title: Identification and characterization of a gain-of-function RAG-1 mutant.
abstract: RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis.
authors.affiliation: Dept. of Medical Microbiology and Immunology, Creighton University Medical Center, 2500 California Plaza, Omaha, NE 68178, USA.
authors.forename: Aleksei N
authors.initials: AN
authors.lastname: Kriatchko
authors.forename: Dirk K
authors.initials: DK
authors.lastname: Anderson
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Swanson
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016654:Genes, RAG-1; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; D011994:Recombinant Proteins; C064658:RAG-1 protein; D004247:DNA