A RAG-1/RAG-2 tetramer supports 12/23-regulated synapsis, cleavage, and transposition of V(D)J recombination signals.
Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.
Swanson
Patrick C
PC
Department of Medical Microbiology and Immunology, School of Medicine, Creighton University, Omaha, Nebraska 68178, USA. pswanson@creighton.edu
D002460:Cell Line; D023902:Chromosome Pairing; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016654:Genes, RAG-1; D024243:HMGB1 Protein / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D021382:Protein Sorting Signals; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D019895:Transposases / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; C499311:RAG2 protein, human; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D019895:Transposases