STIR: Novel Role of PrimPol in Mitochondrial Genome Maintenance (Research Area 8.2 Genetics)

  • Linlin, Zhao (PI)

Grant Details

Description

Extreme conditions in the battlefield such as UV radiation, heat and fatigue can cause the human body to generate an elevated amount of reactive oxygen species (ROS). ROS are toxic substances that can damage the cellular genetic material deoxyribonucleic acid (DNA) as well as its building blocks, deoxyribonucleotide triphosphate (dNTPs). Damage to both DNA and dNTPs are known to cause genomic instability, which is a hallmark of human cancer and neurodegeneration. Mitochondria are subcellular compartments that are particular relevant to ROS induced damage, because approximately 90% of cellular ROS are formed in mitochondria as a part of the normal cellular energy metabolism. Mitochondria have their own DNA that encodes important protein components for energy metabolism. Mitochondrial DNA damage can lead to mitochondrial dysfunctions, which have been associated with more than 200 named disordered and common pathological conditions, such as aging, neurological disorders, diabetes mellitus and cardiovascular disease. Currently there is no effective cure for mitochondrial diseases partly due to the poor understanding the disease pathogenesis. This STIR project seeks to understand how damaged dNTPs affect mitochondrial genomic integrity, a poorly understood subject in the mitochondrial field. Damaged dNTPs are known to cause increased mutagenesis in nucleus because they can be misincorporated into DNA when DNA copies are made. Specifically, this research focuses on a recently discovered mitochondrial DNA polymerase (enzyme that copies DNA), PrimPol, and seeks to understand the contribution of PrimPol to mitochondrial genomic instability via potential misincorporation of damaged dNTPs. A commonly oxidized dNTP is chosen for the current investigation, i.e. 8-oxo-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP). Research in Aim 1 focuses on defining the capability of PrimPol in selecting undamaged nucleotides from damaged nucleotides using enzyme kinetic analysis. Results from Aim 1 demonstrate that 8-oxo-dGMP can be incorporated into DNA by PrimPol by pairing with either template C or template A. The latter is a mutagenic mispair, which is considered as the molecular basis of 8-oxo-dGTPÐinduced A to C mutations. These results suggest that in the presence of damaged nucleotides PrimPol can contribute to the mitochondrial mutagenesis when involved in mitochondrial DNA replication. The Aim 2 of the proposal seeks to determine the threshold concentration of 8-oxo-dGTP that can alter the DNA replication accuracy of PrimPol. To this end, a powerful mass spectrometry-based DNA sequencing method has been developed to analyze the PrimPol synthesized DNA products containing misincorporated oxidized nucleotides. Damaged DNA products are detected when as little as 1% of dGTP is oxidized to 8-oxo-dGTP, a physiological relevant concentration of 8-oxo-dGTP. Together, results from both aims suggest that oxidized nucleotide damage pose a threat to mitochondrial genomic integrity. Thus, considering the important role of PrimPol in mitochondrial DNA maintenance previously revealed by the PrimPol-deficient mouse model, PrimPol potentially contributes to the damaged nucleotide incorporation in the presence of physiological concentrations of oxidized nucleotides. Results from the current research are fundamental to the understanding of mitochondrial genomic instability, which can inform the development of more effective therapeutic strategies for mitochondrial disease. Overall, the outcome of this project brings our understanding of the mitochondrial pathogenesis to a higher level, and can inform the optimization of SoldiersÕ performance and protection.

StatusFinished
Effective start/end date04/8/1501/7/16

Funding

  • U.S. Army: $50,000.00

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