Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammal ian DNA double-strand break repair and immunoglobulin gene rearrangement. T he DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/8 0 kDa heterodimeric DNA-targeting component. We have solved the three-dimen sional structure of DNA-PKcs to similar to 21 Angstrom resolution by analyt ically combining images of nearly 9500 individual particles extracted from cryoelectron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold s ymmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both th e crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs sugge sts that its association with DNA may involve the internalization of double -stranded ends.