Wang J, Dong X, Myung K, Hendrickson E A, Reeves W H
Department of Medicine, Thurston Arthritis Research Center, UNC Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7280, USA.
J Biol Chem. 1998 Jan 9;273(2):842-8. doi: 10.1074/jbc.273.2.842.
The Ku autoantigen is a heterodimer of 70 (p70) and approximately 80 kDa (p80) subunits that is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) complex involved in DNA repair and V(D)J recombination. Binding to DNA ends is critical to the function of DNA-PK, but how Ku interacts with DNA is not completely understood. To define the role of p70 and p80 and their dimerization in DNA binding, heterodimers were assembled by co-expressing the subunits using recombinant baculoviruses. Two p70 dimerization sites, amino acids 1-115 and 430-482, respectively, were identified. Binding of p70 to linear double-stranded DNA could be demonstrated by an immunoprecipitation assay, and required the C-terminal portion (amino acids 430-609), but not interaction with p80. The p70 mutants 1-600, 1-542, 1-115, and 430-600 did not bind DNA efficiently. However, DNA binding of 1-600, 1-542, and 1-115, but not 430-600, was restored by dimerization with p80, indicating that p70 has two DNA binding sites, each partially overlapping one of the dimerization sites. The C-terminal domain can bind DNA by itself, but the N-terminal domain requires dimerization with p80. These observations could be relevant to the multiple functional activities of Ku and explain controversies regarding the role of dimerization in DNA binding.
Ku自身抗原是由70 kDa（p70）和大约80 kDa（p80）亚基组成的异源二聚体，它是参与DNA修复和V（D）J重组的DNA依赖性蛋白激酶（DNA-PK）复合物的DNA结合成分。与DNA末端的结合对DNA-PK的功能至关重要，但Ku与DNA如何相互作用尚未完全清楚。为了确定p70和p80及其二聚化在DNA结合中的作用，使用重组杆状病毒共表达亚基来组装异源二聚体。分别鉴定出两个p70二聚化位点，即氨基酸1-115和430-482。p70与线性双链DNA的结合可通过免疫沉淀试验证实，并且需要C末端部分（氨基酸430-609），但不需要与p80相互作用。p70突变体1-600、1-542、1-115和430-600不能有效地结合DNA。然而，1-600、1-542和1-115（而非430-600）与p80二聚化后恢复了DNA结合能力，这表明p70有两个DNA结合位点，每个位点部分重叠一个二聚化位点。C末端结构域自身可结合DNA，但N末端结构域需要与p80二聚化。这些观察结果可能与Ku的多种功能活性相关，并解释了关于二聚化在DNA结合中作用的争议。
