DEFINING THE MINIMAL DOMAIN OF KU80 FOR INTERACTION WITH KU70

The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ rec ombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subun its. We used the two hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku . Screening a human cDNA library with the Ku heterodimer did not revea l any novel partners. Screening with the individual subunits, we detec ted only Ku70 clones interacting with Ku80 and only Ku80 clones intera cting with Ku70, indicating that these are the primary partners for on e another. Ku80 and Ku70 formed only heterodimers and did not homodime rize. Ku80 was restricted to interacting with just one Ku70 molecule a t a time. The minimal functional interaction domain of Ku80 that inter acted with Ku70 was defined. It consisted of a 28-amino acid region ex tending from amino acid 449 to 477. This region was crucial for intera ction with Ku70, since mutation within this critical site at amino aci ds 453 and 454 abrogated the ability to interact with Ku70. We further more verified that the same region is crucial for interaction with Ku7 0 using in vitro co-translation of both subunits followed by an immuno precipitation with anti Ku70 antibodies. This interaction domain of Ku 80 does not contain any motif previously recognized in protein-protein interactions.