RESUMO

Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.

Assuntos

DNA Helicases/química , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Mutação , Sítios de Ligação , DNA Helicases/genética , Replicação do DNA , DNA Mitocondrial/química , DNA Mitocondrial/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Humanos , Proteínas Mitocondriais/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica