Murine cell line SX9 bearing a mutation in the dna-pkcs gene exhibits aberrant V(D)J recombination not only in the coding joint but also in the signal joint.
We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide "T" (9572) to "C" transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12. 2% as compared with the normal cell line. To confirm the responsibility of the dna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild type dna-pkcs cDNA was observed. SX9 is a novel dna-pkcs-deficient cell line.
Fukumura
R
R
National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba-shi, Chiba 263-8555, Japan.
Araki
R
R
Fujimori
A
A
Mori
M
M
Saito
T
T
Watanabe
F
F
Sarashi
M
M
Itsukaichi
H
H
Eguchi-Kasai
K
K
Sato
K
K
Tatsumi
K
K
Abe
M
M
D019943:Amino Acid Substitution; D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D002384:Catalysis; D004265:DNA Helicases; D018076:DNA, Complementary; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics; D007133:Immunoglobulin Joining Region / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D000072200:Ku Autoantigen; D007930:Leucine / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D011392:Proline / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011836:Radiation Tolerance; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D011392:Proline; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D007930:Leucine