RHINO directs MMEJ to repair DNA breaks in mitosis.

Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, while microhomology-mediated end-joining (MMEJ) has been regarded as a backup mechanism. Through CRISPR/Cas9-based synthetic lethal screens, we identify subunits of the 9-1-1 complex (RAD9A-HUS1-RAD1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncover an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes PLK1 phosphorylation, and interacts with polymerase theta (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs originating in S phase. Our findings offer insights into the synthetic lethal relationship between POLQ and BRCA1/2 and the synergistic effect of Polθ and PARP inhibitors.