Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit modulate RAG-mediated cleavage: implications for the enforcement of the 12/23 rule.
The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity. This observation indicates that DNA repair factors, Ku70/80 and DNA-PKcs, might be present upstream of the DNA cleavage events and not recruited downstream as is currently thought, assigning new nonrepair functions to the DNA-dependent protein kinase.
Sawchuk
Dennis J
DJ
Laboratory of Molecular Immunology and Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.
Mansilla-Soto
Jorge
J
Alarcon
Claudio
C
Singha
Netai C
NC
Langen
Hanno
H
Bianchi
Marco E
ME
Lees-Miller
Susan P
SP
Nussenzweig
Michel C
MC
Cortes
Patricia
P
D034961:Antigens, Nuclear / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D024243:HMGB1 Protein / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013879:Thioredoxins / Q000378:metabolism
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C064658:RAG-1 protein; D013879:Thioredoxins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen