How RAG1/2 evolved from ancestral transposases to initiate V(D)J recombination without transposition.

The RAG1/2 recombinase, which initiates V(D)J recombination in jawed vertebrates, evolved from RNaseH-like transposases such as Transib and ProtoRAG . However, its post-cleavage transposase activity is strictly suppressed. Previous structural studies have focused only on the conserved core domains of RAG1/2, leaving the regulatory mechanisms of the non-core regions unclear. To investigate how RAG1/2 suppresses transposition and regulates DNA cleavage, we determined cryo-EM structures of nearly full-length RAG1/2 complexed with cleaved Recombination Signal Sequences (RSS) in a Signal-End Complex (SEC), at resolutions up to 2.95 Å. Two key structures, SEC-0 and SEC-PHD, reveal distinct regulatory roles of RAG2, which is absent in Transib transposase. SEC-0 displays a closed conformation, revealing that the core RAG2 facilitates sequential DNA cleavage by stabilizing the RSS-cleaved states in a "spring-loaded" mechanism. SEC-PHD reveals how RAG2's non-core PHD and Acidic Hinge (AH) domains, which are absent in ProtoRAG, inhibit target DNA binding in transposition. Histone H3K4me3, which recruits RAG1/2 to RSS sites, does not influence RAG1/2 binding to V, D or J gene segments bordered by RSS . In contrast, the suppressed transposition can be activated by H3K4me3 peptides that dislodge the inhibitory PHD domain . To achieve this de-repression in vivo, however, would require an unlikely close placement of two nucleosomes flanking a target DNA bent by nearly 180°. Our structural and biochemical results elucidate how RAG1 has acquired RAG2 and utilizes its core and non-core domains to enhance V(D)J recombination and suppress transposition.