Abstract
Variable (diversity) joining (V(D)J) recombination is initiated by the introduction of single-strand DNA breaks (nicks) at recombination signal sequences (RSSs). The importance and fate of these RSS nicks for the regulation of the V(D)J rearrangement and their potential contribution to genomic instability are poorly understood. Using two new methodologies, we were able to detect and quantify specific RSS nicks introduced into genomic DNA by incubation with recombination-activating gene proteins in vitro. In vivo, however, we found that nicks mediated by recombination-activating gene (RAG) proteins were detectable only in gene segments associated with RSSs containing 12–base pair spacers but not in those containing 23–base pair spacers. These data support a model of capture rather than synapsis for pairwise RSS cleavage during V(D)J recombination.
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Acknowledgements
We thank E. Robey for Rag1−/− TCRαβ-transgenic mice; Y. Zhuang (Duke University, Durham, North Carolina) for the E2A cell line; and A. Winoto and members of the Schlissel lab for comments. Supported by National Institutes of Health (AI40227 and HL48702 to M.S.S.).
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Supplementary information
Supplementary Fig. 1
Oligo-capture assays of RSS nicks at various rearranging gene-segments. (PDF 91 kb)
Supplementary Fig. 2
Nuclease activity in apoptotic cells does not affect the oligo-capture assay for RSS nicks. (PDF 123 kb)
Supplementary Table 1
5′ phosphorylated and 3′ biotinylated oligo sequences for the oligo-capture assay. (PDF 36 kb)
Supplementary Table 2
Oligonucleotide primers used for LM-PCR. (PDF 18 kb)
Supplementary Table 3
List of nucleotide primers and probes used for sequence detection. (PDF 28 kb)
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Curry, J., Geier, J. & Schlissel, M. Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis. Nat Immunol 6, 1272–1279 (2005). https://doi.org/10.1038/ni1270
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DOI: https://doi.org/10.1038/ni1270
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