Identification of ku and dna-pkcs domains responsible for ku heterodimer and dna-pk holoenzyme assembly

DNA dependent protein kinase (DNA-PK) may play a critical role in DNA double strand break repair in mammalian cells. The DNA-PK holoenzyme is composed of the Ku heterodimer and a large catalytic subunit (DNA-PKcs). Although current understanding of the structure of DNA-PK is limited, it is thought that the Ku heterodimer is responsible for targeting the catalytic subunit to DNA ends. To better understand the structure of DNA-PK we are using a combination of in vivo and in vitro approaches to map the regions of Ku and the DNA-PK catalytic subunit responsible for holoenzyme assembly. The yeast two-hybrid system provides a powerful means of identifying domains responsible for mediating protein-protein interactions. We have identified a region of Ku80, outside of and C-terminal to the putative leucine zipper, that is capable of mediating interaction with Ku70 in the yeast two-hybrid system. Currently, in vitro studies are being used to confirm the ability of this region to interact with Ku70. Analysis of DNA-PKcs fragments in the yeast two- hybrid system are underway to identify regions of the catalytic subunit responsible for interaction with Ku. The association of DNA-PK with DNA is being examined by a combination of election and atomic force microscopy.