Amino acid residues in Rag1 crucial for DNA hairpin formation

• Published in: Nature structural & molecular biology Vol. 13; no. 11; pp. 1010 - 1015
• Main Authors: Roth, David B, Lu, Catherine P, Sandoval, Hector, Brandt, Vicky L, Rice, Phoebe A
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.11.2006
• Subjects: Amino Acid Sequence; Amino acids; Amino Acids, Aromatic - analysis; Amino Acids, Aromatic - genetics; Amino Acids, Aromatic - metabolism; Animals; Biochemistry; Catalytic Domain; Cell receptors; CHO Cells; Conserved Sequence; Cricetinae; DDE; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - metabolism; Homeodomain Proteins - chemistry; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Molecular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrous oxide; Nucleic Acid Conformation; Physiological aspects; Protein folding; Proteins; Residues; Sequence Alignment; Structure-Activity Relationship; T cells; Transposases - chemistry; Transposases - metabolism; VDJ Recombinases - chemistry; VDJ Recombinases - genetics; VDJ Recombinases - metabolism; United States
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb1154

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.