1. Solar UV Radiation UVC <290 nm Absorbed by ozone
UVB – 320 nm ~0.5 % total energy
UVA – 400 nm % total energy

2. UV Spectrum of DNA
Absorbance
250
300
Wavelength (nm)

3. Pyrimidine Photodimers
T<>T
T<>C
C<>C

4. Bipyrimidine (6-4) Photoadducts

5. DNA Photooxidation Mechanisms
Oxidation at
GG(A) doublet P
G Oxidation hν
Hole Transfer
DNA
Type I
Type II
P-. + DNA+. P*
1O2 singlet
oxygen O2
DNA

8. Method of irradiation and visualization of DNA damage
UV irradiation
32P-DNA + PS
Upturned Eppendorf lid
EtOH precipitation
Oligonucleotides 5’-end-labeled with 32P--ATP, purified to remove unincorporated label and the incubated with various concentrations of SP in 5 mM phosphate buffer.
Exposed to 312 nm UVB radiation because maximum absorbance of SP is 300 nm in a droplet contained in an upturned Eppendorf lid.
After precipation of the irradiated DNA with ethanol, samples are treated with hot piperidine to reveal alkali-labile sites.
Cleavage products from irradiated DNA and chemical sequencing reactions are then separated on 16 % sequencing gel by electrophoresis. DNA bands are then visualised by autoradiography.
 Piperidine
Electrophoresis
Autoradiography

9. Single-stranded DNA Singlet oxygen UV dose & [SP] dependent Uniform
G-specific attack
Figure legend
3 – 7. 0, 1, 10, 25, 50, 75 & 100 M SP + 10 kJm-2 UVB
M SP + 7.5, 5, 2.5 and 0 kJm-2 UVB
Uniform attack in ss DNA indicative of damage by singlet oxygen.
Type II photosensitization mechanism.
An increase in G-specific cleavage from 0-10 M SP.
Overcut from M SP and 10 kJm-2.
Decrease in DNA damage from 10-0 kJm-2 at 100 M SP.
Singlet oxygen
UV dose & [SP] dependent

10. Quenching in ss DNA 1 SP + UVB 2 & 3 Quenched by sodium azide & DABCO
4 & 5 Mannitol & SOD have no effect

11. Singlet oxygen damage in ss DNA increases in D2O
H2O
D2O
A: 10 M SP + 10 kJm-2 UVB in H2O.
B: 10 M SP + 10 kJm-2 UVB in D2O.

12. Duplex DNA Specificity for 5’-G of 5’-GG-3’ doublets
3–9. 0, 1.5, 15, 37.5, 75, and 150 M SP + 20 kJm-2 UVB
M SP + 15, 10, 5 and 0 kJm-2 UVB
5’-G of 5’-GA-3’ doublets

13. Conclusions Single-stranded DNA - Type II mechanism
singlet oxygen
Duplex DNA - Type I mechanism
electron transfer - DNA to excited sensitizer
hole migration

14. 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