Dual-site phosphorylation of SLX4 stabilizes the SLX4–MUS81 interface to promote mitotic SMX assembly and genome protection

Faithful genome transmission depends on the timely removal of branched DNA intermediates. This task is executed by the SMX tri-nuclease, a mitosis-specific complex containing three structure-selective endonucleases (SLX1, MUS81-EME1, and XPF-ERCC1) bound to the SLX4 scaffold. A critical step in SMX assembly is the recruitment of MUS81-EME1 to SLX4 in early mitosis, a process tightly regulated by cell-cycle kinases. Mechanistically, CDK1-dependent phosphorylation of SLX4 promotes folding of its SAP domain, which strengthens the SLX4-MUS81 interaction. Here, we define a site-specific phosphorylation code that stabilizes complementary structures in the SLX4-MUS81 interface. Phosphorylation of SLX4 T1571 is required for partial SAP domain folding, pre-organizing the MUS81 binding surface and reducing the entropic penalty of folding upon binding. Co-phosphorylation at T1561 enhances structural stability and promotes intermolecular β-sheet formation with MUS81, providing enthalpic stabilization. Dual-site phosphorylation converts a relatively weak interaction into a high-affinity complex that stimulates MUS81-EME1 nuclease activity. In human cells, both phosphorylation sites are required for robust SMX assembly and genome stability maintenance. Collectively, our findings reveal the structural basis for how CDK1-cyclin B controls SMX assembly in mitosis. More broadly, the SLX4-MUS81 complex illustrates how phosphorylation fine-tunes marginally stable protein interfaces at the boundary of order and disorder.