Mitochondrial DNA replication follows a unidirectional process from two distinct origins: the origin of H-strand replication (OH) in the D-loop and the origin of L-strand replication (OL) downstream of OH. OH initiates synthesis of a daughter H-strand, displacing the parental H-strand. When the leading daughter H-strand passes OL, synthesis of the daughter L-strand begins. RNA primer synthesis is facilitated by transcription factors and proteins including TFAM,POLRMT, and TFB2M. DNA polymerase γ catalyzes strand synthesis, while Twinkle helicase unwinds DNA and TOP1MT relaxes supercoiled DNA. SSBP1 stabilizes single-stranded regions. RNA primers are removed by RNase H1, and MGME1 processes the 5′-end. After gap filling and ligation by DNA ligase III, daughter duplexes are linked at OH as hemicatenanes. TOP3A catalyzes decatenation, allowing daughter molecules to separate.