In vitro V(D)J recombination: signal joint formation.
The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unable to generate signal joints. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent.
Cortes
P
P
Laboratory of Molecular Immunology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021, USA.
Weis-Garcia
F
F
Misulovin
Z
Z
Nussenzweig
A
A
Lai
J S
JS
Li
G
G
Nussenzweig
M C
MC
Baltimore
D
D
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004265:DNA Helicases; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D005803:Genes, Immunoglobulin; D018398:Homeodomain Proteins; D006801:Humans; D007133:Immunoglobulin Joining Region / Q000096:biosynthesis*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection; D045567:VDJ Recombinases
D034961:Antigens, Nuclear; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D011506:Proteins; C499311:RAG2 protein, human; D011994:Recombinant Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen