How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3alpha (Top3alpha) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3alpha-RMI1-RMI2 (BTR) complex. Our data indicate that mtDNA replication termination occurs via a hemicatenane formed at the origin of H-strand replication and that Top3alpha is essential for resolving this structure. Decatenation is a prerequisite for separation of the segregating unit of mtDNA, the nucleoid, within the mitochondrial network. The importance of this process is highlighted in a patient with mitochondrial disease caused by biallelic pathogenic variants in TOP3A, characterized by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3alpha as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.
