Real-time monitoring of RAG-catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex

During V(D)J recombination, the RAG1/2 recombinase is thought to play an active role in transferring newly excised recombination ends from the RAG post-cleavage complex (PCC) to the non-homologous end joining (NHEJ) machinery to promote appropriate antigen receptor gene assembly. However, this transfer mechanism is poorly understood, partly because of the technical difficulty in revealing weak association of coding ends (CEs) with one of the PCCs, coding end complex (CEC). Using fluorescence resonance energy transfer (FRET) and anisotropy measurement, we present here real-time monitoring of the RAG1/2-catalyzed cleavage reaction, and provide unequivocal evidence that CEs are retained within the CEC in the presence of Mg²⁺. By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining. While the latter two CECs exhibit similar stability, the full-length RAG2 renders a less stable CEC unless H3K4me3 peptides are added. Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex. Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.