Purification and functional characterization of human mitochondrial DNA polymerase gamma harboring disease mutations

More than 150 different point mutations in POLG, the gene encoding the human mitochondrial DNA polymerase γ (pol γ), cause a broad spectrum of childhood and adult onset diseases like Alpers syndrome, ataxia-neuropathy syndrome and progressive external ophthalmoplegia. These disease mutations can affect the pol γ enzyme’s properties in numerous ways, thus potentially influencing the severity of the disease. Hence, a detailed characterization of disease mutants will greatly assist researchers and clinicians to develop a clear understanding of the functional defects caused by these mutant enzymes. Experimental approaches for characterizing the wild-type (WT) and mutant pol γ enzymes are extensively described in this manuscript. The methods start with construction and purification of the recombinant wild-type and mutant forms of pol γ protein, followed by assays to determine its structural integrity and thermal stability. Next, the biochemical characterization of these enzymes is described in detail, which includes measuring the purified enzyme’s catalytic activity, its steady-state kinetic parameters and DNA binding activity, and determining the physical and functional interaction of these pol γ proteins with the p55 accessory subunit.