The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for ATP production. Disruption of mitochondrial energy metabolism can occur by genetic and biochemical mechanisms involving nuclear-encoded proteins that are required at the mitochondrial DNA replication fork, which often leads to human disorders and to animal lethality during development. DNA polymerase γ (pol γ), the mitochondrial replicase, and the mitochondrial single-stranded DNA-binding protein (mtSSB) have been the focus of study in our lab for a number of years. Here we describe the purification strategies that we developed for obtaining the recombinant forms of pol γ and mtSSB from bothDrosophila melanogasterand humans. Despite the fact that similar approaches can be used for purifying the homologous proteins, we have observed that there are differences in the behavior of the proteins in some specific steps that may reflect differences in their structural and biochemical properties. Their purification in homogeneous, active form represents the first step toward our long-term goal to understand their biochemistry, biology, and functions at the mitochondrial DNA replication fork.
