The Active DNA-PK Holoenzyme Occupies a Tensed State in a Staggered Synaptic Complex

In the non-homologous end joining (NHEJ) of a DNA double strand break, DNA ends are bound and protected by a large heterotrimeric DNA-PK complex, which synapses across the break to tether the broken ends and initiate the repair program. There is a lack of clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5A (using crosslinking data and available structures), revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry suggests an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, in which the synaptic complex facilitates hierarchical processing of the ends and the assembly of a scaffold, supporting displacement of the catalytic subunit and tension release through kinase activity.