Modeling of the RAG Reaction Mechanism

Summary: In 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. : To better understand human V(D)J recombination, Askary et al. model full-length RAG1/2 nicking and hairpinning time courses to infer the order and rates of RAG binding, synapsis, nicking, and hairpinning steps. This kinetic approach zeroes in on the active fraction of the enzyme population and discerns information that cannot be obtained by other approaches. Furthermore, the authors employ a related approach that provides insight into RAG targeting in the context of chromatin