MUS81 is the catalytic subunit of two structure-specific DNA endonucleases—MUS81-EME1 and MUS81-EME2—that maintain genome stability 12. Both complexes cleave 3'-flaps and nicked Holliday junctions with similar substrate specificity, though MUS81-EME2 exhibits greater activity 13. MUS81-EME2 functions during DNA replication to process stalled replication forks by generating double-strand breaks that can be repaired via homologous recombination, enabling fork restart when obstacles like DNA lesions or interstrand cross-links impede progression 4. MUS81-EME1 operates later in the cell cycle, resolving mitotic recombination intermediates including Holliday junctions formed during homologous recombination 4. MUS81 also participates in resolving R-loop-mediated transcription-replication conflicts through fork cleavage, facilitating replication restart 5. Clinically, MUS81 function varies contextually: in microsatellite-unstable cancers, MUS81 cleaves expanded TA repeats when the WRN helicase is depleted, causing chromosome 11 6. Additionally, MUS81-EME1 functions within the SMX nuclease complex as a backup pathway when the BTR complex cannot resolve DNA intermediates, suggesting therapeutic potential in certain cancer contexts 78.