Activation of Pol theta-mediated end joining begins during mitotic commitment.

Polymerase theta-mediated end-joining (TMEJ) is a salvage DNA repair pathway that anneals and ligates microhomologies flanking a DNA double-strand break (DSB) downstream of aborted homologous recombination to ensure DSBs are resolved before chromosome segregation. Here, we use a sensitive deep-sequencing approach to identify DSB repair outcomes at an endogenous locus. We observe that TMEJ is impaired by depletion or inhibition of DNA2, a component of the DSB resection machinery. Mechanistically we find that DNA2 deficiency influences TMEJ efficiency by slowing S-phase progression, thereby preventing cells from reaching mitotic commitment at the G2/M transition, when we find that TMEJ is optimally licensed to occur. Further interrogation of the cell cycle regulation of TMEJ reinforces a strict requirement for the mitotic kinase PLK1 in enabling TMEJ at the G2/M transition. We surprisingly find CDK1 to be largely dispensable for TMEJ, but important for a small subset of repair events likely occurring in mitosis. These findings have important implications for modulation of TMEJ in response to cell cycle kinase inhibitors that are in active development as anti-tumor agents.