Investigation of Mutagenesis from
Deoxyribonucleotide Pool
Abnormalities
Howard Hughes Medical Institute (HHMI)
Daniel Bai
Dr. Christopher K. Mathews
Biochemistry and Biophysics
Oregon State University
Background
Normal dNTP Pool
dATP pool
dTTP pool
Abnormal dNTP pool
dGTP pool
dCTP pool
= more mutations
Increase in dCTP pool
dATP pool
dTTP pool
dGTP pool
dCTP pool
= more mutations
Symmetric
increase in all 4
pools
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What is a dNTP?
•
What is a dNTP pool?
•
How are dNTP pool abnormalities related to mutagenesis?
Background Continued
umuC, D
dinF
LexA
RecA
LexA
Upon DNA damage
LexA
RecA
uvrB
rNDP reductase
LexA suppressing
repair systems
•
What is the SOS response?
•
How does it work?
•
What is RecA?
SOS Activated
Introduction
rNDP reductase
rNDP
dNDP
rNDP reductase
•
What does ribonucleotide (rNDP) reductase do?
•
How does rNDP reductase related to oxidative stress?
Research Aims
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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.
Research Design
P2 Phage
E-coli B
RecA- mutant E-coli B
• How do we analyze changes in dNTP pools
from oxidative stress?
• What is the treatment?
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?
Past Experiments
John Stenberg
• Worked with the enzyme Lex A
• Little ribonucleotide reductase activity
• Little to no difference in pools
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?
Methods Continued
Scintillation counter
• How is ribonucleotide reductase activity measured?
• How are mutation rates measured?
Current Results
dNTP (pmol)/protein (ug)
E-coli dNTP Pools After Oxidative Stress
1.4
1.2
1
dATP
0.8
0.6
dTTP
0.4
dCTP
dGTP
0.2
0
no trt.
growth w/ trt
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?
trt after 25 min
growth
Results
Decreasing UV
Test for RecA- UV sensitivity
• Successfully transduced E-coli cells
• Observable UV sensitive RecA- cells
Future Work
• Measure dNTP RecA mutant dNTP pools
• Measure mutation rates
• Perform ribonucleotide reductase assay
Acknowledgements
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Howard Hughes Medical Institute
Dr. Christopher K. Mathews
Linda Benson
Indira Rajagopal
Dr. Kevin Ahern