Suwa A, Hirakata M, Takeda Y, Jesch S A, Mimori T, Hardin J A
Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta 30912-3100.
Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6904-8. doi: 10.1073/pnas.91.15.6904.
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.
Ku蛋白是一种自身抗原，由70 kDa和80 kDa的多肽组成。它在游离末端与双链DNA结合。在本研究中，我们检测了抗Ku抗体从不同盐浓度制备的HeLa细胞提取物中免疫沉淀各种结构的能力。在生理条件下，这些抗体鉴定出一种包含Ku蛋白和DNA依赖性蛋白激酶（DNA-PK）的350 kDa组分（p350）的复合物，该复合物似乎在DNA链上紧密结合。在用细胞提取物和生化纯化组分进行的重组实验中，Ku蛋白-p350复合物仅在双链DNA存在的情况下形成。重组后的复合物具有催化活性。与先前的研究一起，这些结果表明，随着DNA-PK全酶的组装，Ku蛋白与DNA相互作用以形成p350的结合位点。
