title: Mutational analysis of all conserved basic amino acids in RAG-1 reveals catalytic, step arrest, and joining-deficient mutants in the V(D)J recombinase.
abstract: Although both RAG-1 and RAG-2 are required for all steps of V(D)J recombination, little is known about the specific contribution of either protein to these steps. RAG-1 contains three acidic active-site amino acids that are thought to coordinate catalytic metal ions. To search for additional catalytic amino acids and to better define the functional anatomy of RAG-1, we mutated all 86 conserved basic amino acids to alanine and evaluated the mutant proteins for DNA binding, nicking, hairpin formation, and joining. We found several amino acids outside of the canonical nonamer-binding domain that are critical for DNA binding, several step arrest mutants with defects in nicking or hairpin formation, and four RAG-1 mutants defective specifically for joining. Analysis of coding joints formed by some of these mutants revealed excessive deletions, frequent use of short sequence homologies, and unusually long palindromic junctional inserts, known as P nucleotides, that result from aberrant hairpin opening. These features characterize junctions found in scid mice, which are deficient for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), suggesting that the RAG proteins and DNA-PKcs perform overlapping functions in coding joint formation. Interestingly, the amino acids that are altered in 12 of our mutants are also mutated in human inherited immunodeficiency syndromes. Our analysis of these mutants provides insights into the molecular mechanisms underlying these disorders.
authors.affiliation: Department of Immunolog, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Leslie E
authors.initials: LE
authors.lastname: Huye
authors.forename: Mary M
authors.initials: MM
authors.lastname: Purugganan
authors.forename: Ming-Ming
authors.initials: MM
authors.lastname: Jiang
authors.forename: David B
authors.initials: DB
authors.lastname: Roth
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016654:Genes, RAG-1; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D019895:Transposases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045567:VDJ Recombinases
chemical_list: D000596:Amino Acids; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; C064658:RAG-1 protein; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019895:Transposases; D045567:VDJ Recombinases; D000409:Alanine