Characterization of ku70 stability and ku70 c-terminal dna end-binding activity

Ku (p80/p70) protein is a crucial component of the DNA-PK complex, which is involved in DNA double-strand break repair. It has been proposed that the Ku protein binds to DNA ends via its 70kD subunit; specifically C-terminal residues 536-609. We found that Ku70 protein expression was impaired in Ku80 mutants, and proposed that Ku70 stability is affected by the absence of Ku80. We overexpressed and purified the C-terminal DNA-binding domain of Ku70 in E.coli. The purified C-terminal domain binds DNA, as demonstrated by a binding assay with DNA-agarose beads, and by Southwestern hybridization. The DNA binding activity will be further studied using gel mobility retardation assays. Experiments involving other DNA conformations, such as nicks and hairpins, are currently underway using the same approaches. Furthermore, the binding is being investigated using AFM and EM. We are also expressing epitope-tagged Ku70 in wildtype and Ku80 mutant cell lines. The turnover of Ku70 in the presence and absence of Ku80 is monitored using pulse-chase labeling. We find that the Ku70 protein is less stable in the absence of Ku80.