RAG enzymes work as a multi-subunit complex to induce cleavage of a single double stranded DNA (dsDNA) molecule between the antigen receptor coding segment and a flanking recombination signal sequence (RSS).
The next step couples these chemical groups, binding the OH-group (on the coding end) to the PO4-group (that is sitting between the RSS and the gene segment on the opposite strand).
RAG-1 was shown to inefficiently induce recombination activity of the VDJ genes when isolated and transfected into fibroblast samples.
[4] Both RAG1 molecules in the closed dimer are involved in the cooperative binding of the 12-RSS and 23-RSS intermediates with base specific interactions in the heptamer of the signal end.
The coding end is highly distorted with one base flipped out from the DNA duplex in the active center, which facilitates the hairpin formation by a potential two-metal ion catalytic mechanism.
These structures elaborate the molecular mechanisms for DNA recognition, catalysis and the unique synapsis underlying the 12/23 rule, provide new insights into the RAG-associated human diseases, and represent a most complete set of complexes in the catalytic pathways of any DDE family recombinases, transposases or integrases.
[8] Active Transib transposons with both RAG1 and RAG2 ("ProtoRAG") has been discovered in B. belcheri (Chinese lancelet) and Psectrotarsia flava (a moth).
Because agnathans (a class of jawless fish) lack a core RAG1 element, it was traditionally assumed that RAG1 invaded after the agnathan/gnathostome split 1001 to 590 million years ago (MYA).
[13] Recent analysis has shown the RAG phylogeny to be gradual and directional, suggesting an evolutionary path that relies on vertical transmission.
It is still unclear what forces led to the development of a RAG1/2-mediated immune system exclusively in jawed vertebrates and not in any invertebrate species that also acquired the RAG1/2-containing transposon.
Current hypotheses include two whole-genome duplication events in vertebrates,[17] which would provide the genetic raw material for the development of the adaptive immune system, and the development of endothelial tissue, greater metabolic activity, and a decreased blood volume-to-body weight ratio, all of which are more specialized in vertebrates than invertebrates and facilitate adaptive immune responses.