Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted VDJ recombination in nonlymphoid cells.
V(D)J recombination occurs efficiently only between gene segments flanked by recombination signals (RSs) containing 12 and 23 base pair spacers (the 12/23 rule). A further limitation "beyond the 12/23 rule" (B12/23) exists at the TCRbeta locus and ensures Dbeta usage. Herein, we show that extrachromosomal V(D)J recombination substrates recapitulate B12/23 restriction in nonlymphoid cells. We further demonstrate that the Vbeta coding flank, the 12-RS heptamer/nonamer, and the 23-RS spacer each can significantly influence B12/23 restriction. Finally, purified core RAG1 and RAG2 proteins (together with HMG2) also reproduce B12/23 restriction in a cell-free system. Our findings indicate that B12/23 restriction of V(D)J recombination is cemented at the level of interactions between the RAG proteins and TCRbeta RS sequences.
Jung
David
D
Howard Hughes Medical Institute, The Children's Hospital, Harvard Medical School, Boston, MA, USA.
Bassing
Craig H
CH
Fugmann
Sebastian D
SD
Cheng
Hwei-Ling
HL
Schatz
David G
DG
Alt
Frederick W
FW
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D005111:Extrachromosomal Inheritance; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D019674:Genes, T-Cell Receptor beta; D018398:Homeodomain Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D016385:TATA Box; D014162:Transfection
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C499308:Rag2 protein, mouse; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein