The Search For Mitochondrial Ribonucleotide Reductase

Daniel BaiDr. Christopher Mathews

Department of Biochemistry and BiophysicsHHMI

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 reductaseRibonucleotide reductase

ribose sugar deoxyribose 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 Optic Disk With Retinal HemorrhageRetinal Hemorrhage

Normal Optic DiskNormal Optic Disk

Progressive External OphthalmoplegiaProgressive External Ophthalmoplegia Mitochondrial DNA depletion myopathyMitochondrial 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 pooldATP pool dTTP pooldTTP pool dCTP pooldCTP pooldGTP pooldGTP pool = more mutations

Increase in dCTP pool

= more mutationsdATP pooldATP pool dTTP pooldTTP pool dCTP pooldCTP pooldGTP pooldGTP pool

Symmetric increase in all 4 pools

dCTP pooldCTP 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

dNMPdNTPdNTP

dNTP

Cytosol

rNDP dNMP

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 Centrifugation Purified Mitochondria

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

Thin Layer ChromatographyThin Layer ChromatographyScintillationScintillation countercounter

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

Ribonucleotide Reductase Activity (pmol dNDP/mg protein hr)

0

5

10

15

20

Yeast Rat liver HeLa cells

Mitochondria

Cytosol

*

Inhibition of Mitochondrial RNR

Mitochondrial and cytosolic RNR each respond differently to dATP and hydroxyurea

Indicative of a new class of RNR

Rat Liver Cytosolic/Mitochondrial RNR Activity(pmol dCDP/ug protein hr)

0

0.4

0.8

1.2

1.6

2

no inhibitors 0.1 mMdATP

0.25mMdATP

1 mM dATP 10 mM dATP 1 mM HU

Mitochondrial RNR

Cytosolic RNR

RNR Activity in Different Tissues

Mito RNR Activity in Different Tissues(pmol dCDP/mg protein hr)

0

50

100

150

200

250

liver muscle heart kidney

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

Combined dNTP pools in Different Tissues (uM)

0

20

40

60

80

100

120

140

liver muscle heart

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