Requirements for DNA hairpin formation in V(D)J recombination

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As is true with some cut and paste transposases such as Tn5 and Hermes, the RAG proteins form a DNA hairpin at one end of the DNA break, via a nicked intermediate. Using abasic DNA substrates, we have shown that different base positions are important for the two steps of cleavage, some bases being essential for nicking while others are involved in hairpin formation. Removal of one particular base in the coding flank greatly enhances hairpin formation, bypassing the normal requirement for a paired complex of two recombination sites. Activation by this abasic substrate is consistent with a mechanism in which one base is flipped out of the double helix and stabilized by protein interactions, thus allowing the sharp DNA bend required for hairpinning. Such a base flip is seen in the crystal structure of the Tn5 post-cleavage complex; in the RAG system it may normally require paired complex formation. We have also searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on its indole ring. We identified W956 of RAG1 as the most essential tryptophan. Mutations at this site greatly inhibited hairpinning, but the reaction could be rescued by the correct abasic substrate. W956, which is near one of the catalytic triad of acidic residues in RAG1, is therefore a likely candidate for interacting with the flipped base during hairpin formation.