Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination

Abstract

V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities. All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR). Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function. The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme. To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression. We find that the Ku70 ؊/؊ cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels. Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku. Significantly, the Ku70 ؊/؊ embryonic stem cells have markedly increased sensitivity to ␥-irradiation relative to Ku70 ؉/؊ or wild-type embryonic stem cells. Furthermore, the Ku70 ؊/؊ cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression. We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can be classified as a member of the x-ray cross-complementation group 6 (XRCC6). Potential differences between the Ku70 ؊/؊ and Ku80 ؊/؊ V(D)J recombination defects are discussed.Immunoglobulin ( In this regard, the completion of the V(D)J recombination reaction can also be viewed as a DSBR process in which generally expressed factors are recruited to repair the specifically broken ends generated by RAG-1 and RAG-2 activity. The three known gene products that are required for the DSBR portion of the V(D)J recombination reaction were identified through studies of ionizing-radiation-sensitive Chinese hamster cell lines and cells from severe combined immunedeficient (Scid) mice (10). There are at least eight x-ray crosscomplementation (XRCC) groups, based on somatic cell hybrid studies (12, 13). Of these, groups XRCC4, XRCC5, and XRCC7, which have specific defects in DSBR (14-17), were also found to have defects in their ability to support V(D)J recombination (18)(19)(20). In this regard, following introduction of RAG-1 and RAG-2 gene expression, cell lines from each complementation group were capable of initiating V(D)J recombination on transiently introduced V(D)J recombination substrates. However, XR-1 and xrs-6 cells (representing the XRCC4 and -5 groups, respectively) were defective in their ability to form both coding and RS joins (18,19), whereas V3 cells (from the XRCC7 group) had impaired ability to form coding joins but were relatively normal in ability to form RS joins (18, 21). V3 cells were further shown to represent the same genetic complementation group as the murine Scid mutation (20).xrs-6 cel...

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