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

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 rNDP dNDP Ribonucleotide reductase ribose sugardeoxyribose sugar

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

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

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 dATP pool dTTP pool dCTP pool dGTP pool = more mutations Increase in dCTP pool = more mutations dATP pool dTTP pool dCTP pool dGTP pool Symmetric increase in all 4 pools dCTP pool = normal pool

Why Do Mitochondria Have RNR?  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 Mitochondria rNDP NdR dNMP dNTP dNTP Cytosol rNDPdNMP NdR

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 Homogenize Differential CentrifugationPurified Mitochondria

RNR Assay  Give the RNR enzyme tritiated H 3 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 Thin Layer Chromatography Scintillationcounter Scintillation counter

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 *

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

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

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

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