The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo.
BACKGROUND
V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both.


RESULTS
We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates transfected into cultured cells. This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo. In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage. The results show that both a 12-spacer and a 23-spacer RSS are required for efficient cleavage. Truncated RAG-1 and RAG-2 proteins, while capable of cleaving at isolated RSS in cell-free systems, also require a 12/23 RSS pair for efficient cleavage in vivo.


CONCLUSIONS
These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length provide evidence for an inefficient, single RSS cleavage pathway that may contribute to aberrant V(D)J rearrangements in vivo.
Steen
S B
SB
Cell and Molecular Biology Program, Baylor College of Medicine, Houston, TX 77030, USA.
Gomelsky
L
L
Roth
D B
DB
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins; D005803:Genes, Immunoglobulin; D018398:Homeodomain Proteins; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008957:Models, Genetic; D011506:Proteins / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011995:Recombination, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007135:Immunoglobulin Variable Region; D011506:Proteins; C499308:Rag2 protein, mouse; D011948:Receptors, Antigen, T-Cell; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein