Comparative Purification Strategies for Drosophila and Human Mitochondrial DNA Replication Proteins: DNA Polymerase γ and Mitochondrial Single-Stranded DNA-Binding Protein

Abstract

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 both Drosophila melanogaster and 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.

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

“…Proteins-Recombinant human and D. melangaster mtSSB proteins were prepared from bacterial cells as described previously (20), except that the glycerol gradient centrifugation step was replaced by gel filtration as follows. Fraction IIb was chromatographed on a Superdex 75 gel filtration column equilibrated with buffer containing 50 mM Tris-HCl, pH 7.5, 8% glycerol, 150 mM KCl, 2 mM EDTA at a flow rate of 1 ml/min at 4°C.…”

“…Fractions containing the mtSSBs were pooled (fraction III) and dialyzed against buffer containing 35 mM Tris-HCl, pH 7.5, 8% glycerol, 100 mM NaCl, 2 mM EDTA, 2 mM dithiothreitol. Recombinant catalytic subunit of human Pol ␥ (Pol ␥␣) and Drosophila Pol ␥ holoenzyme were prepared from Sf9 cells as described previously (20). Recombinant accessory subunit of human Pol ␥ (Pol ␥␤) was prepared from bacterial cells as described previously (20).…”

“…Recombinant catalytic subunit of human Pol ␥ (Pol ␥␣) and Drosophila Pol ␥ holoenzyme were prepared from Sf9 cells as described previously (20). Recombinant accessory subunit of human Pol ␥ (Pol ␥␤) was prepared from bacterial cells as described previously (20). Recombinant E. coli SSB protein was purchased from Affymetrix.…”

Purification and Comparative Assay of Human Mitochondrial Single-Stranded DNA-Binding Protein

“…In addition, mtSSB has been shown to coordinate the functions and stimulate the activities of Pol γ and mtDNA helicase [3–8]. mtSSBs are 13–16 kDa proteins that function as homotetramers [3, 9]. In vivo , depletion of mtSSB protein causes a reduction of mtDNA copy number in cultured cells [10].…”

“…Our group has developed and published protocols to overexpress efficiently mtSSB variants in E. coli cell cultures, and to purify the target protein to near-homogeneity, which has enabled the study of various recombinant mtSSB variants of human and fruit fly origin [3–5, 7, 9, 12]. Here, we present an updated purification scheme to obtain near-homogenous recombinant human mtSSB in two key steps: affinity chromatography using Blue Sepharose and either a cation exchange chromatography using phosphocellulose, and/or sedimentation in a glycerol gradient (Fig.…”