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BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3 Eukaryotic Cells as Phenotypic Indicators: The use of RNAi to modulate gene expression DAY 3. Snapshot of the next four weeks
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BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3Eukaryotic Cells as Phenotypic Indicators:The use of RNAi to modulate gene expressionDAY 3
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
Sunlight Oxidation Food Pollution Cigarette Smoke DNA is constantly being damaged by endogenous and exogenous agents
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
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
Excision Repair Recognition Excision Resynthesis Ligation
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
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?
Mismatch recognition Endonuclease? G T EXO1 G G T C MLH1 PMS2 MSH6 MSH2 PCNA, Pol d, RPA Ligase? G Humans who have inefficient Mismatch Repair are highly prone to colorectal and other cancers!! G T
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
Base Excision Repair AAG/MPG 3MeA DNA glycosylase initiates repair of replication blocking lesions
Human AAG enzyme bound to substrate (aka MPG)
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!!
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
P53 has 18 sites for modification by phosphorylation, acetylation, sumolation
How do deficiencies in p53 ATM ATR Affect human health?
p53 Li-Fraumeni Syndrome Germ line inheritance of mutated p53 genes Cancer Prone
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.