Human DNA-PKcs promotes broken DNA-end structure independence during NHEJ

Non-homologous end joining (NHEJ), which is the predominant DNA double-strand break repair pathway in vertebrates, has been studied extensively, but the role of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is controversial. From genetic and cell-based experiments, DNA-PKcs is presumed to be a critical factor in DNA-end processing, and from structural experiments, DNA-PKcs facilitates DNA-end synapsis. However, these experiments are at odds with biochemical experiments that unequivocally show that DNA-PKcs inhibits NHEJ reactions. Thus, the precise function of DNA-PKcs is currently ambiguous. Here, we revisit this discrepancy using NHEJ reconstitution experiments. Under optimized conditions, we find that DNA-PKcs has a significant role in joining both cohesive and blunt ends in an ATP-dependent manner. Of note, the end joining efficiency of both cohesive vs. blunt ends is indistinguishable. These data suggest that DNA-PKcs promotes a unique productive synaptic complex that is driven in a manner independent of DNA-end structure.