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

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BEH.109 Laboratory Fundamentals in Biological
Engineering. MODULE 3Eukaryotic Cells as
Phenotypic IndicatorsThe use of RNAi to
modulate gene expressionDAY 3
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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
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DNA is constantly being damaged by endogenous and
exogenous agents
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• 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

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• 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

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Excision Repair
Recognition Excision Resynthesis Ligation

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• 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

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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?

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Humans who have inefficient Mismatch Repair are
highly prone to colorectal and other cancers!!
G
T
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• 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

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Base Excision Repair AAG/MPG 3MeA DNA
glycosylase initiates repair of replication
blocking lesions
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Human AAG enzyme bound to substrate (aka MPG)
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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!!
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• 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

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The eukaryotic cell cycle
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Three pronged attack
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P53 has 18 sites for modification by
phosphorylation, acetylation, sumolation
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How do deficiencies in p53 ATM ATR Affect human
health?
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p53

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

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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.
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Ataxia Telangiectasia Cancer Prone
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