In vitro reconstitution of DNA unwinding during replication in human mitochondria
Mitochondrial DNA (mtDNA) plays an important role in mitochondria and its maintenance is critical for sustaining aerobic respiration in cells. However, the details of mtDNA replication mechanism in human mitochondria are still unclear. Three existing models of this process have been put forward to date, which are strand-displacement model, bootlace model and strand-coupled DNA replication model but several contradictions exist among these models. To study the mechanism of mtDNA replication in humans, two proteins, TWINKLE helicase and mitochondrial single-stranded DNA binding protein (mtSSB), that participate in this process were purified and their activity was tested using fluorescein amidite (FAM) labeled DNA and radiolabeled DNA. With these two proteins, part of the mtDNA replication system was rebuilt in vitro. It is found that mtSSB promotes the activity of TWINKLE helicase by protein-protein interaction instead of binding to unwound DNA as claimed in the strand-displacement model. Based on size exclusion chromatography and SDS-PAGE analysis, it is suggested that mtSSB and TWINKLE helicase form a complex during the unwinding process. This experiment builds a platform for further structural study of the mtDNA replication mechanism and protein-protein interaction between mtSSB and TWINKLE helicase.