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Molecular and Cellular Biology, September 2007, p. 6288-6299, Vol. 27, No. 18
0270-7306/07/$08.00+0     doi:10.1128/MCB.00835-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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,¹ Sebastian D. Fugmann,^2, and David G. Schatz^2,3*

Department of Genetics, Yale University School of Medicine, 330 Cedar St., New Haven, Connecticut 06510,¹ Department of Immunobiology, Yale University School of Medicine, 300 Cedar St., New Haven, Connecticut 06510,² Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510³

Received 11 May 2007/ Returned for modification 11 June 2007/ Accepted 9 July 2007

The beyond 12/23 (B12/23) rule ensures inclusion of a Dß gene segment in the assembled T-cell receptor (TCR) ß variable region exon and is manifest by a failure of direct Vß-to-Jß 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 TCRß gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRß locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRß locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jß substrates and to some extent through poor synapsis of Vß and Jß 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 Jß 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.

Published ahead of print on 16 July 2007.

Present address: National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224.