RAG-2 promotes heptamer occupancy by RAG-1 in the assembly of a V(D)J initiation complex.
V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.
Swanson
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PC
Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Desiderio
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D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005803:Genes, Immunoglobulin / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D058619:Maltose-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; C499311:RAG2 protein, human; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein