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 Absorbance 250 300 Wavelength (nm)

Pyrimidine Photodimers T<>T T<>C C<>C

Bipyrimidine (6-4) Photoadducts

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

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

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 8-12. 100 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 25-100 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

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 A: 10 M SP + 10 kJm-2 UVB in H2O. B: 10 M SP + 10 kJm-2 UVB in D2O.

Duplex DNA Specificity for 5’-G of 5’-GG-3’ doublets 3–9. 0, 1.5, 15, 37.5, 75, 112.5 and 150 M SP + 20 kJm-2 UVB 10-13. 150 M SP + 15, 10, 5 and 0 kJm-2 UVB 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