Fig. 2.

Replication of mtDNA by asynchronous strand-displacement synthesis. Initiation of replication occurs through synthesis of an RNA primer from LSP that forms a G-quadruplex with non-template DNA and terminates at CSB2. The replicative mitochondrial DNA polymerase γ (Pol γ) begins DNA synthesis from this primer around O_H, with helicase Twinkle unwinding upstream DNA. The parental L-strand acts as the template for synthesis, with the displaced H-strand being temporarily coated in mitochondrial single-stranded binding protein (mtSSB). Once Twinkle reveals O_L, a stem-loop forms in the ssDNA of the parental H-strand, allowing synthesis of a short RNA primer by POLRMT and subsequent synthesis of the daughter L-strand using the displaced parental H-strand as a template. DNA synthesis proceeds until two complete, hemicatenated mtDNA molecules are produced. RNA primers are removed in a two-nuclease pathway involving RNase H1 and flap endonuclease 1 (FEN1) or FEN1-like activity (not shown), and hemicatenanes are resolved by mitochondrial topoisomerase 3α (Top3α)