Molecular mechanism underlying RAG1/RAG2 synaptic complex formation

Luda S. Shlyakhtenko, Jamie Gilmore, Aleksei N. Kriatchko, Sushil Kumar, Patrick C. Swanson, Yuri L Lyubchenko

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

Two lymphoid cell-specific proteins, RAM and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

Original languageEnglish (US)
Pages (from-to)20956-20965
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number31
DOIs
Publication statusPublished - Jul 31 2009

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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