Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the MUS81-EME1 endonuclease.

The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.