Phosphodiester and N-glycosidic bond cleavage in DNA induced by 4-15 eV electrons

Thin molecular films of the short single strand of DNA, GCAT, were bombarded under vacuum by electrons with energies between 4 and 15 eV. Ex vacuo analysis by high-pressure liquid chromatography of the samples exposed to the electron beam revealed the formation of a multitude of products. Among these, 12 fragments of GCAT were identified by comparison with reference compounds and their yields were measured as a function of electron energy. For all energies, scission of the backbone gave nonmodified fragments containing a terminal phosphate, with negligible amounts of fragments without the phosphate group. This indicates that phosphodiester bond cleavage by 4-15 eV electrons involves cleavage of the C-O bond rather than the P-O bond. The yield functions exhibit maxima at 6 and 10-12 eV, which are interpreted as due to the formation of transient anions leading to fragmentation. Below 15 eV, these resonances dominate bond dissociation processes. All four nonmodified bases are released from the tetramer, by cleavage of the N-glycosidic bond, which occurs principally via the formation of core-excited resonances located around 6 and 10 eV. The formation of the other nonmodified products leading to cleavage of the phosphodiester bond is suggested to occur principally via two different mechanisms: (1) the formation of a core-excited resonance on the phosphate unit followed by dissociation of the transient anion and (2) dissociation of the CO bond of the phosphate group formed by resonance electron transfer from the bases. In each case, phosphodiester bond cleavage leads chiefly to the formation of stable phosphate anions and sugar radicals with minimal amounts of alkoxyl anions and phosphoryl radicals.     

Induction of strand breaks by low-energy electrons (8-68 eV) in a self-assembled monolayer of oligonucleotides: effective cross sections and attenuation lengths

Self-assembled monolayers of 5'-32P-labeled 3'-thiolated oligonucleotides chemisorbed on gold were bombarded by low-energy electrons (LEE) of 8-68 eV. Shorter 5'-32P-oligonucleotides produced by LEE-induced strand breaks were separated with denaturing polyacrylamide gel electrophoresis and quantified by phosphor imaging. The yields of short oligonucleotides (y) decrease exponentially with their length (n), following the equation y=ae-bn, where a and b are constants, which are related to the average effective cross section per nucleotide for DNA strand break (sigmaeff) and the attenuation length (AL=1b) of LEE, respectively. The AL decreases with LEE energies from 2.5+/-0.6 nm at 8 eV to 0.8+/-0.1 nm at 68 eV, whereas sigmaeff increases from (3+/-1)x10(-18) to (5.1+/-1.6)x10(-17) cm2 within the same energy range. The energy dependence of sigmaeff shows a resonance peak of (2.8+/-0.9)x10(-17) cm2 at 18 eV superimposed on a monotonically rising curve. Transient electron attachment to a sigma* anion state of the deoxyribose group, followed by dipolar dissociation into H- and the corresponding positive-ion radical, leading to C-O bond cleavage, is proposed to account for this maximum.     

Induction of strand breaks in DNA films by low energy electrons and soft X-ray under nitrous oxide atmosphere

Five-monolayer (5 ML) plasmid DNA films deposited on glass and tantalum substrates were exposed to Al K_α X-rays of 1.5 keV under gaseous nitrous oxide (N₂O) at atmospheric pressure and temperature. Whereas the damage yields for DNA deposited on glass are due to soft X-rays, those arising from DNA on tantalum are due to both the interaction of low energy photoelectrons from the metal and X-rays. Then, the differences in the yields of damage on glass and tantalum substrates, essentially arises from interaction of essentially low-energy electrons (LEEs) with DNA molecules and the surrounding atmosphere. The G-values (i.e., the number of moles of product per Joule of energy absorbed) for DNA strand breaks induced by LEEs (G_LEE) and the lower limit of G-values for soft X-ray photons (G_XL) were calculated and the results compared to those from previous studies under atmospheric conditions and other ambient gases, such as N₂ and O₂. Under N₂O, the G-values for loss of supercoiled DNA are 103±15 nmol/J for X-rays, and 737±110 nmol/J for LEEs. Compared to corresponding values in an O₂ atmosphere, the effectiveness of X-rays to damage DNA in N₂O is less, but the G value for LEEs in N₂O is more than twice the corresponding value for an oxygenated environment. This result indicates a higher effectiveness for LEEs relative to N₂ and O₂ environments in causing SSB and DSB in an N₂O environment. Thus, the previously observed radiosensitization of cells by N₂O may not be only due to OH radicals but also to the reaction of LEE with N₂O molecules near DNA. The previous experiments with N₂ and O₂ and the present one demonstrate the possibility to investigate damage induced by LEEs to biomolecules under various types of surrounding atmospheres.     

