Requirements for DNA hairpin formation by RAG1/2.
The rearrangement of antigen receptor genes is initiated by double-strand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some "cut-and-paste" transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have 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 the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.
Grundy
Gabrielle J
GJ
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 241, Bethesda, MD 20892, USA.
Hesse
Joanne E
JE
Gellert
Martin
M
D004247:DNA / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C064658:RAG-1 protein; D004247:DNA