The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination

Anna H. Drejer-Teel, Sebastian D. Fugmann, David G. Schatz*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

22 Scopus citations

Abstract

The beyond 12/23 (B12/23) rule ensures inclusion of a Dβ gene segment in the assembled T-cell receptor (TCR) β variable region exon and is manifest by a failure of direct Vβ-to-Jβ gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRβ gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRβ locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRβ locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jβ substrates and to some extent through poor synapsis of Vβ and Jβ substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jβ substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo.

Original languageEnglish
Pages (from-to)6288-6299
Number of pages12
JournalMolecular and Cellular Biology
Volume27
Issue number18
DOIs
StatePublished - 09 2007
Externally publishedYes

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