Wang J, Dong X, Reeves W H
Departments of Medicine, Microbiology and Immunology, Thurston Arthritis Research Center, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7280, USA.
J Biol Chem. 1998 Nov 20;273(47):31068-74. doi: 10.1074/jbc.273.47.31068.
Ku autoantigen, a heterodimer of 70- and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair. Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion. The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent. Both must be intact for end binding activity in gel shift assays. To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses. We show by several criteria that amino acids 371-510 of p80 interact with p70. Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region. In DNA immunoprecipitation assays, amino acids 179-510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179-732 were necessary. Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases.
Ku自身抗原是一种由70 kDa和80 kDa亚基组成的异源二聚体，是DNA修复过程中至关重要的DNA末端结合因子。p70的两个结构域介导DNA结合，一个位于C末端，另一个位于N末端部分。后者必须与p80二聚化才能结合DNA，而前者与p80无关。在凝胶迁移实验中，两者对于末端结合活性都必须完整。为了评估p80在DNA结合中的作用，使用重组杆状病毒将缺失突变体与全长p70共表达。我们通过多项标准表明，p80的氨基酸371 - 510与p70相互作用。p70的两个二聚化结构域都与p80的同一区域结合，但显然是该区域内的不同位点。在DNA免疫沉淀实验中，p80的氨基酸179 - 510是p70依赖p80的DNA结合所必需的，而在凝胶迁移实验中，氨基酸179 - 732是必需的。有趣的是，p80依赖和p80非依赖的DNA结合位点都优先结合DNA末端，这提示了一种模型，即单个Ku异源二聚体可能在物理上使两个断裂的DNA末端并列，便于DNA连接酶将它们重新连接起来。
