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 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 gamma-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.
Gu
Y
Y
Children's Hospital, Boston, MA 02115, USA.
Jin
S
S
Gao
Y
Y
Weaver
D T
DT
Alt
F W
FW
D000818:Animals; D034961:Antigens, Nuclear; D016466:CHO Cells; D006224:Cricetinae; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology*; D005720:Gamma Rays; D018390:Gene Targeting; D007133:Immunoglobulin Joining Region / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D000072200:Ku Autoantigen; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011836:Radiation Tolerance / Q000235:genetics*; D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism / Q000528:radiation effects*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen