Structure of DNA‐dependent protein kinase: implications for its regulation by DNA

DNA double‐strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end‐joining reaction that requires DNA‐PKCS, the catalytic subunit of DNA‐dependent protein kinase. DNA‐PKCS is a 460 kDa serine‐threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 Å resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double‐stranded DNA‐binding proteins, and an enclosed cavity with three openings large enough to accommodate single‐stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA‐PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA‐PKCS binds to double‐ and single‐stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double‐stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double‐ and single‐stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described.