KU70-DEFICIENT EMBRYONIC STEM-CELLS HAVE INCREASED IONIZING RADIOSENSITIVITY, DEFECTIVE-DNA END-BINDING ACTIVITY, AND INABILITY TO SUPPORT V(D)J RECOMBINATION

V(D)J recombination requires both lymphoid-specific and generally expr essed enzymatic activities, All three known generally expressed activi ties involved in V(D)J recombination are also involved in DNA double-s trand break repair (DSBR), Two of these are components of the DNA-depe ndent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic su bunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function, The Ku70 protein is an additional component of DNA-PK; Ku70 forms a he terodimer with Ku80 to generate the DNA end-binding component of tile enzyme, To test putative functions for Ku70, we have used gene-targete d mutation to generate a murine embryonic stem cell line which lacks K u70 expression, We find that the Ku70(-/-) cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship betw een their levels, Correspondingly, these cells lack the nonspecific DN A end-binding activity associated with Ku. Significantly, the Ku70(-/- ) embryonic stem cells have markedly increased sensitivity to gamma-ir radiation relative to Ku70(+/-) or wild-type embryonic stem cells. Fur thermore, tile Ku70(-/-) cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recom bination substrates by enforced RAG-1 and RAG-2 expression. We conclud e that the Ku70 gene product is involved in DSBR and V(D)J recombinati on and confirm that the Ku70 gene can Toe classified as a member of th e x-ray cross-complementation group 6 (XRCC6). Potential differences b etween the Ku70(-/-) and Ku80(-/-) V(D)J recombination defects are dis cussed.