Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies

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Abstract
DNA‐dependent protein kinase (DNA‐PK or the scid factor) and Ku are critical for DNA end‐joining in V(D)J recombination and in general non‐homologous double‐strand break repair. One model for the function of DNA‐PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA‐binding subunit. We find that DNA‐PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double‐stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA‐PK; and (iii) atomic‐force microscopy. Binding of the 18 bp ds DNA to DNA‐PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA‐PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA‐PK and Ku. Atomic‐force microscopy indicates that under such conditions, the DNA‐PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA‐PK.