DNTP Imbalance in Mitochondria Alexandra Frolova Dr. Christopher K. Mathews Laboratory Biochemistry...
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Transcript of DNTP Imbalance in Mitochondria Alexandra Frolova Dr. Christopher K. Mathews Laboratory Biochemistry...
dNTP Imbalance in MitochondriadNTP 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: CancerCardiomyopathiesDegenerative heart, muscle, & neurological disorders
mtDNA mutations lead to accelerated aging in mice.
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.
An imbalance in mtDNA precursor
pools can cause mutations.
c m c m c m c m
dATP dTTP dCTP dGTP
dNT
P p
mol
per
mg
prot
ein
dNTP imbalance in rat heart mitochondria
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.
Cytosol Mitochondria
dNTPs dNTPs
dNMPs dNMPs dNDPs dNTPs
NdRs NdRs dNMPs dNDPs dNTPs
rNDPs rNDPs dNDPs dNTPs
Transport and metabolic pathways of
nucleosides and nucleotides
Project purposeTo understand how various intramitochondrial
enzymes participate in dNTP pool regulation.
To determine which enzyme(s) cause dGTP accumulation.
DNA
DNA polymerase
dGTP
NDP kinase
GDP RNR dGDP
dGMP kinase MTH-1
dGMP
dGK dNT-2
dGuo
Enzymatic pathways of dGTP synthesis
and turnover
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 KinasedNTP + ADP → dNDP (→dNMP) + ATP
Experiment 3: dNMP KinasedNMP + ATP → dNDP + ADP
Methods of mitochondrial preparationMethods 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 analysisSamples 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 chromatogram11 Standards
dNMPs, dNTPs, ANPs
dCM
P
dTM
P
dGM
P AM
P
dAM
P
dCT
P
AD
P
dGT
P
dTT
P
AT
P
dAT
P
Raw data hydrolytic enzyme
dNTP + H2O → dNDP (→ dNMP) +Pi
Raw data NDP kinase
dNTP + ADP → dNDP (→dNMP) + ATP
DatadNMP kinase
dNMP + ATP → dNDP + ADP
ProgressDetermined:
- 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 dNDP + ATP
dGMP + ATP dGDP + ADP
dNMP deoxyribonucleosides
Deoxyribonucleosides + ATP dNTPs + ADP
Dr. Mathews Presentation from 6/21/09
Thank you…Thank you…HHMI
Kevin Ahern
Dr. Christopher Mathews
Linda Benson and Korakod Chimploy