Radiation induced DNA strand breaks measured by a modified method of gel scanning

Gel electrophoresis is an effective method for assaying plasmid DNA fractions, and UV lights with long wavelengths such as 315 nm is used to image the gel. In the present work, the sensitivities of detecting the fluorescence emitted from ethidium bromide (EB) stained DNA bands in the gel illuminated with UV lights of various wavelengths were compared. It was found that, in the range 245 to 320 nm, shorter excitation wavelength had higher detection sensitivity, thus 260 nm was selected for further studies. With this excitation light, as little as 0.7 ng DNA was detected. The fluorescence of DNA-EB bands had a good linear response to DNA quantity in a wide range. In addition, measured via this modified method, the yield of DNA strand breaks and the second-order rate coefficient of the reaction between DNA and √OH radical were consistent with many previous studies.     

Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks

DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of gamma-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for > 4 h in addition to those induced at replication.     

Dehydrogenation of adenine induced by slow (<3 eV) electrons

Low energy (<3 eV) electrons impact to gas phase Adenine generates the dehydrogenated Adenine negative fragments, (A–H)⁻, and an H-atom neutral radical counterpart. Within the energy range of 0.7–2.8 eV, production of (A–H)⁻ arises from Dissociative Electron Attachment (DEA). In addition, a sharp peak is observed at near 0 eV. This peak is identified to arise from dissociative electron transfer reaction of SF₆⁻ (from the calibration gas) with Adenine.     

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.     

Single and multiple photoionisation of H2S by 40-250 eV photons

Multi-electron coincidence measurements on photoionisation of H(2)S have been carried out at photon energies from 40 to 250 eV. They quantify molecular field effects on the Auger process in detail and are in good agreement with the existing theory. Spectra of core-valence double ionisation of H(2)S are presented and partially analysed. Auger decays from the core-valence states produce triply charged product spectra with unexplained and surprising intensity distributions. Triple ionisation by the double Auger process from 2p hole states shows little effect of the molecular field splitting, but includes a substantial contribution from cascade processes, some involving dissociation in intermediate states. The onset of triple ionisation at the molecular geometry is determined as 61 ± 0.5 eV.     

Mass spectrometry investigation of the degradation of polyethylene terephtalate induced by low-energy (<100 eV) electrons

Polyethylene terephtalate (PET) thin films were damaged by low-energy (0–100 eV) electron irradiation to simulate the degradation of this polymer in electronic devices. The products formed were analyzed by mass spectrometry. The emission of anions from the polymer surface is associated with dissociative electron attachment (DEA) and dipolar dissociation (DD) for H⁻, and with DD for O⁻. The monotonic emission rise in O⁻ desorption as a function of incident electron energy is produced by mid-chain C–O–C cleavage, leading to chain scission. The signal of the positive mass fragments showed only a monotonic increase with electron energy. In this case, chemical recombination with hydrogen atoms also leads to chain scission.     

Oxidative DNA strand scission induced by peptides

Cellular oxidative stress promotes chemical reactions causing damage to DNA, proteins, and membranes. Here, we describe experiments indicating that reactive oxygen species, in addition to degrading polypeptides and polynucleotides through direct reactions, can also promote damaging biomolecular cross reactivity by converting protein residues into peroxides that cleave the DNA backbone. The studies reported show that a variety of residues induce strand scission upon oxidation, and hydrogen abstraction occurring at the DNA backbone is responsible for the damage. The observation of peptide-promoted DNA damage suggests that crossreactions within protein/DNA complexes should be considered as a significant cause of the toxicity of reactive oxygen species.     

