Identification of two domains of the p70 Ku protein mediating dimerization with p80 and DNA binding.
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.
Wang
J
J
Department of Medicine, Thurston Arthritis Research Center, UNC Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7280, USA.
Dong
X
X
Myung
K
K
Hendrickson
E A
EA
Reeves
W H
WH
D000911:Antibodies, Monoclonal / Q000276:immunology; D034961:Antigens, Nuclear; D001666:Binding Sites, Antibody; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D006868:Hydrolysis; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen