Abstract
Ku is a heterodimeric protein composed of two subunits of approximately 70 kDa (Ku70) and 80 kDa (Ku80). It is an abundant nuclear DNA-binding factor binding to double-stranded DNA ends without sequence specificity and is involved in DNA double strand breaks (DSBs) repair during ionizing radiation or free radical reaction. Recently, plant Ku protein has been isolated in Arabidopsis thaliana, and its functions are similar to those of mammalian cells. However, the expression of Ku gene is constitutively at high levels in mammalian cells, although several studies suggest that expression of Ku is dependent upon an entry into the cell cycle. In plants, AtKu gene is expressed widely and at low level in plant tissues, and probably induced during plant development. In animals, it has been shown that the DNA-binding activity of Ku heterodimer depends on reduced cysteine residues in vitro, and the Ku70 subunit can weakly binds to the DNA end in the absence of Ku80. In this study, the 8 cysteine residues in AtKu70 were mutated by means of serine-substituted mutagenesis. Mutants were over-expressed in E. coli. The His-tagged fusion protein makes it possible to purify the soluble protein. With the application of immobilized-metal affinity column,Ni2+-NTA (Ni2+-Nitriloacetic acid), and FPLC (Fast performance liquid chromatography) systems to purify the AtKu70, analysis by SDS-PAGE (Sodium dodecylsulphate - polyacrylamide gel electrophoresis) and Western blot reveal that the molecular weight of AtKu70 is about 70 kDa. According to the results of electrophoresis mobility shift assay (EMSA), we found that two mutants, C64S and C84S, have the DNA-binding activity. It indicated that the other 6 mutants could be involved in binding to DNA. In further studies, we will use the biophysical techniques, such as Circular Dichroism (CD), to investigate the structural and functional roles of these 6 mutants.