaks produced per unit of incident radiation energy is approximately constant. Thus, absorption of a 10-eV photon causes DNA strand breaks with maximum efficiency. In addition, the number of electrons liberated from DNA by photons below 10 eV is one or two orders of magnitude higher than the frequency of strand breaks, demonstrating that in this energy range only a small fraction of the ionizations leads to strand breakage in DNA.     

Pulse-radiolytic studies of DNA and polynucleotides in aqueous solution in the presence of oxygen

The kinetic parameters of the formation of single-strand breaks (ssb) induced by OH radicals in presence of oxygen and that of the decay of peroxyl radicals of the polynucleotide have been found to be very similar. The conclusion that the decay of the peroxyl radicals is involved in the rate-determining step of ssb formation is confirmed for poly(U) by a study of the effect of ethanol on ssb formation under conditions of laser pulse excitation. The kinetics of the formation of ssb for poly(U) is complex but is consistent with a first order followed by more complex reactions. This kinetics is compatible with a pathway to ssb formation assuming H abstraction from the sugar moiety by base peroxyl radicals as the rate-determining step in the beginning of the overall reaction.     

Influence of organic ions on DNA damage induced by 1 eV to 60 keV electrons

We report the results of a study on the influence of organic salts on the induction of single strand breaks (SSBs) and double strand breaks (DSBs) in DNA by electrons of 1 eV to 60 keV. Plasmid DNA films are prepared with two different concentrations of organic salts, by varying the amount of the TE buffer (Tris-HCl and EDTA) in the films with ratio of 1:1 and 6:1 Tris ions to DNA nucleotide. The films are bombarded with electrons of 1, 10, 100, and 60?000 eV under vacuum. The damage to the 3197 base-pair plasmid is analyzed ex vacuo by agarose gel electrophoresis. The highest yields are reached at 100 eV and the lowest ones at 60 keV. The ratios of SSB to DSB are surprisingly low at 10 eV (～4.3) at both salt concentrations, and comparable to the ratios measured with 100 eV electrons. At all characteristic electron energies, the yields of SSB and DSB are found to be higher for the DNA having the lowest salt concentration. However, the organic salts are more efficient at protecting DNA against the damage induced by 1 and 10 eV electrons. DNA damage and protection by organic ions are discussed in terms of mechanisms operative at each electron energy. It is suggested that these ions create additional electric fields within the groove of DNA, which modify the resonance parameter of 1 and 10 eV electrons, namely, by reducing the electron capture cross-section of basic DNA units and the lifetime of corresponding transient anions. An interstrand electron transfer mechanism is proposed to explain the low ratios for the yields of SSB to those of DSB produced by 10 eV electrons.