Full-length RAG-2, and not full-length RAG-1, specifically suppresses RAG-mediated transposition but not hybrid joint formation or disintegration.
RAG-1 and RAG-2 initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences flanking a pair of antigen receptor gene segments. Occasionally, the RAG proteins mediate two other alternative DNA rearrangements in vivo: the rejoining of signal and coding ends and the transposition of signal ends into unrelated DNA. In contrast, truncated, catalytically active "core" RAG proteins readily catalyze these reactions in vitro, suggesting that full-length RAG proteins directly or indirectly suppress these undesired reactions in vivo. To discriminate between direct and indirect suppression models, full-length RAG proteins were purified and characterized in vitro. From mammalian cells, full-length RAG-1 is readily purified with core RAG-2 but not full-length RAG-2 and vice versa. Despite differences in DNA binding activity, recombinase containing either core or full-length RAG-1 or RAG-2 possess comparable cleavage, rejoining, and end-processing activity, as well as similar usage preferences for canonical versus cryptic recombination signals. However, recombinase containing full-length RAG-2, but not full-length RAG-1, exhibits dramatically reduced transposition activity in vitro. These data suggest RAG-mediated transposition and rejoining are differentially regulated by the full-length RAG proteins in vivo (the former directly by RAG-2 and the latter indirectly through other factors) and argue that noncore portions of the RAG proteins have little or no direct influence over V(D)J recombinase site specificity.
Swanson
Patrick C
PC
Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska 68178, USA. pswanson@creighton.edu
Volkmer
Dustin
D
Wang
Lei
L
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011946:Receptors, Antigen / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D045567:VDJ Recombinases / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; C499311:RAG2 protein, human; D011946:Receptors, Antigen; D011994:Recombinant Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D045567:VDJ Recombinases