A PLK1-Directed Mitotic Genome Maintenance Network Promotes DNA Synthesis in Mitosis

Polo-like kinase 1 (PLK1) is a master regulator of mitosis and is known to dictate DNA repair pathway choice at this stage of the cell cycle. However, its roles in controlling mitotic DNA damage responses remain incompletely characterised. Here, we used acute PLK1 inhibition as a substrate-trapping strategy to stabilise PLK1–target interactions in mitotic cells and identify PLK1-associated DNA repair factors. Proteomic analysis of endogenous HA-tagged PLK1 complexes revealed interactions with multiple genome stability proteins, including SLX4, RAD52, FANCM, and REV1. We demonstrate that PLK1 binds SLX4 and RAD52 via canonical CDK1-dependent phospho-docking motifs centred on SLX4 Ser1453 and RAD52 Thr300. Mutation of these residues abolished PLK1 binding and, at least for RAD52, prevented PLK1-dependent phosphorylation of mitotic targets. Functional analyses showed that PLK1 docking to SLX4 is dispensable for interstrand crosslink repair but essential for mitotic DNA synthesis (MiDAS), defining a separation-of-function allele. Likewise, disruption of PLK1 docking to RAD52 impaired MiDAS. Together, these findings identify PLK1 as a key coordinator of mitotic genome maintenance pathways required for MiDAS.