1. BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3 Eukaryotic Cells as Phenotypic Indicators: The use of RNAi to modulate gene expression DAY 3

2. Snapshot of the next four weeks We will eliminate the expression of six different genes using RNAi technology, human cells, fluorescent proteins and DNA microarrays

3. Sunlight Pollution Oxidation Food Cigarette Smoke DNA is constantly being damaged by endogenous and exogenous agents

4. DNA Repair Strategies Direct Reversal Photolyase, Methyltransferase, Oxidative demethylase Excision Repair Base excision, nucleotide excision, transcription coupled excision repair, mismatch repair Lesion Avoidance Translesion synthesis, DNA recombination Double strand break repair Homologous recombination, Non-homologous end joining

5. DNA Repair Strategies Direct Reversal Photolyase, Methyltransferase, Oxidative demethylase Excision Repair Base excision, nucleotide excision, transcription coupled excision repair, mismatch repair Lesion Avoidance Translesion synthesis, DNA recombination Double strand break repair Homologous recombination, Non-homologous end joining

6. Excision Repair Recognition Excision Resynthesis Ligation

7. DNA Repair Strategies Direct Reversal Photolyase, Methyltransferase, Oxidative demethylase Excision Repair Base excision, nucleotide excision, transcription coupled excision repair, mismatch repair Lesion Avoidance Translesion synthesis, DNA recombination Double strand break repair Homologous recombination, Non-homologous end joining

9. REPLICATION FIDELITY How many times does the replicative polymerase have to choose the correct nucleotide during one cell division??? Is one mistake in a million choices acceptable? How is fidelity achieved?

12. Humans who have inefficient Mismatch Repair are highly prone to colorectal and other cancers!! G C G T G Mismatch recognition MSH2 MSH6 MLH1 PMS2 Endonuclease? G T EXO1 PCNA, Pol , RPA Ligase? G T

14. DNA Repair Strategies Direct Reversal Photolyase, Methyltransferase, Oxidative demethylase Excision Repair Base excision, nucleotide excision, transcription coupled excision repair, mismatch repair Lesion Avoidance Translesion synthesis, DNA recombination Double strand break repair Homologous recombination, Non-homologous end joining

15. Base Excision Repair AAG/MPG 3MeA DNA glycosylase initiates repair of replication blocking lesions

16. Human AAG enzyme bound to substrate (aka MPG)

18. The absence of the AAG enzyme renders mouse cells very sensitive to the toxic effects of alkylating agents that damage DNA You will try to knock down human AAG with siRNA!!

19. DNA Repair Strategies Direct Reversal Photolyase, Methyltransferase, Oxidative demethylase Excision Repair Base excision, nucleotide excision, transcription coupled excision repair, mismatch repair Lesion Avoidance Translesion synthesis, DNA recombination Double strand break repair Homologous recombination, Non-homologous end joining

20. The eukaryotic cell cycle

25. Three pronged attack

26. P53 has 18 sites for modification by phosphorylation, acetylation, sumolation

27. How do deficiencies in p53 ATM ATR Affect human health?

28. p53 Li-Fraumeni Syndrome Germ line inheritance of mutated p53 genes Cancer Prone

29. THE FIRST SIGNS of ataxia telangiectasia (A-T) usually appear in the second year of life as a lack of balance and slurred speech. It is a progressive, degenerative disease characterized by cerebellar degeneration, immunodeficiency, radiosensitivity (sensitivity to radiant energy, such as x-ray) and a predisposition to cancer.

30. Ataxia Telangiectasia – Cancer Prone