Energy deposition mechanisms and biochemical aspects of DNA strand breaks by ionizing radiation

A theoretical model based on physical, chemical, and biochemical mechanisms has been presented to evaluate the yields of DNA strand breaks (single and double) as a function of linear energy transfer (LET ) or ?dE/dx. Energetic heavy charged particles are considered explicitly to provide a general theory for low- as well as for high-LET radiation. There are three main features of the calculation: (a) track structure considerations for the energy deposition pattern, (b) three-dimensional structure of DNA molecules to provide information on the exact location of damage, and (c) a Monte-Carlo scheme to simulate the diffusion processes of water radicals. To avoid the complexities of a cellular medium, an aqueous solution of DNA is considered in the calculation. When the results of the calculations are compared with experimental measurements of the yields of strand breaks in mammalian DNA (exposed in a cellular complex), reasonable agreement is obtained. However, only those experimental data have been compared where there were no enzyme repair processes. The method of calculation has also been extended to study breaks in higher-order structures of DNA molecules such as chromatin. Specific limitations of the present model have been pointed out for making further improvements.     

Reflection of electrons and photons from solids bombarded by 0.1 - to 100-MeV electrons

Electron and photon reflection ratios (in number and energy) for absorbers bombarded by electrons have been computed with the ITS Monte Carlo system version 3. Electrons of energies from 0.1 to 100 MeV have been assumed normally incident on an effectively semi-infinite absorber. The absorbers considered are elemental solids of atomic numbers from 4 to 92. The data on the electron reflection ratios agree rather well with the experimental data collected from literature except some discrepancies when the number-reflection ratio is small. For photons, the number-reflection ratio increases with increasing energy, but the energy-reflection ratio shows a maximum around 10 MeV. Empirical equations for the electron reflection ratios and the photon energy-reflection ratio are given (for electrons, graphs only).     

10-100 eV Ar+ ion induced damage to D-ribose and 2-deoxy-D-ribose molecules in condensed phase

We report that 10-100 eV Ar+ ion irradiation induces severe damage to the biologically relevant sugar molecules D-ribose and 2-deoxy-D-ribose in the condensed phase on a polycrystalline Pt substrate. Ar+ ions with kinetic energies down to 15 eV induce effective decomposition of both sugar molecules, leading to the desorption of abundant cation and anion fragments, including CH3+, C2H3+, C3H3+, H3O+, CHO+, CH3O+, C2H3O+, H-, O-, and OH-, etc. Use of isotopically labelled molecules (5- 13C D-ribose and 1-D D-ribose) reveals the site specificity for some of the fragment origins, and thus the nature of the chemical bond breaking. It is found that all of the chemical bonds in both molecules are vulnerable to ion impact at energies down to 15 eV, particularly both the endo- and exocyclic C-O bonds. In addition to molecular fragmentation, several chemical reactions are also observed. A small amount of O-/O fragments abstract hydrogen to form OH-. It is found that the formation of the H3O+ ion is related to the hydroxyl groups of the sugar molecules, and is associated with additional hydrogen loss from the parent or adjacent molecules via hydrogen abstraction or proton transfer. The formation of several other cation fragments also requires hydrogen abstraction from its parent or an adjacent molecule. These fragmentations and reactions are likely to occur in a real biomedium during ionizing radiation treatment of tumors and thus bear significant radiobiological relevance.     

Rupture force between the third strand and the double strand within a triplex DNA

The rupture force to separate the third strand and the duplex within a triplex DNA was measured by means of atomic force spectroscopy. The tip and the sample surfaces were functionalized by oligodeoxyribonucleotides 5'-TTCTTCTTTCTTTTCCTTTTCTTTCTTCTTACTTCTCTCTCTC TCTCTCT-SH-3'. The sample surface was hybridized with 5'-AAGAAGAAAGAAAAGGAAAAGAAAGAAGAA-3' to form a double strand DNA on the surface prior to the force measurements. These sequences form triple helices with 30 base pairs under a pH of 5.8 and in the presence of 2.0 mM spermine. Signals of rupture of single and multiple triplex DNA were observed in the force distance curves. Rupture force histograms revealed a force of 42.6 +/- 1.9 pN from 24 independent measurements at a tip velocity of 400 nm/s to separate the third strand from duplex DNA. The velocity dependence of the rupture force quantum indicates a thermal dissociation process similar to that of rupturing a ds-DNA. The number of rupture events was controlled by adding oligonucleotides 5'-AAGAAGAAAGAAAAGGAAAAGAAAGAAGAA-3' either to reduce or to initiate triplex formation.     

