BTBD12 functions as a critical scaffold element for the assembly of a multiprotein complex containing enzymes involved in DNA maintenance and repair.Similar complexes were detected in other human cell lines examined. Domain analysis showed that MSH2-MSH3, ERCC4-ERCC1, and C20ORF94 associated with the N-terminal region of SLX4, while TERF2-TERF2IP, MUS81-EME1, and PLK1 bound the C-terminal region of SLX4. SLX1 associated with the extreme C terminus of SLX4. The SLX4 complex was recruited to sites of DNA damage and repaired 3-prime flap, 5-prime flap, and replication fork structures, consistent with the activities of the SLX4-associated DNA repair factors. The SLX1-SLX4 module specifically promoted symmetrical cleavage of static and migrating Holliday junctions.