Polθ is phosphorylated by Polo-like kinase 1 (PLK1) to enable repair of DNA double strand breaks in mitosis

DNA double strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle 1 . In interphase, DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) 2 . However, these pathways are completely inhibited in mitosis 3–5 , leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta (Polθ) 6 repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Polθ function is activated in mitosis upon phosphorylation by the Polo-like kinase 1 (PLK1). Phosphorylated Polθ is recruited to mitotic DSBs, where it mediates joining of broken DNA ends, while halting mitotic progression. The lack of Polθ leads to a shortening of mitotic duration and defective repair of mitotic DSBs, resulting in a loss of genome integrity. In addition, we identify mitotic Polθ repair as the underlying cause of the synthetic lethality between Polθ and HR. Our findings reveal the critical importance of mitotic DSB repair for maintaining genome stability.