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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2007-02, Vol.104 (9), p.3078-3083
Main Authors: Grundy, Gabrielle J, Hesse, Joanne E, Gellert, Martin
Format: Article
Language:English
Subjects:
3T3 cells
Active sites
Antigens
Biochemistry
Biological Sciences
Chemical bases
Crystal structure
Deoxyribonucleic acid
DNA
DNA - metabolism
DNA Breaks, Single-Stranded
DNA cleavage
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Electrophoretic Mobility Shift Assay
Enzymes
Gels
Genetic mutation
Genetic transposition
Homeodomain Proteins - genetics
Homeodomain Proteins - metabolism
Mutagenesis
Mutation - genetics
Nuclear Proteins - genetics
Nuclear Proteins - metabolism
Nucleic Acid Conformation
Oligonucleotides
Substrates
Transposons
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0611293104