Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

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

doi:10.1074/jbc.m109.028977

Yuri L Lyubchenko, Sushil Kumar + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.028977

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Abstract

Two lymphoid cell-specific proteins, RAG1 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.

Highlights

To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments [1]
To directly observe and analyze RAG-recombination signal sequence (RSS) synaptic complexes assembled on long DNA substrates that more closely model an RSS embedded in chromosomal DNA, we used atomic force microscopy (AFM), given its previously demonstrated success for visualizing synaptic complexes in other systems (10 –14), and its ability to reveal structural details for synaptic complexes that correlate well with independently obtained crystallographic data [15]
We sought to determine whether the PCR-generated DNA substrates support assembly of RAG-RSS complexes detectable by Electrophoretic Mobility Shift Assay (EMSA) under conditions suitable for the analysis of these RAG-RSS complexes using

Summary

Introduction

To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments [1]. We sought to determine whether the PCR-generated DNA substrates support assembly of RAG-RSS complexes detectable by EMSA under conditions suitable for the analysis of these RAG-RSS complexes using

Results

Conclusion