Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date nearly 250 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Mitochondrial DNA replication is assisted by the accessory subunit, encoded by the POLG2 gene. Heterozygous mutations in POLG2 are responsible for several mitochondrial diseases, including progressive external ophthalmoplegia (PEO). We have previously analyzed four such POLG2 mutations with homodimeric preparations of purified recombinant p55 variants and documented their biochemical defects in vitro. However, because the affinity between monomers within the p55 dimer is extremely high (estimated intermolecular Kd of < 0.1 nM) and these mutations occur in heterozygous states, affected individuals would harbor mixtures of variant and wild-type molecules. We expect p55 to occur as 50% heterodimers, 25% wild-type homodimers, and 25% variant homodimers. We report the development of a tandem affinity strategy to isolate p55 heterodimers. The WT/G451E p55 heterodimer impairs polγ function in vitro, demonstrating that the POLG2 c.1352G>A/p.G451E mutation encodes a dominant negative protein. To analyze the subcellular consequence of disease mutations in HEK293 cells, we designed plasmids encoding p55 disease variants tagged with green fluorescent protein (GFP). P205R and L475DfsX2 p55 variants exhibit irregular diffuse mitochondrial fluorescence and unlikeWT p55, they fail to form distinct puncta associated with mtDNA nucleoids. Furthermore, homogenous preparations of P205R and L475DfsX2 p55 form aberrant reducible multimers.We predict that abnormal protein folding or aggregation or both contribute to the pathophysiology of these disorders. Examination of mitochondrial bioenergetics in stable cell lines overexpressing GFP-tagged p55 variants revealed impaired mitochondrial reserve capacity. The T251I mutation in POLG is one of the most common mitochondrial disease-associated mutations found in patients with disorders including progressive external ophthalmoplegia (PEO), as well as Alpers and infantile hepatocerebral syndromes associated with mtDNA depletion. Interestingly, T251I is always found in cis with P587L. To date, it is not understood how these mutations cause disease. To address this we biochemically expressed and purified the WT PolG, T251I, P587L and the T252I/P587L double variants. The WT and T251I enzymes have been assessed for steady state kinetics, exonuclease activity, salt stability and processivity versus NaCl concentration. Preliminary studies reveal a novel activity in the T251I PolG that renders it highly processive and salt tolerant in the absence of the accessory subunit. This mutation may rescue some of the deleterious effects of the P587L mutation and point to a possible mode of therapy. Future studies will extend our analysis to the P587L and T251I/P587L double variant in order to determine the pathological mutation and mechanism leading to disease.
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