Abstract
Abstract Ku is a heterodimeric protein composed of two subunits known as Ku70 (70 kDa) and Ku80 (80 kDa), respectively. It is an abundant nuclear DNA-binding factor binding to double-stranded DNA ends without sequence specificity. After ionizing radiation or free radical reaction Ku is involved in DNA double strand breaks (DSBs) repair through NHEJ pathway. Recently, plant Ku protein has been isolated from 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 while the concentration of AtKu protein is relatively lower in humans. In recent study, Ku protein binding to DNA end in mammalian cells depends on its reduced cysteine residues, and Ku70 can weakly bind to the DNA end alone in the absence of Ku80. In order to determine which cysteine residue of AtKu70 binds to DNA, all 8 cysteine residues in AtKu70 were mutated by serine-substituted mutagenesis. Mutants were expressed in E. coli and purified by immobilized-metal affinity column, Ni2+-NTA and FPLC system. In this study, according to the results of electrophoresis mobility shift assay (EMSA) and circular dichroism (CD) spectrometry, we found that all mutants except C43S decreased their binding affinity, especially C46S and C64S decline significantly. The results indicate that these two cysteine residues are directly responsible for DNA binding capacity. 6