The Search For Mitochondrial Ribonucleotide Reductase

1. The Search For Mitochondrial Ribonucleotide Reductase Daniel Bai Dr. Christopher Mathews Department of Biochemistry and Biophysics HHMI

2. What is Ribonucleotide Reductase? • Ribonucleotide reductases (RNR) provide the building blocks for DNA in all living cells • (RNR) is activated upon DNA damage • It is a key enzyme for DNA repair and replication ribose sugar deoxyribose sugar rNDP dNDP Ribonucleotide reductase

3. What are Mitochondria? • Mitochondria are the powerhouses of eukaryotic cells • Mitochondria are regulators of cellular proliferation and apoptosis – programmed cell death • Functions in: heme synthesis, steroid synthesis, and detoxification • They have their own genomes • Mitochondria mutation rates are on the magnitude of two orders greater than in the nuclear genome Purified Mitochondria

4. Mitochondrial Diseases • 10 types of neuropathies associated with mitochondrial DNA mutations • 22 types of cardiomyopathies caused by abnormal mitochondrial function • Over 500 diseases associated with mitochondrial DNA mutations Optic Disk With Retinal Hemorrhage Normal Optic Disk Progressive External Ophthalmoplegia Mitochondrial DNA depletion myopathy

5. RNR and Mitochondrial Diseases • RNR regulates dNTP pools • Mitochondrial diseases arise from abnormalities in dNTP pools • Abnormal dNTP concentrations cause mitochondrial polymerase γ to make errors dCTP pool dCTP pool dATP pool dTTP pool dGTP pool = more mutations = normal pool Increase in dCTP pool dATP pool dTTP pool dGTP pool dCTP pool = more mutations Symmetric increase in all 4 pools

6. Why Do Mitochondria Have RNR? NdR • Mechanism for dNTP accumulation in mitochondria is unknown. • There are 4 speculated pathways, 3 involve transporting already reduced (deoxy) ribose sugars • Mitochondrial reduction of ribose sugars allows for dNTP pool regulation Cytosol NdR rNDP dNMP rNDP dNMP dNTP Mitochondria dNTP

7. Experimental Methods • Compromise the cellular membrane to free the mitochondria • Differential centrifugation of cellular extract to isolate mitochondria • Sonicate mitochondria to release RNR • Purify the RNR enzyme Organ Differential Centrifugation Purified Mitochondria Homogenize

8. RNR Assay • Give the RNR enzyme tritiated H3 CDP substrate • Use thin layer chromatography to separate out the RNR substrate (CDP) and RNR product (dCDP) • Cut out radiolabeled CDP and dCDP and count the radioactivity on a scintillation counter • Calculate the enzyme activity via the amount of dCDP formed Solvent front dCDP CDP Scintillationcounter Thin Layer Chromatography

9. RNR Activity in Different Species * • RNR activity was compared in 3 types of cells • Yeast had the lowest overall cytosolic and mitochondrial RNR activity • The immortal HeLa cancer cells have the highest cytosolic RNR activity (attributed to the need for rapid nuclear replication) • In yeast and rat liver, the mitochondrial RNR was more active than cytosolic RNR

10. Inhibition of Mitochondrial RNR • Mitochondrial and cytosolic RNR each respond differently to dATP and hydroxyurea • Indicative of a new class of RNR

11. RNR Activity in Different Tissues • Mitochondrial RNR activities varied amongst different tissues • Heart and muscle mitochondrial RNR had 30 fold differences in activity • Liver, muscle, heart, and kidney mitochondria all used RNR for dNDP synthesis • No correlation between RNR activity and dNTP pool sizes

12. Future Work • Check mitochondrial extracts for cytosolic contamination via testing for the presence of cytosolic enzymes • Check cytosolic extract for mitochondrial ETC enzyme activity to ensure mitochondria were not damaged • Compare RNR activity between young and old rat tissues • Purify mitochondrial RNR • Locate the mitochondrial RNR gene in the nuclear genome

13. Acknowledgements • Howard Hughes Medical Institute • Dr. Christopher K. Mathews • Linda Benson • Dr. Indira Rajagopal • Dr. Kevin Ahern