Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated.
Immunoglobulin and T-cell receptor genes are assembled during lymphocyte development by a novel, highly regulated series of gene rearrangement reactions known as V(D)J recombination. All rearranging loci are flanked by conserved heptamer-nonamer recombination signal sequences. Gene rearrangement results in the imprecise fusion of coding sequences and the precise fusion of signal sequences. DNA molecules with double-stranded breaks near signal sequences have been detected in cells undergoing V(D)J recombination of the TCR delta locus. We have devised a ligation-mediated PCR assay that detects broken-ended molecules in purified genomic DNA. Using this assay we found that DNA breaks occurring precisely at the signal sequence-coding sequence junction are a general feature of V(D)J recombination, appearing in association with each type of rearranging immunoglobulin gene segment. We show that a significant fraction of these broken ends are blunt and 5'-phosphorylated. In addition, detection of these broken-ended signal sequences is dependent on the activity of RAG-1 and RAG-2, and is restricted to the G0/G1 phase of the cell cycle. The pattern of broken-ended molecules detected in cells at various stages of development reflects the activity of the V(D)J recombinase at different loci during B- and T-cell development.
Schlissel
M
M
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Constantinescu
A
A
Morrow
T
T
Baxter
M
M
Peng
A
A
D000375:Aging / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000502:physiology; D004268:DNA-Binding Proteins; D015321:Gene Rearrangement / Q000235:genetics*; D018398:Homeodomain Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000502:physiology*; D011995:Recombination, Genetic / Q000235:genetics*; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D011506:Proteins; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases