V(D)J recombination: in vitro coding joint formation.
Antigen receptor genes are assembled through a mechanism known as V(D)J recombination, which involves two different joining reactions: signal and coding joining. Formation of these joints is essential for antigen receptor assembly as well as maintaining chromosomal integrity. Here we report on a cell-free system for coding joint formation using deletion and inversion recombination substrates. In vitro coding joint formation requires RAG1, RAG2, and heat-labile factors present in the nuclear extract of nonlymphoid cells. Both inversion- and deletion-mediated coding joint reactions produce diverse coding joints, with deletions and P nucleotide addition. We also show that deletion-mediated coding joint formation follows the 12/23 rule and requires the catalytic subunit of DNA-dependent protein kinase.
Weis-Garcia
F
F
Laboratory of Molecular Immunology, The Rockefeller University, New York, New York 10021, USA.
Besmer
E
E
Sawchuk
D J
DJ
Yu
W
W
Hu
Y
Y
Cassard
S
S
Nussenzweig
M C
MC
Cortes
P
P
D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D008957:Models, Genetic; D009687:Nuclear Proteins; D011946:Receptors, Antigen / Q000235:genetics*; D011995:Recombination, Genetic; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; D011946:Receptors, Antigen; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA