Modeling of the RAG Reaction Mechanism

•RAG kinetics indicate that RAG synapsis is nearly irreversible•Synapsis is favored prior to any RAG catalysis (nicking)•A RAG octamer is inconsistent with RAG double-strand break formation kinetics•Sequence-specific RAG binding and chromatin contacts are essential for site specificity

SummaryIn vertebrate V(D)J recombination, it remains unclear how the RAG complex coordinates its catalytic steps with binding to two distant recombination sites. Here, we test the ability of the plausible reaction schemes to fit observed time courses for RAG nicking and DNA hairpin formation. The reaction schemes with the best fitting capability (1) strongly favor a RAG tetrameric complex over a RAG octameric complex; (2) indicate that once a RAG complex brings two recombination signal sequence (RSS) sites into synapsis, the synaptic complex rarely disassembles; (3) predict that the binding of both RSS sites (synapsis) occurs before catalysis (nicking); and (4) show that the RAG binding properties permit strong distinction between RSS sites within active chromatin versus nonspecific DNA or RSS sites within inactive chromatin. The results provide general insights for synapsis by nuclear proteins as well as more specific testable predictions for the RAG proteins.

Graphical AbstractFigure optionsDownload full-size imageDownload as PowerPoint slide