Abstract

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-PK_CS, the catalytic subunit of DNA-dependent protein kinase. DNA-PK_CS 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-PK_CS 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-PK_CS 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.