A CENTRAL REGION OF KU80 MEDIATES INTERACTION WITH KU70 IN-VIVO

Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK) , is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 a nd Ku80 respectively, Defects in DNA-PK subunits have been shown to re sult in a reduced capacity to repair DNA double-strand breaks. Assembl y of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit, The regions of the K u subunits responsible for heterodimerization have not been clearly de fined in vivo, A previous study has suggested that the C-terminus of K u80 is required for interaction with Ku70, Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two -hybrid system and an independent mammalian in vivo system, Our two-hy brid study suggests that the central region of Ku80, not its C-terminu s, is capable of mediating interaction with Ku70, To determine if this region mediates interaction with Ku70 in mammalian cells we transfect ed xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 d eletions carrying a nuclear localization signal, Immunoprecipitation f rom transfected cell extracts revealed that the central domain identif ied by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitate s with endogenous xrs-6 Ku70. The central interaction domain maps to t he internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerizat ion with Ku70.