investigation of mutagenesis from deoxyribonucleotide pool abnormalities
DESCRIPTION
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. What is a dNTP? What is a dNTP pool? - PowerPoint PPT PresentationTRANSCRIPT
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Investigation of Mutagenesis from Deoxyribonucleotide Pool
Abnormalities
Howard Hughes Medical Institute (HHMI)
Daniel BaiDr. Christopher K. Mathews
Biochemistry and BiophysicsOregon State University
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Background
• What is a dNTP?
• What is a dNTP pool?
• How are dNTP pool abnormalities related to mutagenesis?
dATP pooldATP pool dTTP pooldTTP pool dCTP pooldCTP pooldGTP pooldGTP pool = more mutations
= more mutationsdATP pooldATP pool dTTP pooldTTP pool dCTP pooldCTP pooldGTP pooldGTP pool
Increase in dCTP pool
Symmetric increase in all 4 pools
Normal dNTP Pool
Abnormal dNTP pool
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Background Continued
• What is the SOS response?
• How does it work?
• What is RecA?
rNDP reductaseuvrB
LexA
LexA
umuC, D
RecA
dinF
LexA suppressing repair systems
Upon DNA damage
LexARecA
SOS Activated
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Introduction
• What does ribonucleotide (rNDP) reductase do?
• How does rNDP reductase related to oxidative stress?
rNDP dNDP
rNDP reductase
rNDP reductaserNDP reductase
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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
• How do we analyze changes in dNTP pools from oxidative stress?
• What is the treatment?
P2 PhageP2 PhageE-coli BE-coli B RecARecA-- mutant E-coli B mutant E-coli B
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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
• How are dNTP pools measured?
• How are oligonucleotides used?
• What is Klenow?
• How does a scintillation counter work?
Complementary Complementary Base pair Base pair T* T* T*T* T* T* A A T* T* T*T* T* T* A… A…
Example
– To measure dATP (analyzed with 3H dTTP)• Oligonucleotide A A A T A A A T…
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Methods Continued
• How is ribonucleotide reductase activity measured?
• How are mutation rates measured?
ScintillationScintillation countercounter
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Current Results
dNTP Pools Changes Under Oxidative Stress
What are the treatments?
Why is there a decrease in dTTP pools?
Chart. 1dNTP pool changes under 3 treatments, each with 2 runs
E-coli dNTP Pools After Oxidative Stress
0
0.2
0.4
0.6
0.8
1
1.2
1.4
no trt. growth w/ trt trt after 25 mingrowth
dN
TP
(p
mo
l)/p
rote
in (
ug
)
dATP
dTTP
dGTP
dCTP
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Results
• Successfully transduced E-coli cells
• Observable UV sensitive RecA- cells
Test for RecA- UV sensitivity
Decreasing UV
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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