TOP1MT (DNA topoisomerase I mitochondrial) is a type IB topoisomerase that releases supercoiling and torsional tension in mitochondrial DNA during replication, transcription, and repair 1. The enzyme transiently cleaves one DNA strand via a catalytic tyrosine residue, forming a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate, allowing the intact strand to rotate and remove supercoils before religation restores the DNA backbone 2. TOP1MT is the sole known mitochondrial-specific topoisomerase in vertebrates, encoded by a nuclear gene and targeted to mitochondria via an N-terminal localization signal 3. Beyond mtDNA integrity maintenance, TOP1MT supports mitochondrial protein synthesis critical for oxidative phosphorylation and tumor growth, with impaired synthesis triggering the ATF4-mediated mitonuclear stress response 4. Aberrant topoisomerase activity generates topoisomerase cleavage complexes and DNA-protein crosslinks (TOP-DPCs), which are repaired by tyrosyl-DNA phosphodiesterases (TDP1) and nucleases, with defects contributing to genomic instability 5. TOP1MT dysregulation associates with multiple cancers and mitochondrial diseases, making it a potential chemotherapy target 2. TOP1MT activity requires divalent cations (Ca²⁺/Mg²⁺) and alkaline pH 1.