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.

[1]  P. Jeggo,et al.  Molecular and biochemical characterization of xrs mutants defective in Ku80 , 1997, Molecular and cellular biology.

[2]  Sandro Santagata,et al.  The Homeodomain Region of Rag-1 Reveals the Parallel Mechanisms of Bacterial and V(D)J Recombination , 1996, Cell.

[3]  Eugenia Spanopoulou,et al.  RAG1 Mediates Signal Sequence Recognition and Recruitment of RAG2 in V(D)J Recombination , 1996, Cell.

[4]  J. Danska,et al.  Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes , 1996, Molecular and cellular biology.

[5]  P. Jeggo,et al.  Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[6]  F. Alt,et al.  The V(D)J recombination defect in the xrs-6 cell line results from a point mutation in the Ku80 gene. , 1996, International immunology.

[7]  F. Alt,et al.  The Igκ 3′ Enhancer Influences the Ratio of Igκ versus Igλ B Lymphocytes , 1996 .

[8]  Molly A Bogue,et al.  Ku86-Deficient Mice Exhibit Severe Combined Immunodeficiency and Defective Processing of V(D)J Recombination Intermediates , 1996, Cell.

[9]  M. Nussenzweig,et al.  Requirement for Ku80 in growth and immunoglobulin V(D)J recombination , 1996, Nature.

[10]  R. Moyzis,et al.  Genomic structure and chromosomal assignment of the mouse Ku70 gene. , 1996, Genomics.

[11]  G. Chu,et al.  Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants , 1996, Molecular and cellular biology.

[12]  Gratien G. Prefontaine,et al.  Sequence-specific DNA binding by Ku autoantigen and its effects on transcription , 1996, Nature.

[13]  F. Alt,et al.  The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination , 1995, Cell.

[14]  S. Jackson,et al.  Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Christina A. Cuomo,et al.  Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps , 1995, Cell.

[16]  D. Weaver,et al.  What to do at an end: DNA double-strand-break repair. , 1995, Trends in genetics : TIG.

[17]  P. Jeggo,et al.  Nomenclature of human genes involved in ionizing radiation sensitivity. , 1995, Mutation research.

[18]  F. Alt,et al.  Potential targets for autosomal SCID mutations. , 1995, Current opinion in immunology.

[19]  D. Ward,et al.  Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Ramsden,et al.  Initiation of V(D)J recombination in a cell-free system , 1995, Cell.

[21]  M. Oshimura,et al.  Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  F. Alt,et al.  Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation , 1995, Cell.

[23]  M. Oettinger,et al.  DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect , 1995, Science.

[24]  H. Band,et al.  Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[25]  P. Jeggo,et al.  DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Lieber,et al.  Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. , 1994, Science.

[27]  N. Tuteja,et al.  Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen. , 1994, The EMBO journal.

[28]  F. Alt,et al.  Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. , 1994, Science.

[29]  J. Leszyk,et al.  DNA-dependent ATPase from HeLa cells is related to human Ku autoantigen. , 1994, Biochemistry.

[30]  G. Chu,et al.  A DNA end-binding factor involved in double-strand break repair and V(D)J recombination , 1994, Molecular and cellular biology.

[31]  R. Getts,et al.  Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. , 1994, The Journal of biological chemistry.

[32]  C. Kahn,et al.  The insulin signaling system. , 1994, The Journal of biological chemistry.

[33]  M. Lieber,et al.  V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair , 1993, Molecular and cellular biology.

[34]  F. Alt,et al.  Impairment of V(D)J recombination in double-strand break repair mutants. , 1993, Science.

[35]  S. Jackson,et al.  The DNA-dependent protein kinase: Requirement for DNA ends and association with Ku antigen , 1993, Cell.

[36]  Rudolf Jaenisch,et al.  Targeted mutation of the DNA methyltransferase gene results in embryonic lethality , 1992, Cell.

[37]  K. Rajewsky,et al.  Generation and analysis of interleukin-4 deficient mice. , 1991, Science.

[38]  P. Jeggo,et al.  Genetic analysis of ionising radiation sensitive mutants of cultured mammalian cell lines. , 1991, Mutation research.

[39]  M. Schlissel,et al.  Wild-type V(D)J recombination in scid pre-B cells. , 1990, Molecular and cellular biology.

[40]  R. Yenofsky,et al.  A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[41]  David Baltimore,et al.  The V(D)J recombination activating gene, RAG-1 , 1989, Cell.

[42]  W. Reeves,et al.  Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. , 1989, The Journal of biological chemistry.

[43]  Joins,et al.  Isolation of scid pre‐B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins. , 1989, The EMBO journal.

[44]  Naomi Rosenberg,et al.  The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination , 1988, Cell.

[45]  F. Alt,et al.  The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism , 1988, Cell.

[46]  M. Lieber,et al.  Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals , 1987, Cell.

[47]  J. Steitz,et al.  Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. , 1986, The Journal of biological chemistry.

[48]  A. Giaccia,et al.  Cell cycle-dependent repair of double-strand DNA breaks in a γ-ray-sensitive Chinese hamster cell , 1985, Somatic cell and molecular genetics.

[49]  P. Jeggo,et al.  X-ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA-damaging agents. , 1983, Mutation research.

[50]  S. Tonegawa.  Somatic generation of antibody diversity , 1983, Nature.

[51]  M. Lieber,et al.  Molecular Analysis of DNA Rearrangements in the Immune System , 1996, Current Topics in Microbiology and Immunology.

[52]  F. Alt,et al.  Activities involved in V(D)J recombination. , 1992, Current topics in microbiology and immunology.

[53]  Bosma Mj.  B and T cell leakiness in the scid mouse mutant. , 1992 .

[54]  A. J. Varghese,et al.  Cell cycle responses of two X-ray sensitive mutants defective in DNA repair. , 1989, International journal of radiation biology.

[55]  S. Nesnow,et al.  The genetic toxicology of Gene-Tox non-carcinogens. , 1988, Mutation research.