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. 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. An imbalance in mtDNA precursor pools can cause mutations.

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

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 and nucleotides

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

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

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 +H 2 0 Deoxyguanosine + P i MTH-1 (mutT homolog) dGTP + H 2 O dGMP + PP i

10. Experiment layout 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 + H 2 O → dNDP (→ dNMP) +P i 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. Example of chromatogram 11 Standards dNMPs, dNTPs, ANPs dCMP dTMP dGMP AMP dAMPdCTP ADP dGTP dTTP ATP dATP

14. Raw data hydrolytic enzyme dNTP + H 2 O → dNDP (→ dNMP) +P i

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

16. Data dNMP kinase dNMP + 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