Identification of Two Domains of the p70 Ku Protein Mediating Dimerization with p80 and DNA Binding*
- From the Departments of Medicine, Microbiology and Immunology, Thurston Arthritis Research Center, UNC Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7280 and the §Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912
Abstract
The Ku autoantigen is a heterodimer of 70 (p70) and ∼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.
Footnotes
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↵* This work was supported in part by Grants R01-AR40391 (to W. H. R.), R01-AI35763 (to E. A. H.), and T32-AR7416 from the United States Public Health Service.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Supported by a Postdoctoral Fellowship from the Arthritis Foundation.
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↵¶ Leukemia Scholar of America.
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↵‖ To whom correspondence should be addressed: Division of Rheumatology and Immunology, University of North Carolina at Chapel Hill, 3330 Thurston Bldg., CB 7280, Chapel Hill, NC 27599-7280. Tel.: 919-966-4191; Fax: 919-966-1739; E-mail:reeves.thurston{at}mhs.unc.edu.
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↵1 The abbreviations used are: ds, double-stranded; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; m.o.i., multiplicity of infection; NET, NaCl-EDTA-Tris; bv, baculovirus.
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↵2 J. Wang, unpublished data.
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- Received August 27, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
