Investigation of Mutagenesis from Deoxyribonucleotide Pool Abnormalities

1
Investigation of Mutagenesis from
Deoxyribonucleotide Pool Abnormalities

• Howard Hughes Medical Institute (HHMI)
• Daniel Bai
• Dr. Christopher K. Mathews
• Biochemistry and Biophysics
• Oregon State University

2
Background
Normal dNTP Pool
Abnormal dNTP pool
dCTP pool
dATP pool
dTTP pool
dGTP pool
more mutations
Increase in dCTP pool
dATP pool
dTTP pool
dCTP pool
dGTP pool
more mutations
Symmetric increase in all 4 pools

• What is a dNTP?
• What is a dNTP pool?
• How are dNTP pool abnormalities related to
  mutagenesis?

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Background Continued
umuC, D
dinF
LexA
LexA
RecA
LexA
Upon DNA damage
RecA
rNDP reductase
uvrB
LexA suppressing repair systems
SOS Activated

• What is the SOS response?
• How does it work?
• What is RecA?

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Introduction
rNDP reductase
rNDP
dNDP
rNDP reductase

• What does ribonucleotide (rNDP) reductase do?
• How does rNDP reductase related to oxidative
  stress?

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Research Aims

• To analyze the effects of oxidative stress on
  dNTP concentrations.
• To understand the correlation between pool
  imbalances and the mechanisms of mutagenesis.
• To study the activity of the enzyme
  ribonucleotide reductase under oxidative stress.
• To measure rates of mutation after oxidative
  stress.

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Research Design
P2 Phage
RecA- mutant E-coli B
E-coli B

• How do we analyze changes in dNTP pools from
  oxidative stress?
• What is the treatment?

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Questions to be answered

• Questions to be answered
• Will RecA mutants contain different dNTP pools
  compared to the wild type?
• What effects does this particular dNTP change
  have on mutagenesis?
• How does the activity of ribonucleotide reductase
  change in response to oxidative stress?
• In cells with abnormal dNTP pools, are the
  mutations resulting from DNA replication or DNA
  repair?

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Past Experiments

• John Stenberg
• Worked with the enzyme Lex A
• Little ribonucleotide reductase activity
• Little to no difference in pools

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Methods

• Example
• To measure dATP (analyzed with 3H dTTP)
• Oligonucleotide? A A A T A A A T

• Complementary Base pair ? T T T A T T
  T A

• How are dNTP pools measured?
• How are oligonucleotides used?
• What is Klenow?
• How does a scintillation counter work?

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Methods Continued
Scintillation counter

• How is ribonucleotide reductase activity
  measured?
• How are mutation rates measured?

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Current Results
Chart. 1 dNTP pool changes under 3 treatments,
each with 2 runs

• dNTP Pools Changes Under Oxidative Stress
• What are the treatments?
• Why is there a decrease in dTTP pools?

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Results
Decreasing UV
Test for RecA- UV sensitivity

• Successfully transduced E-coli cells
• Observable UV sensitive RecA- cells

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Future Work

• Measure dNTP RecA mutant dNTP pools
• Measure mutation rates
• Perform ribonucleotide reductase assay

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Acknowledgements

• Howard Hughes Medical Institute
• Dr. Christopher K. Mathews
• Linda Benson
• Indira Rajagopal
• Dr. Kevin Ahern