Faculty Bibliography

The Rag1 and Rag2 proteins initiate V(D)J recombination by introducing site-specific DNA double-strand breaks. Cleavage occurs by nicking one DNA strand, followed by a one-step transesterification reaction that forms a DNA hairpin structure. A similar reaction allows Rag transposition, in which the 3'-OH groups produced by Rag cleavage are joined to target DNA. The Rag1 active site DDE triad clearly plays a catalytic role in both cleavage and transposition, but no other residues in Rag1 responsible for transesterification have been identified. Furthermore, although Rag2 is essential for both cleavage and transposition, the nature of its involvement is unknown. Here, we identify basic amino acids in the catalytic core of Rag1 specifically important for transesterification. We also show that some Rag1 mutants with severe defects in hairpin formation nonetheless catalyze substantial levels of transposition. Lastly, we show that a catalytically defective Rag2 mutant is impaired in target capture and displays a novel form of coding flank sensitivity. These findings provide the first identification of components of Rag1 that are specifically required for transesterification and suggest an unexpected role for Rag2 in DNA cleavage and transposition

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases