Defining the functional domains of Ku p70 subunit using deletion mutant proteins *
The Ku autoantigen has been recently found to play a crucial role in double strand break (DSB) repair of chromosomal DNA and V (D) J recombination of immunoglobulin genes. Ku is an abundant heterodimeric nuclear protein consisting of p70 and p86 subunits, which is localized at the nucleosome linker of the chromosome. The manner in which DNA recognizcs Ku is thought to be quite unique ; it binds to DSB once, and freely moves to the internal portion of double stranded DNA (dsDNA). Although physical contact of the p70 subunit with DNA has been demonstrated, its molecular basis has not been well investigated. In this report, to identify the functional domains on p70 subunits, we produced recombinant p70 deletion mutants by using the Baculovirus system. The results showed that aa392-466 on p70 was crucial for a functional Ku heterodimer. The leucine zipper like motifs and helix-turn-hclix like motif arc not necessary for either heterodimerization or the DNA binding property of Ku. We also investigated nuclear localization of those mutants by immunofluorescence microscopy, and found that aa483-583 was crucial for nuclear localization of p70. No homologous region between these above sequences and other known DNA binding proteins was found by database analysis. These data may be related to the unique function of Ku. (Accepted on April 13, 1999) Kawasaki Igakkaishi 25(1) : 35 - 46, 1999
