Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase.
V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.
Department of Immunology, M929, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.
M M
MM
Purugganan
S
S
Shah
J F
JF
Kearney
D B
DB
Roth
D000818:Animals; D034961:Antigens, Nuclear; D016466:CHO Cells; D002467:Cell Nucleus / Q000201:enzymology; D006224:Cricetinae; D004265:DNA Helicases; D004253:DNA Nucleotidylexotransferase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007133:Immunoglobulin Joining Region / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D009711:Nucleotides; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004253:DNA Nucleotidylexotransferase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen