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dNTP Imbalance in Mitochondria

dNTP Imbalance in Mitochondria. Alexandra Frolova Dr. Christopher K. Mathews Laboratory Biochemistry and Biophysics. Rates of mutation in mtDNA are 10-100 fold higher than in nDNA . mtDNA mutations are linked to various human diseases: Cancer Cardiomyopathies

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dNTP Imbalance in Mitochondria

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  1. dNTP Imbalance in Mitochondria Alexandra Frolova Dr. Christopher K. Mathews Laboratory Biochemistry and Biophysics

  2. Rates of mutation in mtDNA are 10-100 fold higher than in nDNA. • mtDNA mutations are linked to various human diseases: • Cancer • Cardiomyopathies • Degenerative heart, muscle, & neurological disorders • mtDNA mutations lead to accelerated aging in mice.

  3. An imbalance in mtDNA precursor pools can cause mutations. • Mathews’ lab has determined that there exists a high concentrations of dGTP relative to the other dNTPs in mtDNA pools in various mammalian tissues. Song, S., Z F. Pursell, W.C. Copeland, M.J. Longley, T.A. Kunkel, and C.K. Mathews (2005) DNA Precursor Asymmetries in Mammalian Tissue Mitochondria and Possible Contributions to Mutagenesis via Reduced Replication Fidelity. Proc. Natl. Acad. Sci. USA 102, 4990-4995.

  4. dNTP imbalance in rat heart mitochondria dNTP pmol per mg protein c m c m c m c m dATP dTTP dCTP dGTP

  5. Excess of one dNTP can cause misinsertion and/or inhibited proofreading, which can lead to substitution mutations. • Reactive oxygen species (ROS) can oxidize dGTP to form mutagenic 8-oxo-dGTP. • Various polymerases will wrongly insert 8-oxo-dGTP opposite template A leading to A-T to C-G transversions. Pursell, Z.F., J.T. MacDonald, C.K. Mathews, and T.A. Kunkel (2008) Trace Amounts of 8-oxo-dGTP in Mitochondrial Pools Reduce DNA Polymerase γ Replication Fidelity. Nucl. Ac. Res. 36, 2174-2181.

  6. Transport and metabolic pathways of nucleosides andnucleotides

  7. Project purpose • To understand how various intramitochondrial enzymes participate in dNTP pool regulation. • To determine which enzyme(s) cause dGTP accumulation.

  8. Enzymaticpathways of dGTP synthesis and turnover DNA DNA polymerase dGTP NDPkinase GDP RNR dGDP dGMP kinase MTH-1 dGMP dGK dNT-2 dGuo

  9. Enzymatic pathways that may influence dGTP levels • NDP Kinase dGTP + ADP dGDP + ATP • dGMP Kinase dGMP + ATP dGDP + ADP  • NT2 mitochondrial 5’-nucleotidase dGMP +H20 Deoxyguanosine + Pi • MTH-1 (mutT homolog) dGTP + H2O dGMP + PPi

  10. Experimentlayout • Incubate mixtures of the four dNTPs with mitochondria extract and various substrates to: • monitor enzyme activity • determine which enzymatic steps are critical for maintaining dNTP pool stability. Experiment 1: Hydrolytic dNTP breakdown • dNTP + H2O → dNDP (→ dNMP) +Pi Experiment 2: NDP Kinase • dNTP + ADP → dNDP (→dNMP) + ATP Experiment 3: dNMP Kinase • dNMP + ATP → dNDP + ADP

  11. Methods of mitochondrial preparation • Isolate rat liver mitochondria using homogenization and differential centrifugation. • Prepare mitochondria extract by using sonication. • Add detergent η-dodecyl-β-maltoside. • Centrifuge 15K for 30 mins.

  12. Methods of sample analysis • Samples analyzed using High Performance Liquid Chromatography (HPLC) . • Column used was a C-18 Reverse Phase Column. • Sample components separated using linear gradient. • Buffer A: 8mM TBA-OH, 10mM monobasic K phosphate, 0.25% methanol, pH 7.0 • Buffer B: 2mM TBA-OH, 100mM monobasic K phosphate, 30% methanol, pH 7.0

  13. Exampleof chromatogram11 StandardsdNMPs, dNTPs, ANPs AMP dGMP dGTP dATP dTMP ADP ATP dTTP dAMP dCTP dCMP

  14. Raw data hydrolytic enzymedNTP + H2O → dNDP (→ dNMP) +Pi

  15. Raw data NDP kinasedNTP + ADP → dNDP (→dNMP) + ATP

  16. DatadNMP kinasedNMP + ATP → dNDP + ADP

  17. Progress • Determined: • - concentrations of substrates and reactants, • - incubation times, • - HPLC elution program • necessary for detecting activity of hydrolytic enzyme and NDPK. • Made progress towards: • creating a functional method for examining dNMPK activity using HPLC. • - acquiring publishable data.

  18. Future objectives • dNTP + ADP dNDP + ATP • dGMP + ATP dGDP + ADP • dNMP deoxyribonucleosides • Deoxyribonucleosides + ATP dNTPs + ADP

  19. Dr. Mathews Presentation from 6/21/09

  20. Thank you… • HHMI • Kevin Ahern • Dr. Christopher Mathews • Linda Benson and Korakod Chimploy

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