DNA-DEPENDENT PROTEIN-KINASE (KU PROTEIN-P350 COMPLEX) ASSEMBLES ON DOUBLE-STRANDED DNA

The Ku protein is an autoantigen that consists of 70- and 80-kDa polyp eptides. It associates with double-stranded DNA at free ends. In the p resent study, we examined the ability of anti-Ku antibodies to immunop recipitate various structures from extracts of HeLa cells prepared at different salt concentrations, Under physiological conditions, these a ntibodies identified a complex containing the Ku protein and the 350-k Da component (p350) of DNA-dependent protein kinase (DNA-PK), which ap peared to be closely associated on the DNA strand. In reconstitution e xperiments with cell extracts and biochemically purified components, t he Ku protein-p350 complex formed only in the presence of double-stran ded DNA. The reconstituted complex was catalytically active. Together with previous studies, these results indicate that the Ku protein inte racts with DNA to create a binding site for p350 as the DNA-PK holoenz yme assembles.