Faculty Bibliography

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position -1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex

Hypomorphic RAG mutants with severely reduced V(D)J recombination activity cause Omenn Syndrome (OS), an immunodeficiency with features of immune dysregulation and a restricted TCR repertoire. Precisely how RAG mutants produce autoimmune and allergic symptoms has been unclear. Current models posit that the severe recombination defect restricts the number of lymphocyte clones, a few of which are selected upon Ag exposure. We show that murine RAG1 R972Q, corresponding to an OS mutation, renders the recombinase hypersensitive to selected coding sequences at the hairpin formation step. Other RAG1 OS mutants tested do not manifest this sequence sensitivity. These new data support a novel mechanism for OS: by selectively impairing recombination at certain coding flanks, a RAG mutant can cause primary repertoire restriction, as opposed to a more random, limited repertoire that develops secondary to severely diminished recombination activity

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