RAG-2 promotes heptamer occupancy by RAG-1 in the assembly of a V(D)J initiation complex

Patrick Swanson, Stephen Desiderio

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-l; how RAG-2 collaborates with RAG- 1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-l, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.

Original languageEnglish
Pages (from-to)3674-3683
Number of pages10
JournalMolecular and Cellular Biology
Volume19
Issue number5
StatePublished - May 1999
Externally publishedYes

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Genetic Recombination
Protein Sorting Signals
DNA
V(D)J Recombination
Antigen Receptors
Proteins
Electrophoretic Mobility Shift Assay

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

RAG-2 promotes heptamer occupancy by RAG-1 in the assembly of a V(D)J initiation complex. / Swanson, Patrick; Desiderio, Stephen.

In: Molecular and Cellular Biology, Vol. 19, No. 5, 05.1999, p. 3674-3683.

Research output: Contribution to journalArticle

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