1. Interaction of 0-15 eV electrons with DNA: Resonances, diffraction and charge transfer The presented results represent the work of many scientists especially: Marc Michaud Sylwia Ptasinska Badia Boudaiffa Michael Huels Pierre Cloutier Darel Hunting Hassan Adoul-Carime Xiaoning Pan Luc Parenteau Andrew Bass Frederick Martin Yi Zheng Richard Wagner Xifeng Li Michael Sevilla Laurent Caron This work was funded by:

2. Secondary electrons (SE) are the most abundant species produced by ionizing radiation. Most of the energy distribution of SE is composed of LEE The most probable energy lies below 10 eV. Cross sections for LEE damage to biomolecules are large owing to the formation of transient anions. There are many processes driven by LEE, but why are they important in relation to radiobiological damage?

3. DNA and sub-units Guanine Cytosine Thymine Adenine tetrahydrofuran (THF) 3-hydroxy- tetrahydrofuran  -tetrahydrofuryl alcohol + H2O+ H2O

4. What sort of damage is induced in DNA by LEE? From LEE impact experiments on thin films of DNA and its basic constituents, we know that they produce: Base, phosphate and sugar modifications Base, phosphate and sugar modifications Single strand breaks Single strand breaks Base release Base release Combination Combination Double strand breaks Multiple strand breaks Crosslinks What are the mechanisms of LEE damage?

5. Apparatus for product analysis "Electron stimulated desorption of H¯ from thin films of thymine and uracil" M.-A. Hervé du Penhoat et al., J. Chem. Phys. 114, 5755 (2001).

6. LEE Damage to Plasmid DNA M.A. Huels et al., J.A.C.S. 125, 4467 ( 2003)

7. H - desorption from films of linear and plasmid DNA X. Pan et al., Phys. Rev. Lett. 90, 208102 (2003). ESD of H - Yield function similar to DSB damage Anion yields from linear and supercoiled DNA are very similar H - yield consistant with those of sub-units, especially Thymine and THF ESD signal at low electron dose consistant with a one-step reaction

8. Conclusions from comparisons of LEE stimulated desorption of anion from random and oriented DNA films and subunits of DNA H¯ arises from DEA to the bases with a minor contribution from the sugar O¯ arises from DEA to the phosphate group OH¯ arises from DEA to the protonated phosphate group Question Question: Do strand breaks and other damages occur via transient anion formation on the subunits?

9. Model Target System Reference standards for fragment species obtained using: micrococcal nuclease – (3’ terminal phosphate - GCp, GCAp) Phosphodiesteras (P1) – (5’- phosphate: pT, pAT, pCAT) alkaline phosphatase to remove terminal phosphates GCATGCAT

10. Alkoxyl anion + phosphoryl radical Carbon-centered sugar radical + phosphate anion Proposed pathways of phosphodiester bond cleavage of DNA by low-energy electrons LEE irradiation of tetramers gave non-modified fragments containing a terminal phosphate group (A) while those without a phosphate group were minor (B).

11. LEE damage to DNA – Intro/Summary DNA damage induced by LEE below 15 eV occurs principally by the formation of transient anions of the subunits. The contribution from direct scattering increases with energy. Anion ESD yields of: H¯ arises from the bases with a small contribution from the backbone, O¯ from the phosphate group, and OH¯ from a protonated phosphate group. Other anions have been observed. Anion ESD yields arise from DEA below 15 eV. Two major pathways of LEE reactions in DNA: cleavage of the N- glycosidic bond (base release) and the phosphodiester bond (strand break). Phosphodiester bond breaks by C-O bond rather than P-O bond rupture. Between 0-5 eV, SSB are produced with a cross section of about E-14 cm 2 for 3,000 bp, similar values are found at 10 and 100 eV.

12. Sub-excitation energy electron damage to DNA Barrios et al J. Phys. Chem. 106, 7991 (2002) - Electron capture by cytosine and transfer to dissociative C-O bond Li et al JACS 125, 13668 (2003) - Scission of 5’ and 3’ C-O bond by electron attachment. Endothermic by ~0.5 eV Dablowska et al Eur. Phys. J. D 35, 429 (2005) – Proton transfer mechanism of DNA strand breaks induced by excess electrons.

13. Gu et al., Nucl. Acids Res. 1-8 (2007) (in press)

14. [SU¯] (E o ) [SU][SU]* DEA ++ e¯ (E o ) e¯ (E<<E o ) e¯ c e¯ t e¯ c e¯ t 1 2 3 SU=subunit base sugar phosphate water +diffraction

15. Upper curve (Martin et al, Phys. Rev. Lett. 93, 068101 (2004)): From ETS data, sum of capture cross sections for the four bases normalized to the second peak of the DNA damage yield (full squares) and shifted by 0.4 eV. Lower curve (Denifl et al, Chem. Phys. Lett. 377, 74 (2003)): DEA cross section from gaseous Thymine with no energy shift. Capture cross section of the bases vs single SB

16. Electron transfer in DNA 1 2 3 4 5 6 X LEE induced cleavage reactions greatly impeded next to the abasic site below 6 eV. There is a shift of electron transfer to direct attachment from low to high electron energy. Electron transfer of LEE occurs from base moiety to the sugar-phosphate backbone in DNA.

17. Percentage distribution of damage by sites of cleavage, induced by 6, 10 and 15 eV electrons. *Xp was not detected by HPLC and the yield was considered to lie below the detection limit. Total damage = SB + base release = 100

18. Yield functions: GCAT vs GCXT For strand break, a resonance shows at around 10 eV. Presence of an abasic site greatly decreases the yield of strand break and base release in DNA (three times less).

19. On average 25% decrease for abasic Same results for H- and O- desorption No diffraction Since OH- and O- originate from the backbone, these anions arise from e- transfer unless there is a change in the resonance parameters

20. [base¯] (E o ) [base] [base]* DEA ++ e¯ (E o ) e¯ (E<<E o ) e¯ c e¯ t e¯ c e¯ t 1 2 3 +diffraction At higher energies, there is little coherence. Thus, creation of an abasic site has little effect on the branching ratios for electron emission in the continuum or within DNA At low energies, transfer within DNA becomes much larger, but strongly depends on diffraction and hence is considerably decreased by formation of an abasic site

21. Neutral particle desorption from a single DNA strand zCN (black squares) zOCN and/or H 2 NCN (white circles) zH and H 2 desorption also observed zRatio CN/OCN is constant zResonance structures superimposed in linearly increasing background H. Abdoul Carime et al., Surf. Sci. 451, 102 (2000). Isocyanic acid

23. Opinion of the presenter Shape resonances have high cross-section and can lead to DEA (the only bond breaking process). Shape resonances have high cross-section and can lead to DEA (the only bond breaking process). Electron transfer is high. Electron transfer is high. Below 3-4 eV Above the energy threshold for electronic excitation Core excited shape resonances have a high cross section for decay into their parent neutral state and direct inelastic scattering may be significant. The magnitude of the DEA is not necessarily large compared to autoionization. There is little coherent enhancement of the electron wavefunction at the primary impact energy. Above the energy threshold for electronic excitation Core excited shape resonances have a high cross section for decay into their parent neutral state and direct inelastic scattering may be significant. The magnitude of the DEA is not necessarily large compared to autoionization. There is little coherent enhancement of the electron wavefunction at the primary impact energy. Proton transfer has to be re-examined in the context of the present data and hypothesis

24. Are transient negative ions formed within the 0-15 eV linked directly to stable anions of the bases or other SU?Are transient negative ions formed within the 0-15 eV linked directly to stable anions of the bases or other SU? If so how?If so how? Possible mechanisms Possible mechanisms : 1.Vibrational stabilization triggered by the change in DNA configuration by the extra charge. The extra energy (<2eV) of the electron is dispersed in vibrational excitation of DNA and then transfered to the surrounding medium. Does not work for core-excited resonances. 2.Electron-emission decay of a core-excited shape resonance followed by vibrational stabilization. 3.Proton transfer stabilization. Neutralizes the anion charge while leaving a site with a ground state electron. 4.Superinelastic vibrational or electronic electron transfer. [Lu, Bass and Sanche, Phys. Rev. Lett. 88, 17601 (2002)].

25. E Superinelastic electron transfer EoEo  E ph H2OH2O EE 0 eV ERER EE 1.0 eV

28. | O ‌‌‌‌‌| O = P ─ O¯ H + (O 18 H) | O | Site of formation