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.