Initiation of V(D)J recombination in a cell-free system.
Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-length, blunt, 5'-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.
van Gent
D C
DC
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
McBlane
J F
JF
Ramsden
D A
DA
Sadofsky
M J
MJ
Hesse
J E
JE
Gellert
M
M
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins; D005803:Genes, Immunoglobulin; D018398:Homeodomain Proteins; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011995:Recombination, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA