Solar UV Radiation

• UVC <290 nm Absorbed by ozone

• UVB 290 – 320 nm ~0.5 % total energy

• UVA 320 – 400 nm 6% total energy

UV Spectrum of DNA

250 300Wavelength (nm)

Absorbance

Pyrimidine Photodimers

NH

NH

NH

NH

CH3CH3

H H

O

O

O

O

NH

NH

NH

N

CH3H

H H

O

O

NH2

O

N

NH

NH

N

H H

H H

NH2

O

NH2

O

T<>T T<>C C<>C

N

NH

NH

NH

O

O

CH3

O

H

NH2

N

NH

NH

NH

O

O

CH3

O

H

OH

CH3

T(6-4)C T(6-4)T

Bipyrimidine (6-4) Photoadducts

DNA Photooxidation Mechanisms

P

P*Type II

O2

1O2 singlet oxygen

DNA

G Oxidation

hν

Type I

DNA P-. + DNA+.

Hole Transfer

Oxidation atGG(A) doublet

N

N

H2NN

N

NN

N

HN

HHO

N

dR

N

dR

O OH2N

H2N

H

dR

O

O O

Imidazolone Oxazolone Spiroiminodihydantoin

N

N

S

HO3S

H

HOOC

SUPROFEN

PBSA

O

32P-DNA + PSUV irradiation

EtOH precipitation

Piperidine

Electrophoresis

Autoradiography

Upturned Eppendorf lid

Method of irradiation and visualization of DNA damage

Single-stranded DNA

• Singlet oxygen

• UV dose & [SP] dependent

Uniform G-specific attack

Quenching in ss DNA

1 SP + UVB

2 & 3 Quenched by sodium azide & DABCO

4 & 5 Mannitol & SOD have no effect

Singlet oxygen damage in ss DNA increases in D2O

H2O

D2O

Duplex DNA

Specificity for 5’-G of 5’-GG-3’ doublets

5’-G of 5’-GA-3’ doublets

Conclusions

• Single-stranded DNA - Type II mechanism– singlet oxygen

• Duplex DNA - Type I mechanism– electron transfer - DNA to excited sensitizer– hole migration

MALDI-TOF Analysis of DNA

• Requires picomole amounts of sample

• Mass accuracy to ~0.1%

• Generate +ve and –ve ion spectra

• UV-absorbing matrix for <100 base pairs

• Applications in DNA sequencing