Electron attachment-induced DNA single strand breaks: C3'-O3' sigma-bond breaking of pyrimidine nucleotides predominates

A detailed understanding of DNA strand breaks induced by low energy electrons (LEE) is of crucial importance for the advancement of many areas of molecular biology and medicine. To elucidate the mechanism of DNA strand breaks by LEEs, theoretical investigations of the electron attachment-induced C3'-O3' sigma-bond breaking of the pyrimidine nucleotides have been performed. Calculations of 2'-deoxycytidine-3'-monophosphate and 2'-deoxythymidine-3'-monophosphate in their protonated form (denoted as 3'-dCMPH and 3'-dTMPH) have been carried out with the reliably calibrated B3LYP/DZP++ theoretical approach. Our results demonstrate that the transfer of the negative charge from the pi*-orbital of the radical anion of pyrimidines to the DNA backbone does not pass through the N1-glycosidic bond. Instead, the migration of the excessive negative charge through the atomic orbital overlap between the C6 of pyrimidine and the C3' of ribose most likely represents a pathway that subsequently leads to the strand breaks. The proposed mechanism of the LEE-induced single strand breaks in DNA assumes that the formation of the base-centered radical anions is the first step in this process. Subsequently, these electronically stable radical anions may undergo either C-O bond breaking or N-glycosidic bond rupture. The present investigation of 3'-dCMPH and 3'-dTMPH yields an energy barrier of 6.2-7.1 kcal/mol for the C3'-O3' sigma-bond cleavage. This is much lower than the energy barriers required for the C5'-O5' sigma-bond and the N1-glycosidic bond break. Therefore, we conclude that the C3'-O3' sigma-bond rupture dominates the LEE-induced single strand breaks of DNA.     

Water induced weakly bound electrons in DNA

We have studied the effect of humidity on the electronic properties of DNA base pairs. We found that the hydrogen links of the nucleobases with water molecules lead to a shift of the pi electron density from carbon atoms to nitrogen atoms and can change the symmetry of the wave function for some nucleobases. As a result, the orbital energies are shifted which leads to a decrease in the potential barrier for the hole transfer between the G-C and A-T pairs from 0.7 eV for the dehydrated case to 0.123 eV for the hydrated. More importantly, the pi electron density redistribution activated by hydration is enhanced by the intrastrand interactions. This leads to a modification of the nucleobase chemical structures from the covalent type to a resonance structure with separated charges, where some pi electrons are not locked up into the covalent bonds. Within the (G-C)(2) sequences, there is overlapping of the electronic clouds of such unlocked electrons belonging to the stacked guanines, that significantly increases the electron coupling between them to V(DA)=0.095 eV against the V(DA)=0.025 eV for the dehydrated case. Consequently, the charge transfer between two guanines within the (G-C)(2) sequences is increased by 250 times due to hydration. The presence of nonbonded electrons suppress the band gap up to approximately 3.0 eV, that allows us to consider DNA as a narrow band gap semiconductor.     

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.     

Detection of telomere damage as a result of strand breaks in telomeric and subtelomeric DNA

Telomeres are the tandem repetitive DNA sequences at both ends of a chromosome with a repeating unit of TTAGGG. The integrity of a telomere is crucial to chromosomal stability and cellular viability. Damages to telomere DNA disrupt telomere integrity and accelerate telomere shortening. We describe a method for the assessment of strand breaks in the telomere/subtelomere region in cultured cells. Cells were embedded in agarose plugs and subjected to lysis and alkaline treatment to relax the DNA double helix. The telomere fragments as the result of strand breaks in the telomere/subtelomere region were then separated from the genomic DNA by electrophoresis, blotted onto membranes, and detected by a probe specific to the telomere sequence. Because of the large content of the telomere in human cells and the fact that telomere DNA is much more prone to damage than the bulk genomic DNA, the analysis may serve as a good indication of general DNA damage as well.