Topoisomerase 3a is required for decatenation and segregation of human mitochondrial DNA

How mitochondrial DNA (mtDNA) replication is terminated, and the newly formed genomes are separated, remains unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3a (Top3a) has been repurposed from the nuclear Holliday junction-dissolving BTR complex to fulfil this function. At the end of mtDNA replication a hemicatenane is formed at the origin of H-strand replication and Top3a 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, characterised by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3a as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.