The Search For Mitochondrial
Ribonucleotide Reductase
Daniel Bai
Dr. Christopher Mathews
Department of Biochemistry and Biophysics
HHMI
What is Ribonucleotide
Reductase?
ribose sugar
deoxyribose sugar
rNDP
dNDP
Ribonucleotide reductase
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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
What are Mitochondria?
Purified Mitochondria
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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
Mitochondrial Diseases
Normal Optic Disk
Progressive External Ophthalmoplegia
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Optic Disk With
Retinal Hemorrhage
Mitochondrial DNA depletion myopathy
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
RNR and Mitochondrial
Diseases
dATP pool
dTTP pool
dGTP pool
dCTP pool
dCTP
pool
= =more
normal
mutations
pool
Increase in dCTP pool
dATP pool
dTTP pool
dGTP pool
dCTP pool
= more mutations
Symmetric
increase in all 4
pools
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RNR regulates dNTP pools
Mitochondrial diseases arise from abnormalities in dNTP pools
Abnormal dNTP concentrations cause mitochondrial polymerase γ
to make errors
Why Do Mitochondria Have
RNR?
NdR
Cytosol
NdR
rNDP
rNDP
dNMP
dNTP
Mitochondria
dNTP
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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
dNMP
Experimental Methods
Organ
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Homogenize
Differential Centrifugation
Purified Mitochondria
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
RNR Assay
Solvent front
dCDP
CDP
Thin Layer Chromatography
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Scintillation counter
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
RNR Activity in Different
Species
Ribonucleotide Reductase Activity
(pmol dNDP/mg protein hr)
20
15
Mitochondria
Cytosol
10
5
0
Yeast
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Rat liver
HeLa cells
*
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
Rat Liver Cytosolic/Mitochondrial RNR Activity
(pmol dCDP/ug protein hr)
2
Mitochondrial RNR
1.6
Cytosolic RNR
1.2
0.8
0.4
0
no inhibitors
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0.1 mM
dATP
0.25mM
dATP
1 mM dATP 10 mM dATP
1 mM HU
Mitochondrial and cytosolic RNR each respond differently
to dATP and hydroxyurea
Indicative of a new class of RNR
RNR Activity in Different
Tissues
Combined dNTP pools in Different Tissues
(uM)
Mito RNR Activity in Different Tissues
(pmol dCDP/mg protein hr)
140
250
120
200
100
80
150
60
100
40
20
50
0
0
liver
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muscle
heart
kidney
liver
muscle
heart
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
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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
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Howard Hughes Medical Institute
Dr. Christopher K. Mathews
Linda Benson
Dr. Indira Rajagopal
Dr. Kevin Ahern