Abstract
Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is largely inactivated in mitosis. Instead, mitotic cells rely on alternative repair processes such as microhomology-mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). How these mitotic DNA repair pathways are functionally regulated remains unclear. Here we reveal that the CIP2A-TOPBP1 complex plays an essential regulatory role by facilitating the mitotic recruitment of both SMX complex components and Polθ to mitotic chromatin. Recruitment of the SMX complex components is driven by CDK1-dependent phosphorylation of SLX4 at Thr1260, enabling its interaction with TOPBP1 BRCT domains 1/2, thereby promoting MiDAS. Concurrently, CIP2A promotes efficient mitotic localisation of Polθ to facilitate MMEJ. The simultaneous functional disruption of both MiDAS and MMEJ pathways upon CIP2A loss provides rationale for the synthetic lethality observed in BRCA1 or 2-deficient cells. These findings position the CIP2A-TOPBP1 axis as a central regulatory hub for mitotic DNA repair, highlighting therapeutic opportunities in tumours characterised by HR deficiency or elevated replication stress.