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)
87
9
5
43
2
16
•OH
dRDeoxyguanosine
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 poolsIMP
AMP GMP
GDPADP
dADP dGDP
dATP 60μM dGTP 10μM
CTP UTP UMP
UDPCDP
dUDPdCDP
dCTP 30μM dTTP*60μM
rNDP reductase
NDP kinase
Hypothesis: 8-oxo-dGTP is not mutagenic at intracellular levels.
method
HPLC
UV detection EC detection
mutTmutT-
Extract nucleotides
mpA
Linear gradient
mpB
Identify/quantify nucleotides
52-278M E.coliB
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
Detection of 8-oxo-dGTP
52-278M
52-278M + 60fmol spike 8-oxo-dGTP
E.coliB +420mV
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)
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