DNA polymerase λ autoinhibition is relieved via Ku interaction during non-homologous end joining.

DNA ends are frequently damaged during the formation of DNA double-strand breaks (DSBs). These ends must be repaired to enable ligation during non-homologous end joining (NHEJ). NHEJ uses several end processing factors to repair DNA ends within the short-range synaptic complex (SRC), including Polymerase λ (Pol λ) which performs gap fill-in. Pol λ possesses a Ku Binding Motif (KBM) within its BRCT domain that interacts with Ku and recruits it to the SRC. Here, we show that in addition to its role in recruitment, Ku also stimulates Pol λ polymerase activity at DSBs. Using a structural prediction approach and biochemical assays, we identify and characterize an autoinhibitory intramolecular interaction between the N-terminal BRCT and C-terminal catalytic domains of Pol λ. Furthermore, single-molecule approaches reveal that Ku increases both the binding rate of Pol λ to primer-template DNA and the rate of nucleotide incorporation, demonstrating that Ku releases Pol λ autoinhibition and stimulates its polymerase activity within the SRC during NHEJ. Combined, these data highlight how intricate protein-protein interactions within the SRC complex are critical to regulate end-processing and maximize the fidelity of DSB repair.