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 polypeptides. It associates with double-stranded DNA at free ends. In the present study, we examined the ability of anti-Ku antibodies to immunoprecipitate various structures from extracts of HeLa cells prepared at different salt concentrations. Under physiological conditions, these antibodies identified a complex containing the Ku protein and the 350-kDa component (p350) of DNA-dependent protein kinase (DNA-PK), which appeared to be closely associated on the DNA strand. In reconstitution experiments with cell extracts and biochemically purified components, the Ku protein-p350 complex formed only in the presence of double-stranded DNA. The reconstituted complex was catalytically active. Together with previous studies, these results indicate that the Ku protein interacts with DNA to create a binding site for p350 as the DNA-PK holoenzyme assembles.
Suwa
A
A
Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta 30912-3100.
Hirakata
M
M
Takeda
Y
Y
Jesch
S A
SA
Mimori
T
T
Hardin
J A
JA
D034961:Antigens, Nuclear; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen