RESUMO
The replicative mitochondrial DNA (mtDNA) helicase is essential for mtDNA replication and maintenance of the mitochondrial genome. Despite substantial advances that have been made in its characterization, there is still much to be understood about the functional roles of its domains and its interactions with the other components of the minimal mitochondrial DNA replisome. Critical to achieving this is the ability to isolate the enzyme in a stable, active form. In this chapter we describe a modified, streamlined purification strategy for recombinant forms of the enzyme. We also present assays to assess its helix unwinding activity and the stimulatory effects of the mitochondrial single-stranded DNA-binding protein (mtSSB). Finally, we describe a concentration/buffer exchange method that we have employed to achieve greater enzyme stability and appropriate conditions for biochemical and biophysical studies.
Assuntos
DNA Helicases/genética , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Animais , Linhagem Celular , DNA Primase/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Mitocondriais/metabolismo , Células Sf9RESUMO
The mitochondrial single-stranded DNA-binding protein (mtSSB) coordinates the function of replisome components at the mitochondrial replication fork. In recent years, it has been demonstrated that mtSSB stimulates the activities of DNA polymerase γ (Pol γ) and mitochondrial DNA (mtDNA) helicase in a concentration-dependent manner. Here we present a new approach to purify the human mtSSB and our standard assays to evaluate its biochemical properties, including a Gel Mobility Shift Assay (GMSA) to assess single-stranded DNA (ssDNA) binding activity, and an assay to assess SSB stimulation of Pol γ activity.
Assuntos
Replicação do DNA/genética , DNA Mitocondrial/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Mitocondriais/genética , DNA Helicases/metabolismo , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems.