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Analysis of 8-oxo-dGTP, a
mutagenic nucleotide, at
physiological levels in E.coli
Jordan Kane Boutilier
Mentor: Dr. Christopher Mathews
Department of Biochemistry and Biophysics
Oregon State University
Reactive oxygen species (ROS)
• Generated via cellular respiration
• Most ROS are free radicals that contain
unpaired electrons
• Oxidative Stress
• Oxidative damage
• Causes [nucleo]base modification
ROS induced mutagenesis:
7,8-dihydro-8-oxoguanine (8-oxodG)
•OH
6
1
5
7
4
9
8
2
3
dR
Deoxyguanosine
Alternative base pairings observed for (8-oxo-G).
Normal G:C
Watson Crick
base pair
7,8-dihydro-8-oxoguanine forms
stable base pair with adenine
MutT removes 8-oxo-dGTP from
Nucleotide pools
Is 8-oxo-dGTP a critical substrate
for MutT?
• mutT mutants can display a mutator
phenotype during anaerobic growth
• 8-oxo-dGTP is poor substrate for DNA
polymerase
• Physiological levels: dNTP precursor pools
Biosynthesis of precursor pools
IMP
CTP
UTP
CDP
UMP
UDP
AMP GMP
ADP
GDP
rNDP reductase
dCDP
dUDP
dADP
dGDP
NDP kinase
dCTP 30μM
dTTP*60μM
dATP 60μM dGTP 10μM
a
g
c
t
Hypothesis: 8-oxo-dGTP is not
mutagenic at intracellular levels.
method
52-278M
E.coliB
mutT-
mutT
mpA
mpB
Linear
gradient
HPLC
Identify/quantify
nucleotides
Extract
nucleotides
UV detection
EC detection
Calibration technique
HPLC Elution Profile of Standards
Approach
• Comparison of crude extracts of E. coli
mutT wild type and mutant strains
• preliminary quantification of 8-oxo-dGTP at
physiological levels
E.coliB +420mV
Detection of 8-oxo-dGTP
52-278M
52-278M + 60fmol spike 8-oxo-dGTP
Significance
• 8-oxo-dGTP at physiological levels is
extremely low
• Comparison to other dNTPs
• Most likely not mutagenic at this level
• Not the critical substrate for MutT
Acknowledgments
•
•
•
•
•
•
Dr. Mathews and Lab
Dr. Tory Hagen and Lab
Dr. Kevin Ahern
Mary Lynn Tassotto
HHMI
National Science Foundation
(Undergraduate research supplement)