title: Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination.
abstract: We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Japan.
authors.forename: F
authors.initials: F
authors.lastname: Nagawa
authors.forename: K
authors.initials: K
authors.lastname: Ishiguro
authors.forename: A
authors.initials: A
authors.lastname: Tsuboi
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: A
authors.initials: A
authors.lastname: Ishikawa
authors.forename: T
authors.initials: T
authors.lastname: Takemori
authors.forename: A J
authors.initials: AJ
authors.lastname: Otsuka
authors.forename: H
authors.initials: H
authors.lastname: Sakano
mesh_terms: D000818:Animals; D018983:DNA Footprinting; D004254:DNA Nucleotidyltransferases; D004268:DNA-Binding Proteins / Q000235:genetics*; D016654:Genes, RAG-1; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011995:Recombination, Genetic; D045567:VDJ Recombinases
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; D011948:Receptors, Antigen, T-Cell; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases