Identification of two domains of the p70 Ku protein mediating dimerization with p80 and DNA binding

Jingsong Wang, Xingwen Dong, Kyungjae Myung, Eric A. Hendrickson, Westley H. Reeves

Research output: Contribution to journalArticle

66 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)842-848
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number2
DOIs
StatePublished - Jan 9 1998

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