title: An interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex.
abstract: V(D)J recombination assembles functional antigen receptor genes during lymphocyte development. Formation of the recombination complex containing the recombination activating proteins, RAG1 and RAG2, is essential for the site-specific DNA cleavage steps in V(D)J recombination. However, little is known concerning how complex formation leads to a catalytically-active complex. Here, we combined limited proteolysis and mass spectrometry methods to identify regions of RAG1 that are sequestered upon association with RAG2. These results show that RAG2 bridges an interdomain boundary in the catalytic region of RAG1. In a second approach, mutation of RAG1 residues within the interdomain boundary were tested for disruption of RAG1:RAG2 complex formation using fluorescence-based pull down assays. The core RAG1 mutants demonstrated varying effects on complex formation with RAG2. Interestingly, two mutants showed opposing results for the ability to interact with core versus full length RAG2, indicating that the non-core region of RAG2 participates in binding to core RAG1. Significantly, all of the RAG1 interdomain mutants demonstrated altered stoichiometries of the RAG complexes, with an increased number of RAG2 per RAG1 subunit compared to the wild type complex. Based on our results, we propose that interaction of RAG2 with RAG1 induces cooperative interactions of multiple binding sites, induced through conformational changes at the RAG1 interdomain boundary, and resulting in formation of the DNA cleavage active site.
authors: Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73190.
authors: Jennifer N
authors: JN
authors: Byrum
authors: Shuying
authors: S
authors: Zhao
authors: Negar S
authors: NS
authors: Rahman
authors: Lori M
authors: LM
authors: Gwyn
authors: William
authors: W
authors: Rodgers
authors: Karla K
authors: KK
authors: Rodgers
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D045567:VDJ Recombinases / Q000737:chemistry / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D046912:Multiprotein Complexes; C499308:Rag2 protein, mouse; C064658:RAG-1 protein; D045567:VDJ Recombinases
keywords: RAG1; RAG2; V(D)J recombination; flow cytometry; limited proteolysis; mass spectrometry; protein-protein interactions