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Cell-free V(D)J recombination

Nature volume 388, pages 488–491 (1997)Cite this article

Abstract

V(D)J recombination generates diversity in the immune system through the lymphoid-specific assembly of multiple gene segments into functional immunoglobulin and T-cell receptor genes (for reviews, see refs 1, 2). The first step in V(D)J recombination is cleavage of DNA at recombination signal sequences. Cleavage produces a blunt DNA end on each signal sequence and a hairpin end on adjacent coding gene segments3,4, and can be reproduced in vitro by using purified RAG1 and RAG2 proteins5,6. The later steps involve processing and joining of the cleaved DNA ends, and until now have been studied only in cells. Here we reconstitute the complete V(D)J recombination reaction in a cell-free system. We find that the RAG proteins are not only involved in cleavage, but are also needed in the later steps for efficient joining of coding ends. Joining is largely directed by short pieces of identical sequence in the coding flanks, but addition of human DNA ligase I results in greater diversity. Coding junctions contain short deletions as well as additions complementary to a coding flank (P nucleotides). Addition of non-templated nucleotides into coding junctions is mediated by terminal deoxyribonucleotidyl transferase. The cell-free reaction can therefore reproduce the complete set of processing events that occur in cells.

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Figure 1: Requirements for cell-free V(D)J recombination.
Figure 2: Effect of RAG proteins in joining reactions.
Figure 3: a, b, Effect of DNA ligases on formation of coding junctions.
Figure 4: Effect of ligase I (L-I) and TdT on signal junction formation.

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Acknowledgements

We thank T. Lindahl for purified human ligase IV and for clones expressing recombinant human DNA ligases I and III, and our colleagues for helpful discussions. D.A.R. was supported by fellowships from the MRC of Canada and the Cancer Research Institute, USA, and T.T.P. was supported by a Helen Hay Whitney postdoctoral fellowship.

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Authors and Affiliations

  1. Laboratory of Molecular Biology, Building 5, Room 241

    Dale A. Ramsden, Tanya T. Paull & Martin Gellert

  2. National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892-0540, Maryland, USA

    Dale A. Ramsden, Tanya T. Paull & Martin Gellert

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  1. Dale A. Ramsden
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Correspondence to Martin Gellert.

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Ramsden, D., Paull, T. & Gellert, M. Cell-free V(D)J recombination. Nature 388, 488–491 (1997). https://doi.org/10.1038/41351

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