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Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis

Nature Immunology volume 6, pages 1272–1279 (2005)Cite this article

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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Figure 1: Oligo-capture assay for RSS nicks.
Figure 2: Limit of detection and specificity of the oligo-capture assay.
Figure 3: DNA nicks at Vκ RSS-12 but not Jκ RSS-23 sequences in pro–B cell lines induced to rearrange the Igk locus.
Figure 4: DNA nicks and breaks in the Igh locus.
Figure 5: DNA nicks at Jα50 RSS-12 but not Vα13 RSS-23 or Vκ RSS-12 sequences in thymocyte genomic DNA.
Figure 6: Alternative assays for RAG1 and RAG2 activity on RSS-12 and RSS-23 DNA.

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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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Authors and Affiliations

  1. Department of Molecular and Cell Biology, Division of Immunology, University of California, Berkeley, 94720-3200, California, USA

    John D Curry, Jamie K Geier & Mark S Schlissel

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  1. John D Curry
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  2. Jamie K Geier
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  3. Mark S Schlissel
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Correspondence to Mark S Schlissel.

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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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