Analysis of 8-oxo-dGTP, a mutagenic nucleotide, at physiological levels in E.coli Jordan Kane Boutilier Mentor: Dr. Christopher Mathews Department of Biochemistry.

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Presentation transcript:

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 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 AMPGMP GDPADP dADPdGDP dATP 60μM dGTP 10μM CTPUTP UMP UDPCDP dUDPdCDP dCTP 30μM dTTP*60μM rNDP reductase NDP kinase

Hypothesis: 8-oxo-dGTP is not mutagenic at intracellular levels.

method HPLC UV detectionEC detection mutTmutT - Extract nucleotides mpA Linear gradient mpB Identify/quantify nucleotides ME.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 M M + 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)