Analysis of regions of RAG-2 important for V(D)J recombination.
The recombinase activating genes RAG-1 and RAG-2 operate together to activate V(D)J recombination, and thus play an essential role in the generation of immune system diversity. As a first step in understanding the function of the RAG-2 protein, we have tested a series of deletion and insertion mutations for their ability to induce V(D)J joining of a variety of model substrates. Mutants were assayed for their ability to induce deletional and inversional V(D)J joining, thereby testing their proficiency at forming both signal and coding joints, and, in some cases, for their ability to carry out recombination of both extrachromosomal and integrated recombination substrates. All these reactions were affected similarly by any one mutation. Although the RAG-2 protein shows extensive evolutionary conservation across its length, we found that the carboxy-terminal portion of RAG-2, including an acidic region, is dispensable for all forms of recombination tested. In contrast, all mutations we created in the N-terminal region severely decreased recombination. Thus, the core active region required for V(D)J recombination is confined to the first three-quarters of the RAG-2 protein.
Cuomo
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Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Oettinger
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MA
D000595:Amino Acid Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins; D005347:Fibroblasts; D015321:Gene Rearrangement / Q000235:genetics*; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009687:Nuclear Proteins; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011506:Proteins; C499311:RAG2 protein, human; D011948:Receptors, Antigen, T-Cell; C064655:V(D)J recombination activating protein 2