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?
ribose sugar
deoxyribose sugar
rNDP
dNDP
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

3
What are Mitochondria?
Purified 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

4
Mitochondrial Diseases
Optic Disk With Retinal Hemorrhage
Normal Optic Disk
Progressive External Ophthalmoplegia

• 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

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RNR and Mitochondrial Diseases
dCTP pool
dCTP pool
dATP pool
dTTP pool
dGTP pool
more mutations
normal pool
Increase in dCTP pool
dATP pool
dTTP pool
dCTP pool
dGTP pool
more mutations
Symmetric increase in all 4 pools

• RNR regulates dNTP pools
• Mitochondrial diseases arise from abnormalities
  in dNTP pools
• Abnormal dNTP concentrations cause mitochondrial
  polymerase ? to make errors

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

• 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

7
Experimental Methods
Organ
Differential Centrifugation
Purified Mitochondria
Homogenize

• 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

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RNR Assay
Solvent front
dCDP
CDP
Scintillation counter
Thin Layer Chromatography

• 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

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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

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Inhibition of Mitochondrial RNR

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

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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

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Acknowledgements

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