Electrons and DNA: From Electron Attachment to DNA Strand Breaks

Michael SevillaChemistry DepartmentOakland University

What are the mechanisms of electrons reaction with DNA?

DNA and Electrons

• A variety of studies show that aqueous electrons add to the DNA bases and do not cause strand breaks.

• The base anion radicals undergo protonation reactions.

• Only nonsolvated electrons with kinetic energy (LEE) cause DNA strand breaks.

• At low temperatures electron in DNA are stable for long times.

-5

-3

-1

1

3

5

7

9

11

0 2 4 6 8 10 12 14

D (bp)

log 1

0(k)

(Å-1)

0.75

0.92

1.4

2.8

Vacuum

pdIdCpdIdC

DNA (salmon sperm)

pdAdTpdAdT

Electron Tunneling Decay Constant in DNA at 77 K

k = 1011exp(-D)

Z Cai and M Sevilla, in "Topics in Current Chemistry 237: Long Range Transfer in DNA II", Gary Shuster, Ed., Springer-Verlag, pages 103-128, 2004 .

DNA irradiation Studies

Irradiate hydrated DNA at 77K in frozen solutions • Direct Ionization of DNA results• Identify initial radicals by Electron Spin Resonance

Spectroscopy

Ionizing radiationDNA•+ + DNA•–DNA

Oxidative path Reductive path

"The Chemical Consequences of Radiation Damage to DNA" D. Becker and M. D. Sevilla, in Advances in Radiation Biology, Vol.17, (J. Lett, Ed.) Academic Press, 121-180 (1993).

RADICAL COMPOSITION (-irradiated DNA) ESR Spectra

• The composition of DNA radicals (77 K) is ca.:

• 25% (C-•) C(N3)H• • 25% T-•• 40% G+•• 10% Sugar•All to ±5%

C(N3)H•

Effect of scavengers on G-values

L. I. Shukla, R. Pazdro, D. Becker and M. D. Sevilla, Radiation Research: Vol. 163, 59-602 (2005).

Sug•

Oxidation pathway: formation of neutral sugar radical(s) from

sugar cation radical(s)

• Deprotonation from C1’, (C2’), C3’, C4’, C5’ may occur.

•

CH2 O BHH

HO

H

O

•CH2 O B

HH

HO

H

O

H+ + BH+

B+ 1'

2'3'

4'

5'

L. I. Shukla, R Pazdro, D Becker and M D Sevilla, RAD. RES.163, 591–602 (2005)

How do electrons damage DNA?Clues from

Ion Beam Irradiation of DNA

ca. 100 MeV/nucleon

O+8, Ar+18, Kr+36, Xe+54

D Becker, A Bryant-Friedrich, C Trzasko and M D Sevilla, Rad Res 160, 174 (2003)

PARTRAC Calculations, Hauptner et al (2006)

Cross Section of Heavy Ion Track Cross Section of Heavy Ion Track

Aloke Chatterjee and John Magee, J. Phys. Chem. Vol. 84, 3629-3536 (1980)

High Yields of Sugar radicals found in ion-beam irradiated DNA

D. Becker, A. Bryant-Friedrich, C. Trzasko, and M. D. Sevilla, Radiation Research, 160, 174-185 (2003) M. Bowman, D.Becker, M. D. Sevilla and J. Zimbrick, Radiation Research: Vol. 163, 447-454 (2005).

Excited States Important?

Yields of Sugar Radicals Increased in core of ion beam

irradiated DNA

● Sugar Radical Yield per Joule in track core

more than in the penumbra.

● New sugar phosphate species found in ion beam irradiated DNA

D. Becker, A. Bryant-Friedrich, C. Trzasko, and M. D. Sevilla, Radiation Research, 160, 174-185 (2003)

Ar ion Irradiated DNAPhosphoryl radicals - strand break radical(s)QuickTime™ and aGraphics decompressorare needed to see this picture.

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-0.022

-0.017

-0.012

-0.007

-0.002

0.003

0.008

0 100 200 300 400 500 600 700 800 900 1000

ROPO2• –

8

A

B

20.0 mT

CH2

O B

HH

HO

OPH

O–

O

• •

CH2O B

HH

HO

O

P

H

O–

O

2nd Strand Break radical found in ion beam irradiated DNA

N• from DNA

Simulation using theoretical spectral

parameters

BaseO

H

HH

HH

OPO

O-

O

•

C3’dephos●

LEE Induced Strand Scission + e

–

O CH2

baseO

O

P O–

O

–O

R

2 –

O CH2

baseO

–O

O CH2

baseO

A B

•

+ +•

ROPO2–• C3'

dephos

O–

R

P O–

O

O

R

P O–

O

O

O CH2

baseO

O

R

P O–

O

O•

dry

(5%) (95%)

Path B bond scissionPath A bond scissionLEE

D. Becker, A. Bryant-Friedrich, C. Trzasko, and M. D. Sevilla, Radiation Research, 160, 174-185 (2003)

What about Excited states?• We know excitation of DNA holes forms sugar

radicals

Visible excitation converts G•+ to Sugar Radicals

A Adhikary, S Collins, D Khanduri, and M D Sevilla, JPC B 2007, 111, 7415-7421 A Adhikary, A Kumar and M D Sevilla, Rad Res 2006 165, 479–484

O

ROCH2

H

RO H

H

G

H

1'

2'3'4'

5'

•

LEE Reaction with DNA:Strand Break Formation

• LEE capture at the base or the sugar phosphate backbone?

• What energies are needed?

• Are excited states involved?

B. Boudaiffa, P. Cloutier, D. Hunting, M. A. Huels, L. Sanche,

Science 2000, 287, 1658

F. Martin, P.D. Burrow, Z. Cai, P. Cloutier, D.J. Hunting, L. Sanche, Phys. Rev. Lett. 93, 068101-1 (2004)

DNA Strand break induced by low energy electrons

DNADNA

Modeling the LEE Induced DNA Strand Break

• Li, X.; Sevilla, M. D.; Sanche, L.; J. Am. Chem. Soc., 2003, 125, 13668

Sugar-Phosphate-Sugar model

-30.0

-20.0

-10.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

C-O distance (Å)

Re

lati

ve

E (

kc

al/m

ole

)

3'C-O: neutral

5'C-O: anion

5'C-O: neutral

3'C-O: anion

The Potential Energy Surfaces

DNADNA

Basis set dependence of Spin Distribution

3-21G(d) 6-31G(d) 6-31+G(d)

Xifeng Li and Michael D. Sevilla, in Theoretical Treatment of the Interaction of Radiation with Biological Systems. J. R. Sabin and E. Brandas, Eds., Advances in Quantum Chemistry, Elsevier, Volume 52, 59-88 (2007).

New DFT Calculations on 5’-dTMPH Anion Radical

What about the vertical surface?A. Kumar; M. Sevilla, J. Phys. Chem. B 2007, 111, 5464-5474

Recent Previous Work : J. Simons, Acc. Chem. Res. 2006, 39, 772-779 Bao, X.; Wang, J.; Gu, J.; Leszczynski, J. Proc. Nat. Acad. Sci.U.S.A. 2006, 103, 5658. Gu, J.; Wang, J.; Leszczynski, J. J. Am. Chem. Soc. 2006, 128,9322.

Adiabatic Barriers of 7 kcal/mol for 3’- C-O bond and 14 kcal/mol for 5’-C-O bond

LEE Induced strand break in 5'-dTMPH as a model: Vertical or Adiabatic pathways?

vertical

adiabatic

B3LYP/6-31G* calculated adiabatic and vertical potential surfaces (PES) of C5'-O5' bond dissociation of 5'-dTMPH radical anion. The singly occupied molecular orbital (SOMO) is also shown

1.45 1.5 1.6 1.7 1.8 1.9 2.0

B3LYP/6-31G*

kcal/mol

26.0

24.0

22.0

20.0

18.0

16.0

14.0

12.0

10.0

8.0

6.0

4.0

2.0

0.0

-8.0

C5'-O5' (Å)

Vertical PES

16.5 17.6

22.5

25.5 24.4

20.7

15.5

0.7

4.8

10.8

14.81.78 Å

-7.4

TS

Adiabatic PES

B3LYP/6-31++G** calculated adiabatic and vertical potential energy surfaces (PES) of C5'-O5' bond dissociation of 5'-dTMPH radical anion.

B3LYP/6-31++G**

kcal/mol

4.85.7

10.9

18.0

21.7

19.1

14.4

0.6

4.5

10.2

13.5

1.78 Å

0.5

-5.8

20.0

18.0

16.0

14.0

12.0

10.0

8.0

6.0

4.0

2.0

0.0

-2.0

-4.0

-6.0

8.2 kcal/mol

22.0

TS

Adiabatic PES

Vertical PES

1.45 1.5 1.6 1.7 1.8 1.9 2.0C5'-O5'(Å)

B3LYP/6-31G** optimized geometries of neutral and anionic radical of 5'-dTMP with Na+ and 11 H2O.

A. Kumar; M. Sevilla, J. Phys. Chem. B 2007, 111, 5464-5474

5'-dTMPNa + 11 H2O (Neutral) 5'-dTMPNa + 11 H2O (Anion radical)

Na+ Na+

DFT (B3LYP/6-31G**) calculated adiabatic potential energy surface (PES) of C5'-O5' bond dissociation of hydrated (11 H2O) 5'-dTMP radical anion with Na+ as a counter ion.

Na+

Na+

Na+

Na+

B3LYP/6-31G**

kcal/mol

33.0

30.0

27.0

24.0

21.0

18.0

15.0

12.0

9.0

6.0

3.0

0.0

-6.0

1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.5C5'-O5' (Å)

15.4

28.9

11.0

-3.3

Na+

Proposed mechanism of single strand break (SSB) due to attachment of LEE with 5'-dTMPH molecule

Why C-O not P-O?

• Experimentally only cleavage at the C-O bond is found. Y Zheng, P Cloutier, D Hunting, J R Wagner, L Sanche, JCP, 124, 064710 (2006).

• The loss of phosphate anion is the driving force for the reaction because the phosphate radical has a such a large EA.

J. Simons, Acc. Chem. Res. 2006, 39, 772-779

Base Release in Nucleosides Induced by Low Energy Electrons

OHCH2

OOH N

NH

O

O

CH3

OHCH2

CH O

OHN NH

CH

O

O

CH3

- -+.

X. Li, L. Sanche and M. Sevilla, Radiation Research, 165, 721 (2006)

J. Gu, Y. Xie and H. F. Schaefer, III, J. Am. Chem. Soc. 127, 1053–1057 (2005).

Potential Energy Surfaces (PESs) for C-N bond dissociation in Nucleoside Anion Radicals: Calculated with DFT (b3lyp, 6-31+G(d))

0.0

5.0

10.0

15.0

20.0

25.0

1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0

C-N1 distance (Å)

Rel

ativ

e E

ner

gy

(kca

l/mo

l)dT

dC

-8.0

-4.0

0.0

4.0

8.0

12.0

16.0

20.0

24.0

1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3

C1'-N distance (Å)

Re

lative

En

erg

y (

kca

l/m

ol)

dG

dA1

dA2

dA and dG anion radicals

dT and dC Anion Radicals

X. Li, L. Sanche and M. Sevilla, Radiation Research, 165, 721 (2006)

LEE Induced Excited States

• LEE induced resonances found experimentally suggest excited state involvement.

• What are the available excited state levels?

• We performed TD-DFT calculations to aid

our understanding in 5’-dTMP-●

J. Simons, Acc. Chem. Res. 2006, 39, 772-779

J. Berdys, I. Anusiewicz, P. Skurski, J. Simons JACS (2004)

Excited States of Sugar Phosphate Portion

Molecular orbital Energies and MO plots of neutral 5'-dTMPH. B3LYP/6-31G*. Scaled values by method of Modelli and Jones JPC 2006. *Experimental VOEs of thymine (Aflatooni et al. J. Phys. Chem. A (1998) 102, 6205 )

MOs Orbital Energy (eV) Scaled VOE (eV)

LUMO + 4 (σ3*)

LUMO + 3 (σ2*)

LUMO + 2 (σ1*)

LUMO + 1 (π2*)

LUMO (π1*)

HOMO (π)

1.78 2.64

1.27 2.23

0.73 1.80

0.43 1.56 (1.71)*

-0.84 0.53 (0.29)*

-6.24 ca. - 5 eV

Summary

• Strand break formation by LEE induced C-O bond scission is the lowest energy pathway in comparison to C-N, N-H or C-H bond scissions.

• LEE induced anion excited states likely provide a facile route to strand breaks.

Ph.D. Students. Amitava Adhikary Deepti Khanduri

Anil Kumar Sean Collins David Becker Alyson EngleLata Shukla Tom Casey Collaborators Leon Sanche Xifeng Li

AcknowledgmentsThis research is supported by NIH NCI RO1CA045424

DNA Radiation Chemistry Group at Oakland University

OU ESR GROUP