1. dNMP Kinase Activity in Mitochondria and Its Role in Mitochondrial Mutagenesis  Brian M. Blair  Dr. Christopher K. Mathews  Department of Biochemistry and Biophysics  Oregon State University  HHMI Research 2007 http://www.nsf.gov/news/overviews/biology/assets/interact08.jpg

2. Why Is Research on mtDNA Metabolism Important?  Semiautonomous eukaryotic organelle  Responsible for ATP synthesis  Functions linked to:  Apoptosis  Aging process  Sensitivity to anti- HIV drugs  Contain their own genome:  mtDNA  mtDNA has 10-100 fold higher mutation rate than nuclear DNA  Mutations passed from generation to generation  Less effective mtDNA repair mechanism  mtDNA mutations = disease http://en.wikipedia.org/wiki/Intermembrane_space_of_mitochondria http://www.ccc.columbia.edu/Mitochondrial_Diseases/mito/round.gif

3. Mitochondrial Disease  Researchers have now discovered over 40 types of mitochondrial disease  40,000-70,000 Americans affected  Many age-related diseases involve defects of mitochondrion Diseases involving altered mitochondrial function:  Diseases involving altered mitochondrial function:  Parkinson’s Disease  Alzheimer’s Disease  Type 2 Diabetes  Various Cancers  Neurodegenerative Disorders  Cardiomyopathies

4. Deoxyribonucleoside Triphosphates (dNTPs)  Four mtDNA precursors:  dATP  dGTP  dCTP  dTTP  dNTP pool asymmetries = mtDNA mutagenesis Deoxyguanosine triphosphate Deoxyadenosine triphosphate Deoxythymidine triphosphate Deoxycytidine triphosphate

5. Metabolic Routes to Intramitochondrial dNTPs  Pathways involving dNMPs  Formation by salvage route  Are the pathways involving dNMPs significant in forming the dNTP pool asymmetries?

6. Purpose  Design an assay to measure dNMP phosphorylation to dNDP within the mitochondrion  Measure enzymatic activity of dNMP kinase  Brain, Liver, Heart, Skeletal Muscle, and Kidneys  3)Measure the dNMP kinase activity using dTMP, dGMP, dCMP, and dAMP as substrates  The dNMP kinase activity will vary within the different mammalian tissue mitochondria.  Analysis of this activity will help explain the different uptake pathways in dNMP metabolism and possible reasons behind dNTP asymmetry. Hypothesis #1

7. dNMPdNDP dNMP Kinase ATPADP A -P-P-P* dN -P-P *

8. Method 1: TLC Assay  Develop assay using T 4 infected E. coli HB101/pBK5 recombinant  ATP-γ-P 33 to trace activity of dNMP kinase  Rxn Mixture:  0.2 M Tris-HCl, pH 7.8; 0.02 M MgCl 2 ; 0.02 M ATP; 2.0 mM dTMP  Use 50 µL rxn mixture with substrate, 0.1 µCi ATP- γ-P 33, 10 µL rat mitochondrial extract, water to 100 µL  Run on TLC in 0.5 M LiCl and 5% Na 2 B 4 O 7, pH 7.0 aqueous solvent  Measure cpm of dNDP and calculate specific activity of enzyme

9. Results of TLC Data  No substrate control consistently has more counts than the with dTMP substrate 1) Test ATP-γ-P 33 for contamination 2) Original problem still present  Results:  Still co-migration occurs  TLC assay cannot be used  Other attempts to fix:  Remove small molecules or pre- existing substrates from extract  Run TLC in 10 different solvent systems

10. Method 2: HPLC Assay  0.2 M Tris-HCl, pH 7.8; 0.02 M MgCl 2 ; 0.02 M ATP; 2.0 mM dTMP  Use 50 µL rxn mixture with substrate, 10 µL rat mitochondrial extract, water to 100 µL  Dilute 20-fold and run in HPLC  Run standards to label nucleotides during rxn

11. Nucleotide Standards ATP ADP AMP dTTP dTDP dTMP

12. 0’ Rxn30’ Rxn 60’ Rxn120’ Rxn 0 dTMP Substrate dTMPADP ATP ADP ATP dTMP Thy 1? 2?

13. Hypothesis #2  dTMP is getting broken down to thymidine and P i as part of the 5’-deoxynucleotidase activity and regulation pathway Results of Reactions

14. 1mM Thymidine 0 ATP 0’0 ATP 60’ dTMP Thy

15. Explanation of dTMP Data  Removal of ATP blocks formation of dTMP from thymidine  Result:  All dTMP gets converted to thymidine and P i  Activity of 5’- deoxynucleotidase high in rat liver mitochondria

16. Summary Developed assay using thin layer chromatography to measure enzymatic activity of dNMP kinase Co-migration of unknown products at dTDP designated area Used HPLC to visualize elution times and peak areas; I found two unknown reaction products formed Proved activity of 5’-deoxynucleotidase forming thymidine from dTMP in absence of ATP; activity is high in rat liver mitochondria

17. Future Research Goals  Continue to perfect the HPLC assay  Pursue the other reactions that are occurring and find the reason behind this occurrence  Use mass spectrometer to determine molecular weight of unknown products  Measure the dNMP kinase activity of the four different deoxynucleotides within the different tissue mitochondria

18. Acknowledgements  Howard Hughes Medical Institute  Dr. Christopher K. Mathews  Linda Benson  Dr. Kevin Ahern  Oregon State University  Funding:  Howard Hughes Medical Institute