Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination.
In addition to creating the DNA double strand breaks that initiate V(D)J recombination, the RAG proteins are thought to play a critical role in the joining phase of the reaction. One such role, suggested by in vitro studies, might be to ensure the structural integrity of postcleavage complexes, but the significance of such a function in vivo is unknown. We have identified RAG1 mutants that are proficient in DNA cleavage but defective in their ability to interact with coding ends after cleavage and in the capture of target DNA for transposition. As a result, these mutants exhibit severe defects in hybrid joint formation, hairpin coding end opening, and transposition in vitro, and in V(D)J recombination in vivo. Our results suggest that the RAG proteins have an architectural function in facilitating proper and efficient V(D)J joining, and a protective function in preventing degradation of broken ends prior to joining.
Tsai
Chia-Lun
CL
Department of Molecular Biophysics and Biochemistry, Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Drejer
Anna H
AH
Schatz
David G
DG
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics*; D016654:Genes, RAG-1; D018698:Glutamic Acid / Q000737:chemistry; D018398:Homeodomain Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D010641:Phenotype; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000502:physiology; D011995:Recombination, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D018698:Glutamic Acid; D012694:Serine; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D003545:Cysteine; D000409:Alanine