Post on 15-Dec-2015
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