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