A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.
The variable portions of antigen receptor genes are assembled from component gene segments by a site-specific recombination reaction known as V(D)J recombination. The RAG1 and RAG2 proteins are the critical lymphoid cell-specific components of the recombination enzymatic machinery and are responsible for site-specific DNA recognition and cleavage. Previous studies had defined a minimal, recombinationally active core region of murine RAG1 consisting of amino acids 384 to 1008 of the 1,040-residue RAG1 protein. No recombination function has heretofore been ascribed to any portion of the 383-amino-acid N-terminal region that is missing from the core, but it seems likely to be of functional significance, based on its evolutionary conservation. Using extrachromosomal recombination substrates, we demonstrate here that the N-terminal region enhances the recombination activity of RAG1 by up to an order of magnitude in a variety of cell lines. Deletion analysis localized a region of the N terminus critical for this effect to amino acids 216 to 238, and further mutagenesis demonstrated that a small basic amino acid motif (BIIa) in this region is essential for enhancing the activity of RAG1. Despite the fact that BIIa is important for the interaction of RAG1 with the nuclear localization factor Srp-1, it does not appear to enhance recombination by facilitating nuclear transport of RAG1. A variety of models for how this region stimulates the recombination activity of RAG1 are considered.
McMahan
C J
CJ
Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.
Difilippantonio
M J
MJ
Rao
N
N
Spanopoulou
E
E
Schatz
D G
DG
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D011995:Recombination, Genetic / Q000235:genetics*; D017384:Sequence Deletion
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C064658:RAG-1 protein