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Analysis of 8-oxo-dGTP, a mutagenic nucleotide, at physiological levels in E.coli

Analysis of 8-oxo-dGTP, a mutagenic nucleotide, at physiological levels in E.coli. 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.

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Analysis of 8-oxo-dGTP, a mutagenic nucleotide, at physiological levels in E.coli

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  1. Analysis of 8-oxo-dGTP, a mutagenic nucleotide, at physiological levels in E.coli

  2. 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

  3. ROS induced mutagenesis: 7,8-dihydro-8-oxoguanine (8-oxodG) •OH 6 1 5 7 8 2 4 9 3 dR Deoxyguanosine

  4. 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

  5. MutT removes 8-oxo-dGTP from Nucleotide pools

  6. 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

  7. Biosynthesis of precursor pools IMP UMP AMP GMP CTP UTP CDP UDP ADP GDP rNDP reductase dCDP dUDP dADP dGDP NDP kinase dCTP 30μM dATP 60μM dGTP 10μM dTTP*60μM

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

  9. method 52-278M E.coliB mutT- mutT mpA mpB Linear gradient HPLC Identify/quantify nucleotides Extract nucleotides UV detection EC detection

  10. Calibration technique

  11. HPLC Elution Profile of Standards

  12. Approach • Comparison of crude extracts of E. coli mutT wild type and mutant strains • preliminary quantification of 8-oxo-dGTP at physiological levels

  13. E.coliB +420mV Detection of 8-oxo-dGTP 52-278M 52-278M+ 60fmol spike 8-oxo-dGTP

  14. 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

  15. 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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