Analysis of the Ku antigen-dependent activation of reporter genes in yeast and characterization of the nuclear import signals of Ku antigen.

Abstract

Ku antigen (Ku70/Ku80) is a heteromeric, DNA binding protein that has been implicated in DNA repair, V(D)J recombination, tumour suppression, growth, cell cycle control, telomere maintenance and the regulation of gene transcription. Ku is a DNA binding subunit of the DNA-dependent protein kinase and functions to recruit and stimulate the catalytic subunit at appropriate DNA target sites. I present the unexpected finding that expression of human Ku fused to an ectopic activation domain broadly activated reporter genes integrated into the yeast genome independent of the reporter gene, yeast minimal promoter and reporter gene integration site. Interestingly, expression of various Ku70 deletion mutants alone or in combination with Ku80 revealed that the regions of Ku identified to be required for activation share a striking resemblance to portions of Ku needed to bind DNA. Furthermore, a mutation in Ku70 that negates the Ku70-dependent activation property in yeast also disrupts the ability of Ku70 monomers to associate tightly with the nucleus in mammalian cells. Moreover, I demonstrate that human Ku80 cannot substitute for the yeast homologue (HDF2) to rescue the temperature sensitive phenotype of a yeast strain deficient in Ku80 but expression of a variant human Ku heterodimer partially rescues the phenotype. By contrast, the characteristic slow growth displayed by HDF2 deficient yeast at 30°C could not be rescued by expression of the same human Ku heterodimer. Also, I determined that Ku70 contains a bipartite nuclear localization signal between amino acids 539--556, while the core of the nuclear localization signal of Ku80 is a basic motif from 565--569. Each nuclear localization signal mediated the nuclear transfer of the individual Ku subunits and Ku heterodimer, while site directed mutagenesis of both nuclear localization signals resulted in a Ku heterodimer that was localized to the cytoplasm. Lastly, I show that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization reduce the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.