dNTP Imbalance in Mitochondria
Alexandra Frolova
Dr. Christopher K. Mathews Laboratory
Biochemistry and Biophysics
 Rates of mutation in mtDNA are 10-100 fold higher than in
nDNA.
 mtDNA mutations are linked to various human diseases:
 Cancer
 Cardiomyopathies
 Degenerative heart, muscle, & neurological disorders
 mtDNA mutations lead to accelerated aging in mice.
An imbalance in mtDNA precursor pools
can cause mutations.
Mathews’ lab has determined that there exists a high
concentrations of dGTP relative to the other dNTPs in mtDNA
pools in various mammalian tissues.
Song, S., Z F. Pursell, W.C. Copeland, M.J. Longley, T.A.
Kunkel, and C.K. Mathews (2005) DNA Precursor
Asymmetries in Mammalian Tissue Mitochondria and
Possible Contributions to Mutagenesis via Reduced
Replication Fidelity. Proc. Natl. Acad. Sci. USA 102,
4990-4995.
dNTP pmol per mg protein
dNTP imbalance in rat heart
mitochondria
c
m
dATP
c
m
dTTP
c
m
dCTP
c
m
dGTP
Excess of one dNTP can cause misinsertion and/or inhibited
proofreading, which can lead to substitution mutations.
Reactive oxygen species (ROS) can oxidize dGTP to
form mutagenic 8-oxo-dGTP.
Various polymerases will wrongly insert 8-oxo-dGTP opposite
template A leading to A-T to C-G transversions.
Pursell, Z.F., J.T. MacDonald, C.K. Mathews, and T.A. Kunkel
(2008) Trace Amounts of 8-oxo-dGTP in Mitochondrial
Pools Reduce DNA Polymerase γ Replication Fidelity.
Nucl. Ac. Res. 36, 2174-2181.
Transport and metabolic pathways of
nucleosides and nucleotides
Cy tosol
Mitochondria
dNTPs
dNTPs
dNMPs
dNMPs
NdRs
NdRs
rNDPs
rNDPs
dNDPs
dNMPs
dNDPs
dNTPs
dNDPs dNTPs
dNTPs
Project purpose
 To understand how various intramitochondrial enzymes
participate in dNTP pool regulation.
 To determine which enzyme(s) cause dGTP
accumulation.
Enzymatic pathways of
dGTP synthesis and
turnover
DNA
DNA polymerase
dGTP
NDP kinase
GDP
RNR
dGDP
dGMP kinase
MTH-1
dGMP
dGK
dGuo
dNT-2
Enzymatic pathways that may influence
dGTP levels
 NDP Kinase
dGTP + ADP
dGDP + ATP
 dGMP Kinase
dGMP + ATP
dGDP + ADP
 NT2 mitochondrial 5’-nucleotidase
dGMP +H20
Deoxyguanosine + Pi
 MTH-1 (mutT homolog)
dGTP + H2O
dGMP + PPi
Experiment layout
 Incubate mixtures of the four dNTPs with mitochondria
extract and various substrates to:
 monitor enzyme activity
 determine which enzymatic steps are critical for
maintaining dNTP pool stability.
Experiment 1: Hydrolytic dNTP breakdown
 dNTP + H2O → dNDP (→ dNMP) +Pi
Experiment 2: NDP Kinase
 dNTP + ADP → dNDP (→dNMP) + ATP
Experiment 3: dNMP Kinase
 dNMP + ATP → dNDP + ADP
Methods of mitochondrial preparation
 Isolate rat liver mitochondria using homogenization and
differential centrifugation.
 Prepare mitochondria extract by using sonication.
 Add detergent η-dodecyl-β-maltoside.
 Centrifuge 15K for 30 mins.
Methods of sample analysis
 Samples analyzed using High Performance Liquid
Chromatography (HPLC) .
 Column used was a C-18 Reverse Phase Column.
 Sample components separated using linear gradient.
 Buffer A: 8mM TBA-OH, 10mM monobasic K phosphate,
0.25% methanol, pH 7.0
 Buffer B: 2mM TBA-OH, 100mM monobasic K
phosphate, 30% methanol, pH 7.0
Example of chromatogram
dATP
ATP
dGTP
dTTP
dCTP
ADP
AMP
dAMP
dCMP
dGMP
dTMP
11 Standards
dNMPs, dNTPs, ANPs
Raw data
hydrolytic enzyme
dNTP + H2O → dNDP (→ dNMP) +Pi
Peak Area vs. Incubation Time
2500000
Peak Area
2000000
dCTP
1500000
dGTP
dTTP
1000000
dATP
500000
0
0
10
20
Incubation time (minutes)
30
40
Raw data
NDP kinase
dNTP + ADP → dNDP (→dNMP) + ATP
Peak Area vs. Incubation Time
800000
700000
Peak Area
600000
dCTP
500000
dGTP
400000
dTTP
300000
dATP
200000
100000
0
0
10
20
Incubation time (minutes)
30
40
Data
dNMP kinase
dNMP + ATP → dNDP + ADP
Progress
 Determined:
- concentrations of substrates and reactants,
- incubation times,
- HPLC elution program
necessary for detecting activity of hydrolytic
enzyme and NDPK.
 Made progress towards:
- creating a functional method for examining
dNMPK activity using HPLC.
- acquiring publishable data.
Future objectives
 dNTP + ADP
 dGMP + ATP
 dNMP
dNDP + ATP
dGDP + ADP
deoxyribonucleosides
 Deoxyribonucleosides + ATP
dNTPs + ADP
 Dr. Mathews Presentation from 6/21/09
 HHMI
Thank you…
 Kevin Ahern
 Dr. Christopher Mathews
 Linda Benson and Korakod Chimploy