1. A Photobiology Primer

2. Sunlight Ozone layer and atmosphere Skin DNA damage ROS generation DNA repair Cell death Mutation Altered gene expression Other effects Considerations in Studying UVR Effects

3. Part I: The UVR spectrum UVR reaching the earth UVR penetration of skin UVR light sources in the lab Part II: UVR-induced DNA damage Direct Indirect The action spectrum Part III: DNA repair Photoreactivation BER NER Mismatch repair Post-replication repair Part IV: DNA damage versus mutation Types of mutations UVR-induced mutations Mutation fixation Hallmark mutations Others

4. Part I The UVR spectrum –UVR reaching the earth –UVR penetration of skin UVR light sources in the lab

6. UVR Subdivisions

7. The Spectrum of Sunlight

8. UVR Reaching the Earth’s Surface

9. UVR Penetration into the Skin

11. UVR Light Sources Sunlight Kodacel/FS40 FS40

12. Sunlight UVA 340

14. Part II UVR-induced DNA damage –Direct –Indirect The action spectrum

15. UVC UVB UVA Direct DNA damage Indirect DNA damage Wavelength Dependence of UVR- induced DNA Damage

16. The Action Spectrum

17. Direct UVR-induced DNA Damage Base changes –Cyclobutane pyrimidine dimers –6,4-Photoproducts Crosslinks (protein, DNA) Photosensitization (psoralen) Photolysis (BrdU)

19. Differential Susceptibility CPD TT > CT > TC > CC 68 : 13 : 16 : 3 6,4 PP TC/CC >> TT CT not susceptible C5 methylated cytosine not susceptible CPD induction several fold higher than 6,4 PP

20. Indirect DNA Damage: ROS Intracellular –Mitochondrial respiration –Peroxisome metabolism –Enzymatic synthesis of NO –Phagocytic leukocytes Extracellular –Radiolysis of water (ionizing radiation, near UV) –Heat –Drugs

21. Generated by –Absorption of energy –Monovalent reduction –Fenton reaction –Enzymatic activity Include –Radicals (unpaired electrons) –Molecules –Ions ROS Characteristics

22. Some Reactive Oxygen Species

23. Stable Free (di)radical Unpaired electrons with parallel spins Highly reactive Not a free radical Paired electrons with opposite spins Generation of Singlet Oxygen

24. ROS Can Be Generated by Reduction of Molecular Oxygen

25. Superoxide dismutase H 2 O 2 + triplet O 2 Fe Hydroxyl radical Hydroxyl radical Singlet O 2 + Hydroxide ion NO Peroxynitrate Monvalent reduction H2O2H2O2 Reactions of Superoxide Radical

26. The Fenton Reaction

27. ROS Detoxification Other antioxidants

28. Base damage –Thymine glycol –8-oxodG Damage to sugar–phosphate backbone –Fragmentation –Base loss –Strand break Imidazole ring opening ROS Damage to DNA

29. Blocks replication Causes mispairing ROS Base Damage

30. Part III DNA repair –Photoreactivation –Base excision repair (BER) –Nucleotide excision repair (NER) –Mismatch repair –Post-replication repair

31. Photoreactivation Found in many species up to and including marsupials Not demonstrated in placental mammals There are both CPD and 6,4 PP photolyases Can be used to study DNA damage dependence of UVR effects

32. Base Excision Repair Two forms: short patch and long patch Mostly for repair of non- bulky adducts DNA glycosylases that recognize CPDs and 6,4 PP not found in mammals Major pathway for repair of DNA adducts due to ROS

33. Human DNA Glycosylases Enzyme Size (amino acid residues) Gene location at chromosome Altered base removed from DNA UNG31312q23-q24U and 5-hydroxyuracil TDG41012q24.1U or T opposite G, ethenocytosine hSMUG 1 27012q13.1-q14U (preferentially from single-strand DNA) MBD45803q21U or T opposite G at CpG sequences hOGG13453p258-oxo G opposite C, formamidopyrimidine MYH5211p32.1-p34.3A opposite 8-oxo G hNTH131216p13.2-p13.3 Thymine glycol, cytosine glycol, dihydrouracil, formamidopyrimidine MPG29316p (near telomere)3-MeA, ethenoadenine, hypoxanthine

35. Nucleotide Excision Repair Major mammalian form of CPD and 6,4 PP repair in mammals Loss of function mutations can result in xeroderma pigmentosum

39. Mismatch Repair Substrates Base:base mismatch –Non-instructive DNA adduct –Nucleotide misincorporation Insertion/deletion loops –Slippage

40. MutSα: base-base and insertion-deletion mismatches MutSβ: insertion-deletion mismatches only

42. Post-replication Repair Lesion bypass (error prone) Homologous recombination (error free)

43. Post-replication Recombination Repair During replication, the replication machinery skips over the region with the dimer, leaving a gap; the complementary strand is replicated normally. The two newly synthesized strands are shown in red. Strand exchange between homologous strands occurs. Recombination is completed, filling in the gap opposite the pyrimidine dimer, but leaving a gap in the other daughter duplex. This last gap is easily filled, using the normal complementary strand a template.

44. Double-strand Break Repair Homologous recombination (error free) Non-homologous end-joining (error prone)

45. Double-strand Break Repair

46. Part IV DNA damage versus mutation Types of mutations UVR-induced mutations –Mutation fixation –Hallmark mutations –Others

47. DNA Damage

48. Lesion is induced Unrepaired/misrepaired lesion miscodes Mutation fixation occurs Mutation Fixation

49. Types of Mutation

50. Substitution Deletion Insertion Normal

51. Base Substitutions Transition Y ↔ Y, R ↔ R T ↔ C G ↔ A Transversion Y ↔ R T ↔ G C ↔ A

52. A GT C

53. UVR Signature Mutations Mutations –C to T transition –CC to TT tandem transition Location –Dipyrimidine sequences –Pyrimidine stretches

54. The A Rule Preferential incorporation of A opposite a non- instructive DNA lesion TT dimers thus encode AA sequence and no mutation arises Due to character of the DNA polymerase TT → AA ^

55. Mutations Due to ROS-induced DNA Damage G:C → T:A transversion