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A Photobiology Primer

A Photobiology Primer. Considerations in Studying UVR Effects. Sunlight. Ozone layer and atmosphere. Skin. ROS generation. Other effects. DNA damage. DNA repair. Cell death Mutation Altered gene expression. Part III: DNA repair Photoreactivation BER

claudia-mullen
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A Photobiology Primer

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  1. A Photobiology Primer

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

  3. 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 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

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

  5. UVR Subdivisions

  6. The Spectrum of Sunlight

  7. UVR Reaching the Earth’s Surface

  8. UVR Penetration into the Skin

  9. UVR Light Sources FS40 Kodacel/FS40 Sunlight

  10. Sunlight UVA 340

  11. Part II • UVR-induced DNA damage • Direct • Indirect • The action spectrum

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

  13. The Action Spectrum

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

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

  16. 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

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

  18. Some Reactive Oxygen Species

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

  20. ROS Can Be Generated by Reduction of Molecular Oxygen

  21. Reactions of Superoxide Radical Monvalent reduction Superoxide dismutase NO Hydroxyl radical H2O2 Fe Peroxynitrate H2O2 + triplet O2 Singlet O2 + Hydroxide ion Hydroxyl radical

  22. The Fenton Reaction

  23. ROS Detoxification Other antioxidants

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

  25. ROS Base Damage Blocks replication Causes mispairing

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

  27. 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

  28. 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

  29. Human DNA Glycosylases

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

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

  32. MutSα: base-base and insertion-deletion mismatches • MutSβ: insertion-deletion mismatches only

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

  34. 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.

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

  36. Double-strand Break Repair

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

  38. DNA Damage

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

  40. Types of Mutation

  41. Normal Substitution Deletion Insertion

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