Molecular mechanism underlying RAG1/RAG2 synaptic complex formation.
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.
Shlyakhtenko
Luda S
LS
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Gilmore
Jamie
J
Kriatchko
Aleksei N
AN
Kumar
Sushil
S
Swanson
Patrick C
PC
Lyubchenko
Yuri L
YL
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D023902:Chromosome Pairing / Q000187:drug effects; D004247:DNA / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects; D011995:Recombination, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C499308:Rag2 protein, mouse; C064658:RAG-1 protein; D004247:DNA; D008274:Magnesium; D002118:Calcium