A biochemically defined system for coding joint formation in V(D)J recombination.
V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.
Lu
Haihui
H
Norris Comprehensive Cancer Center, Room 5428, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089, USA.
Shimazaki
Noriko
N
Norris Comprehensive Cancer Center, Room 5428, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089, USA.
Raval
Prafulla
P
Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
Gu
Jiafeng
J
Norris Comprehensive Cancer Center, Room 5428, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089, USA.
Watanabe
Go
G
Norris Comprehensive Cancer Center, Room 5428, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089, USA.
Schwarz
Klaus
K
Institute for Clinical Transfusion Medicine and Immunogenetics, Ulm and Institute for Transfusion Medicine, University Hospital Ulm, 89081 Ulm, Germany.
Swanson
Patrick C
PC
Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
Lieber
Michael R
MR
Norris Comprehensive Cancer Center, Room 5428, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089, USA. Electronic address: lieber@usc.edu.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004789:Enzyme Activation; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics*; D006801:Humans; D007313:Insecta; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011995:Recombination, Genetic / Q000235:genetics*
D004247:DNA; D051747:DNA-Activated Protein Kinase