Unified Mechanism for the Generation of Isolated and Clustered DNA

Jan 6, 2017 - Clustered DNA damages are the most detrimental modifications induced by ionizing radiation in cells and several mechanisms have been ...
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Unified Mechanism for the Generation of Isolated and Clustered DNA Damages by a Single Low Energy (5−10 eV) Electron Yu Shao,† Yanfang Dong,† Darel Hunting,‡ Yi Zheng,*,† and Léon Sanche‡ †

Research Institute of Photocatalysis, State Key Laboratory of Photocatalysis on Energy and Environment, Fuzhou University, Fuzhou 350002, P.R. China ‡ Group in the Radiation Sciences, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, QC Canada J1H 5N4 S Supporting Information *

ABSTRACT: Clustered DNA damages are the most detrimental modifications induced by ionizing radiation in cells and several mechanisms have been proposed for their formation. We report measurements of such damages induced by a single low energy electron via the formation of the two major core-excited resonances of DNA located at 4.6 and 9.6 eV. Cross-links and single and double strand breaks (SSBs and DSBs) are analyzed by gel electrophoresis. Treatment of irradiated samples with Esherichia coli base excision repair endonucleases reveals base damages (BDs). DSBs resulting from such treatments arise from clustered damages consisting of at least two BDs or one BD accompanied by a SSB. The total DNA damages induced by 4.6 and 9.6 eV electrons are 132 ± 32 and 201 ± 36 × 10−15 electron−1 molecule−1, comprising 43% and 52% BDs, respectively. We propose a unifying mechanism to account for these clustered damages, DSBs, and single BDs, as well as all previously measured isolated lesions.

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INTRODUCTION

Various in situ techniques of microanalysis exist to detect damage induced by LEEs to short single DNA strands under vacuum conditions.14−18 However, the complexity and large dimensions of biological DNA make it difficult to directly measure damage in vacuo.13,18 Usually, modifications are quantified ex-vacuo by electrophoresis, after LEE bombardment of supercoiled plasmid DNA.18 The technique can only reveal immediate conformation variations of the initial form, including interduplex cross-links and single strand breaks (SSBs) and DSBs. For this reason, the clustered damages identified are restricted to DSBs and intermolecular cross-links, whereas the majority of the most detrimental clustered damages are not revealed. In fact, about 200 manuscripts (PubMed) report SSBs and/or DSBs of biological DNA induced by LEEs, but only the recent work of Sahbani et al.11 addressed non-DSB clustered damages. The lesions were induced at a fixed energy (10 eV), in layered 1,3-diaminopropane−DNA films prepared under atmospheric conditions. In these experiments, the diaminopropane molecules, which are structurally similar to amino acids, act as intercalates to produce a uniform well-layered film. The difficulties in measuring LMDS are essentially due to their complexity. LMDS can consist of a DSB, multiple base damages (BDs) in the same or opposite strand, a SSB with BD in the same or opposite strand and a multitude of combinations of

Irradiation of DNA with high energy particles or photons generates both isolated and clustered damages.1−3 Clustered damages are defined as multiple (≥2) closely spaced lesions, including strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites, which occur within 1−2 helical turns of the DNA helix (i.e., within a 10−20 base-pair (bp) region).4−6 Accumulated evidence in the literature indicates these local multiply damaged sites (LMDS) are more challenging for the cell to repair than individual, widely dispersed lesions. Thus, such damages are regarded as potentially lethal or mutagenic and related to diverse human diseases.7,8 Since secondary low energy electrons (LEEs) are major initial products of high energy radiation,9,10 the DNA damages they induce could be responsible for a large portion of cell dysfunction and death in radiotherapy. Recently, Sahbani et al.11 related conformational and base DNA damage to the decrease in ampicillin resistance of Escherichia coli transformed by plasmid DNA irradiated with 10 eV electrons. Later, they reported12 such reduced resistance for irradiation with 0.5 to 18 eV electrons. Two peaks at 5.5 and 9.5 eV clearly appeared in the loss of transformation efficiency.12 These peaks corresponded to maxima in the yields of double strand breaks (DSBs) induced by LEEs,13 which were due to the formation of transient negative ions (TNIs). The results of Sahbani et al. established a correlation between LEE-induced DNA damages, TNIs, and cell biological function. © XXXX American Chemical Society

Received: December 1, 2016 Revised: January 3, 2017 Published: January 6, 2017 A

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EDTA); it consisted of 95% supercoiled, 3% cross-links, 1% SSB and 1% concatemeric forms. The concentration of DNA was determined by ultraviolet (UV) absorption at 260 nm with a BioTek Epoch spectrophotometer, according to 1 OD = 50 ng/μL. The quantity of impurities including proteins and salts in the plasmid solution were estimated by measuring the ratio of UV absorption from 260 to 280 nm. The ratio was 1.9, which corresponds to purity greater than 95%.30 The TE buffer was separated from DNA using the G-50 medium, in order to obtain DNA with only its structural salt. Sephadex G-50 is a suitable medium for separation of molecules having a molecular weight larger than 3 × 104 g mol−1 from molecules with a molecular weight smaller than 1500 g mol−1.31 LEE Irradiation. The DNA samples were deposited on substrates consisting of 450 ± 50 nm films of tantalum vacuum evaporated onto a 0.4 mm thick silicon wafer. Prior to sample deposition, the tantalum surface was cleaned in pure ethanol and ddH2O and dried with a flow of dry nitrogen. Ten μL of the DNA solution (i.e., 320 ng) were deposited onto the cleaned tantalum surface. After freezing at −65 °C for 7 min in a glovebox containing an atmosphere of 99% pure dry nitrogen, the samples were lyophilized (freeze-dried) under a pressure of 7 mTorr by a hydrocarbon-free carbon-vane pump for 2 h. Assuming that the molecules were uniformly distributed on the surface with a radius of 2 ± 0.5 mm, with a density of DNA of 1.7 g cm−3,32 the average thickness of the films was approximated to be 15 nm (five monolayers (ML)). Such a thickness has been widely used in DNA-LEE experiments.18 On the basis of the average film thickness of 15 nm, which is of the order of the penetration depth (5−20 nm) of 4−15 eV electrons in liquid water or amorphous ice,33 most electrons impinging on the film are expected to be drained to the metal substrate. The freeze-dried samples were transferred directly from the glovebox under pure nitrogen to the LEE irradiation chamber also filled with pure nitrogen. In this manner, the f ilms were never exposed to environmental humidity and contaminants. Afterward, the irradiation chamber was subsequently evacuated for ∼24 h, with a turbomolecular pump, to reach a base pressure of 2 × 10−8 Torr at ambient temperature. The potential between the substrate (ground) and the center of the filament of the LEE gun was set at 5 or 10 V. The energy scale was calibrated by taking, as the zero electron energy reference, the onset of electron transmission through the films. At this onset, the potential between the point of emission of the filament and the substrate was 0.4 V, indicating the potential change due to the different work functions between the connections and the DNA-vacuum interface. Absolute electron energies of 4.6 ± 0.3 and 9.6 ± 0.3 eV were obtained by subtracting 0.4 V from each voltage measured between the filament and the metal substrate. At each energy, the samples were bombarded with an incident electron beam of only 6 nA during 5 to 30 s, to keep the fluence as low as possible. For each set of parameters, eight samples were bombarded including the control samples, which were not irradiated to serve as the zero fluence data point in exposureresponse curves. The DNA films were recovered from the tantalum substrates under atmospheric conditions with 20 μL of ddH2O; the efficiency was 91%. The DNA solution was divided into three portions: one for no enzyme treatment, the other two for enzyme treatment. Enzyme Treatment. The DNA was treated with E. coli base excision repair endocuclease III (Nth) and formamidopyrimidine N-glycosylase (Fpg) (Trevigen Inc.).34,35 The DNA

these lesions. Such damages, DSBs, and more complex LMDS may be difficult to repair and can thus lead to replication fork collapse, which is detrimental to cell survival.8 Whereas in the previous BD study with 10 eV electrons,11,12 plasmid supercoiled DNA was irradiated at relatively high fluences needed to measure cellular response, the present results were recorded at considerably lower fluences with electrons of 4.6 and 9.6 eV. The low fluence insures that the induced damage results from a single electron interaction. Electrons of 4.6 eV form a single or a group of TNIs leading to SSBs, whereas the resonances at 9.6 eV cause both SSBs and DSBs.13 DNA damages including cross-links, SSBs, DSBs, and the supercoiled configuration are analyzed by agarose gel electrophoresis. We measure base modifications that lead to a strand break upon treatment of irradiated samples with the repair enzymes, endocuclease III and formamidopyrimidine Nglycosylase (Nth and Fpg).19 These enzymes transform an oxidized pyrimidine or purine into a strand break, respectively. Any increase in DSBs after such treatment can therefore be assigned to cluster damage associated with at least two localized base modifications or a BD and SSB on opposite strands. From extrapolation of exposure-response curves to zero fluence, the yields of all damages arising from a single-electron interaction are reported. Below the energy of the first electronically excited state of the bases, the LEE mechanisms leading to a BD, an abasic site or a SSB in DNA, via the initial formation of a shape resonance, are fairly well established.20−29 For small DNA units consisting of two or less bases and sugar−phosphate moieties, two models have been proposed. According to the model of Simon20 and Gu et al.,21 the captured electron, occupying a previously unfilled orbital of a base, transfers to the phosphate group, where the C−O bond is ruptured via dissociative electron attachment (DEA). In the model of Sevilla and co-workers, the additional electron localizes directly on the P−O bond, causing its rupture via DEA. The experimental results of Li et al.26 indicate that for such small pieces of DNA the later model is more adequate; but as the chain of a short strand lengthens electron capture by a base followed by electron transfer to the phosphate group becomes dominant. This result was corroborated by Martin et al.22 who measured SSBs induced in plasmid DNA by 0.5 to 4.5 eV electrons. Above this energy, core excited resonances around 5 and 10 eV have been identified as the TNIs responsible for causing SSBs and DSBs.13 However, due to limitations in treating theoretically such twoelectron one-hole states and their decay within DNA, no clear quantum mechanical description has emerged yet to explain the details of the mechanism of damage. As in the case of shape resonances at lower energy,18,22,26 single base damage or abasic sites most likely occur via DEA (i.e., from dissociation of the core-excited anion), whereas SSBs result from electron transfer from a base to the phosphate group, where the C−O bond is broken via DEA.13,18 Considering the present results, we now face the challenge of developing a unifying mechanism to explain how a single electron hitting DNA can lead to all types of isolated and clustered damages observed so far. Such a mechanism is presented in this paper.

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EXPERIMENTAL SECTION Preparation of Plasmid DNA. Plasmid DNA (pGEM3Zf(−), 3197 bp) was extracted from E. coli DH5 and purified with a HiSpeed plasmid Maxi kit (QIAGEN). The purified plasmid DNA was eluted in buffer TE (10 mM Tris and 1 mM B

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The Journal of Physical Chemistry C repair enzymes, Nth and Fpg, can specifically recognize, remove several modified pyrimidines and purines, respectively, by hydrolysis of the glycosidic bond, and form essentially strand breaks.36,37 For instance, Fpg can excise 8-oxo-7,8-dihydroguanine and subsequently form apurinic/apyrimidinic (AP) site by β-elimination, resulting in a 1-nucleotide gap displayed as a strand break in the electrophoresis analysis.38 According to enzyme response curves, DNA was mixed with one unit of Fpg or 0.5 unit of Nth, and incubated at 37 °C for 60 min. One unit is defined as the cleavage at an AP site of a 34 base-pair oligonucleotide at a rate of 1 pmol/h. The reactions were stopped by addition of 0.5 M EDTA buffer and kept on ice prior to the further analysis of DNA damages. Untreated DNA was also kept at 37 °C for 60 min in order to reveal any heat labile effects. Quantification of DNA Conformation Variations. The DNA configurations supercoiled, concatemeric, nicked circular (SSB) and linear (DSB) were analyzed by running the samples on 1% agarose gel in 1X TAE buffer at 100 V for 7 min, followed by 75 V for 90 min. The samples and the agarose gels were stained with SYBR Green I at concentrations of 100× and 10000×, respectively. The gels were then scanned by a STORM 860 laser scanner (Molecular Dynamics) adjusted for the blue fluorescent mode at an excitation wavelength of 450 nm and PMT voltage of 800 in the normal sensitivity mode. The binding efficiencies of SYBR Green I for the same amount (80 ng) of supercoiled and linear DNA was measured and the correction factor was determined to be 1.5. This factor arises from the weaker binding of SYBR Green I to supercoiled DNA than to the nicked circular and linear forms. The different DNA bands on the gel were analyzed by the ImageQuant 5.0 (Molecular Dynamics) software. Examples of the scanned images are shown in the supplement, where we also explain how the bands were identified. Effective yields for DNA Damages and the Formation of Specific Lesions. The effective yields of various DNA damages were obtained by extrapolation of the exposureresponse curves to zero fluence. Since the cross reactivity of Nth and Fpg for enzyme-sensitive sites is low, the enzymesensitive sites (ess) recognized by both enzymes were assumed to be independent.39 The sum of base lesions leading to respective DNA damages, were obtained by addition of both yields of enzyme-sensitive sites as following:

Figure 1. Exposure-response curves for cross-links, SSBs, DSBs and loss of supercoiled in 5 ML DNA films (■) induced by 4.6 eV electrons together with the parallel treatment with Fpg (●) and Nth (▲) enzymes. The dashed lines are exponential fits and the solid lines the initial slopes. The DSBs were fitted with a linear function. Each data point is the result of eight identical bombardment procedures, and the error bars are the standard deviation.

