DNA polymerase Lambda is anchored within the NHEJ synaptic complex via Ku70/80

Non-homologous end joining (NHEJ) is the predominant pathway by which double-strand DNA breaks (DSBs) are repaired in mammals. To enable final break closure, various NHEJ end-processing factors respond to the chemistry of the damaged DNA ends. Amongst these factors is DNA polymerase lambda (Pol λ), a member of the Pol X family. How members of the Pol X family engage with the NHEJ complex is unknown. Here, we present cryo-EM structures of Pol λ in complex with the Ku70/80 DSB sensor whilst engaged with the DNA-PK holoenzyme in a long-range synaptic complex. These structures reveal a specific interaction site between Ku70/80 and the Pol λ BRCT domain. The functionality of this interaction is assessed by generating point mutations on either side of the Pol λ BRCT:Ku70/80 interface. Using these mutants in two orthogonal assays in cells (live protein recruitment at biphoton laser-damaged nuclear sites and transfection with an original gap-filling reporter plasmid) defines the molecular basis and essentiality of the BRCT domain for the recruitment and activity of the Pol λ within the NHEJ complex. Ultimately, these data explain the role of this interaction in cell survival to DSBs. Finally, we propose a unified model for the interaction of the three Pol X family members bearing BRCT domains with the same site of Ku70/80.