How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3α (Top3α) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3α-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 Top3α 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 Top3α as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.

如何终止 mtDNA 复制以及如何分离新形成的基因组仍然未知。我们在这里证明了拓扑异构酶 3α (Top3α) 的线粒体同种型实现了这一功能，独立于其作为 Holliday 结解析 BLM-Top3α-RMI1-RMI2 (BTR) 复合物的组成部分的核作用。我们的数据表明，mtDNA 复制终止是通过在 H 链复制起点形成的半链烷发生的，而 Top3α 对于解析这种结构是必不可少的。分离是线粒体网络内分离 mtDNA 的分离单元，即类核的先决条件。该过程的重要性在由TOP3A中双等位基因致病变异引起的线粒体疾病患者中得到强调，以肌肉受限的 mtDNA 缺失和慢性进行性眼外肌麻痹 (CPEO) 加综合征为特征。我们的工作将 Top3α 确立为 mtDNA 复制机制的重要组成部分和 mtDNA 分离机制的第一个组成部分。