Figure 2. Exposure-response curves for cross-links, SSBs, DSBs and loss of supercoiled in 5 ML DNA films (■) induced by 9.6 eV electrons together with the parallel treatment with Fpg (●) and Nth (▲) enzymes. The dashed lines are exponential fits and the solid lines the initial slopes. The DSBs were fitted with a linear function. Each data point is the result of eight identical bombardment procedures, and the error bars are the standard deviation.

Y (base lesion)ess = Y (Nth) + Y (Fpg) − 2[Y (DNA) + Y (heat)]

Y(Nth) and Y(Fpg) are defined as the effective yields of various lesions from respective exposure curves with Nth and Fpg treatment, while Y(DNA) indicates the yields of DNA damages in the absence of enzyme treatment. Y(heat) is the yield of heat liable sites that arose from samples incubated at 37 °C for 60 min without enzymes. The total yields of DNA damages are calculated as Y(total) = Y(Nth) + Y(Fpg) − Y(DNA) − 2Y(heat).

independent bombardments recorded at a given fluence. The percentage of the supercoiled configuration, formation of SSBs and cross-links exhibit an exponential behavior as a function of number of incident electrons, as previously observed without enzyme treatment.13 The majority of the loss of the supercoiled configuration (LS) arises from SSB formation, while the formation of cross-links and DSBs are at least 1 order of magnitude lower.13 The enzyme digestion of respective DNA samples clearly leads to increasing LS, SSB and cross-links yields with a larger contribution from Fpg. The latter may be due to a different sensitivity of the two enzymes to their respective base lesions19 and/or to the production of more BDs from TNIs formed with purines.

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RESULTS AND DISCUSSION Exposure-response curves for the percentage of the supercoiled configuration and formation of cross-links, SSBs, DSBs induced by 4.6 and 9.6 eV electron impact on a 5 monolayers (ML) DNA films are shown in Figure 1 and 2, respectively; the results obtained with Fpg and Nth treatments are also included. Each data point in the yields vs fluence curves is the average of eight C

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Table 1. Effective Yields (in 10−15 electron−1 molecule−1) of the Supercoiled Configuration and Formation of Cross-Links, SSBs, and DSBs Induced by 4.6 and 9.6 eV Electrons in DNA Films with Thickness of 5 Monolayers (ML)a energy (eV)

condition

cross-links

4.6

true heat labile + fpg + nth true heat labile + fpg + nth

10 ± 5 65 ± 13 n.d.b 7±2 13 ± 7 109 ± 28 13 ± 8 81 ± 19 12 ± 8 81 ± 13 n.d. 11 ± 3 20 ± 6 158 ± 38 15 ± 6 123 ± 28 Base Lesions Revealed by Enzymes

9.6

SSBs

DSBs n.d. n.d. 5.5 4.1 7.6 n.d. 15.6 12.1

± 2.0 ± 1.2 ± 2.3 ± 4.9 ± 2.3

supercoiled −75 −9 −127 −98 −96 −10 −178 −140

± ± ± ± ± ± ± ±

14 2 32 10 16 3 27 19

energy(eV)

cross-links

isolated base modification (SSBs)

clustered damage (DSBs)

total base modifications

% of BD to total damage

4.6 9.6

7±6 12 ± 7

44 ± 12 97 ± 23

9.6 ± 0.8 12.4 ± 3.0

−57 ± 18 −106 ± 20

43 ± 19 52 ± 17

a

The corresponding base damages (BDs) revealed only by the enzymes are listed in the lower portion. The errors are calculated from a linear regression fit analysis of the slope near zero fluence. bn.d.: nondetected.

of cluster damage, because our repair enzymes are less efficient at incising at modified bases, when other damage is present. For example, the binding of Fpg to 8-oxoguanine is inhibited, if it is opposite (or almost opposite) to a strand break or AP site on the complementary strand.43 According to experimental results, between 4 and 15 eV, damage to biological DNA or single DNA strands results essentially from the initial capture of an electron by the positive electron affinity of an electronically excited state of a base.10,13,18,25,26 The core-excited TNIs thus formed can decays by DEA or autoionization. The latter process can leave the base in an electronically excited state.18 In order to explain the formation of LMDS involving damage on both strands f rom a single electron, the electron and the electronic excitation must separate, with one causing damage on a strand and the other on the complementary strand. A priori, the excitation or electron can transfer to the opposite strand, but electron transfer is much more probable, since a one-electron jump is much more likely than a simultaneous two-electron jump.44 The later transition is required in a transfer of electronic excitation. We explain in Scheme 1, how all damages observed so far can be formed from an initial core-excited TNI on a base. The possible electron pathways (1−4) are shown with the initial electronically excited base in bracket and the additional electron in subscript, to represent a core-excited resonance. The latter can dissociate producing a single lesion (i.e., a BD or an abasic site) or autoionize. Autoionization can occur via electron transfer to the phosphate group causing rupture the C−O bond via DEA. LMDS involving two adjacent BDs are created by dissociation of the electronic excited state on one strand and DEA of the electron transferred to the opposite base (path no. 1). Alternatively, further transfer of the additional electron to the phosphate group of the complementary strand produces a BD in the initial site and a strand break by rupture of the C−O bond via DEA in the opposite strand (path no. 2). If the transferred electron hops from base to base (path no. 3), a LMDS can still be created, as long as within 20 base-pairs from its initial capture, it forms a dissociative base anion or transfers to a phosphate group. Pathway 4 (i.e., electron transfer to the phosphate unit, while leaving the base in a dissociative state in the same strand) results in a LMDS located within a singlestrand, as observed experimentally by Li et al.45 As suggested by Luo et al.,13 reaction of a base radical with a nearby DNA can produce an interduplex cross-links.

The increase in SSBs with enzyme treatment can be interpreted as arising from isolated BDs in DNA. The rise of DSBs after reaction with the enzymes indicates the formation of LMDS. The increase of damages for the control samples (data points at zero dose) after enzyme treatment shows that DNA is sensitized by the manipulations, most likely by contact with the tantalum substrate. At 4.6 eV, DSBs could barely be detected due to small yields and our detection limit. In contrast, after Fpg and Nth treatments the curves clearly display linearly increasing DSB yields. Such DSBs are induced by the combination of one or more BDs recognized by Fpg and Nth. The minimum damage leading to such DSBs is therefore two BDs or one SSB and a BD occurring on opposite strands within 10−20 bps. The same damages are revealed upon enzyme treatment of samples irradiated with 9.6 eV electrons. As previously reported, at this energy, DSBs are detected without enzyme treatment.13 The increase in cross-links after enzyme treatment is likely to be caused by removal of a guanine lesion, followed by reaction of the enzyme-induced lesion with thymine or cytosine of a nearby DNA.40 As shown by Rezaee et al.,41,42 extrapolation to zero fluence coupled with a low incident current density is expected to produce initial slopes in fluence-response curves, representing most accurately values of the yields caused by the interaction of a single electron, irrespective of film charging. Furthermore, this procedure automatically eliminates any background contribution arising from manipulation of the sample. By dividing the initial slope by the original percentage of supercoiled DNA, the effective yields of supercoiled DNA, SSBs, cross-links and DSBs are calculated and listed in the upper section of Table 1; the various DNA conformations related to BDs revealed by enzyme treatment are summarized in the lower section. The errors in the corresponding yields are calculated from a linear regression fit analysis of the slope near zero fluence. Within experimental error, the contributions of BDs to LS correlates fairly well with the sum of cross-links, SSBs and DSBs, arising from enzyme treatment. Surprisingly, the percentages of the yields of base lesions to total DNA damage are as high as 43 and 52%. The DSBs revealed by enzymes are 1.6−2.3 times larger than those from untreated DNA. Since no DSB is detected with 4.6 eV irradiation, all DSBs detected after enzyme treatment arise from the 4.6 eV resonance present in the yields of SSBs, rather than the resonance in the yield function of DSBs located at 5.6 eV.13 The present measurements probably underestimate the levels D

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The Journal of Physical Chemistry C Scheme 1. Diagram of Possible Electron (e−) Pathways Yielding a LMDS, Following the Formation of a CoreExcited Transient Anion on a Basea

bond rupture.20−23,25,26 Over long times, protonation or attack by H2O of the abasic site can lead to dissociation of C−O bonds.50 However, within resonance lifetimes (10−12−10−14 s), through-bond electron transfer could occur via π*−σ* configuration interaction.20 This processes would result in an anion with an additional electron in a dissociative σ* orbital of the sugar−phosphate chain, causing rupture a C−O bond.20,59 This mechanism is consistent with numerous analyses of various damages produced by the impact of electrons with energies below 12 eV on plasmid DNA13,18,22 and small oligonucleotides.10,15,18,25,26

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SUMMARY We have reported the first measurements of single and multiple BDs in lyophilized plasmid DNA films induced by 4.6 and 9.6 eV electrons. At these energies, molecular dissociation occurs mostly via the formation of core-excited TNIs formed by electron capture in an unfilled orbital of a base. The multiple BDs were related to clustered lesions occurring within one or two turns of the DNA helix. These lesions are highly detrimental to cells, since they are difficult to repair and can lead to replication fork collapse.8 The additional BDs revealed by enzymatic treatment indicate that in previous measurements13 a large portion (43% and 52%) of the LEE-induced modifications to biological DNA were not observed. From these new results and previous ones on conformational plasmid damages, we presented a unifying mechanism, which could explain from the capture of a single electron by a DNA base, the production of any of the isolated or clustered lesions measured so far. Considering the recent advances in theoretical treatment of excess electrons in electronic excited states by time-dependent density functional theory24,59 and other approaches,54 we hope that the electron transfer mechanism proposed in this paper may help in future description and understanding of LEE-induced damage to DNA.

a Key: (1) e− transfer to the base on the opposite strand, forming two adjacent BDs; (2) further e− transfer to the phosphate unit, causing a strand break via C−O bond breakage (SSB with opposite BD); (3) subsequent e− hopping between bases, giving BD or SSB at a farther location; (4) e− transfer to the phosphate unit of the strand hosting the transient anion, leading to C−O bond scission (i.e., SSB with nearby BD on the same strand).

Pyrimidine and purine base radical sites can be transferred to the sugar moiety by H atom abstraction.46,47 In absence of oxygen, the sugar radical at the C4′ position can lead directly to a strand break via phosphodiester bond cleavage.48 Such DNA radicals induced by ionizing radiation have been abundantly detected by electron spin resonance.49 Furthermore, any abasic site produced by rupture the N-glycosidic bond from a dissociating electronic state of a base may transform by subsequent reactions into a strand break.50 We therefore expect pathway 1 followed by 2 in Scheme 1 to also lead to a DSB. The mechanism of Scheme 1 requires the electronic excited state of the base to be dissociative in the Franck−Condon region, as well as DEA on a base and electron transfer to the phosphate group to be highly probable. Electron-energy-loss spectra (EELS) indicate that the 9.6 eV TNIs can decay by autoionization causing electronic excitation of a base via a multitude of π → π* transitions.51−53 The 4.6 eV resonance decays into the lowest electronically excited states of the bases, such as those resulting from the transitions 13A(π2 → π3*) and 13A″(n2 → π3*) of thymine52 and 13A′ (π → π*) of cytosine.51 At both energies, the cross sections are on the order of 10−16− 10−17 cm2.51−53 Computational evidence supports the view that such transitions can lead to dissociative states.54−56 The present observation of a high level of BDs supports these theoretical predictions. According to EELS, an electron autodetaching from a base TNI could have energies varying from zero to 5 eV, depending on the resonance and final electronic state.51−53 Upon transfer to the opposite base (path no. 1), with such low energy, the electron is likely to form a shape resonance and occupy a previously unfilled orbital of the opposite base, where it would have a high propensity to undergo DEA or transfer to the phosphate group.18,20−22,25,26 In fact, DEA to the pyrimidines and adenine below 4 eV reaches huge cross sections around 10−15 cm2.57,58 Some theoretical studies show that electrons with energy below ca. 2 eV can attach exclusively to the π* orbitals of a base.20,21,23 The latter can trigger N-glycosidic

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.6b12110. Description of the identification of DNA conformations from bands in the electrophoresis data and examples of scanned images of electrophoresis for 4.6 and 9.6 eV electron irradiations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(Y.Z.) E-mail: [email protected]. ORCID

Yi Zheng: 0000-0002-8870-201X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided by the Canadian Institutes of Health Research (MOP81356), National Key Technologies R & D Program of China (2014BAC13B03), National Basic Research Program of China (973 Program: 2013CB632405), and the NNSF of China (21673044). We would like to thank Prof. Richard Wagner for helpful comments and suggestions. E

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