title: Real-time analysis of RAG complex activity in V(D)J recombination.
abstract: Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Zagelbaum
authors.affiliation: Department of Pathology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Biochemistry & Molecular Biology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Molecular Microbiology & Immunology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Section of Molecular & Computational Biology, Department of Biological Sciences, University of Southern California (USC), Los Angeles, CA 90089.
authors.forename: Noriko
authors.initials: N
authors.lastname: Shimazaki
authors.affiliation: Department of Pathology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Biochemistry & Molecular Biology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Molecular Microbiology & Immunology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Section of Molecular & Computational Biology, Department of Biological Sciences, University of Southern California (USC), Los Angeles, CA 90089.
authors.forename: Zitadel Anne
authors.initials: ZA
authors.lastname: Esguerra
authors.affiliation: Department of Pathology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Biochemistry & Molecular Biology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Molecular Microbiology & Immunology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Section of Molecular & Computational Biology, Department of Biological Sciences, University of Southern California (USC), Los Angeles, CA 90089.
authors.forename: Go
authors.identifier: 0000-0003-3911-3021
authors.initials: G
authors.lastname: Watanabe
authors.affiliation: Department of Pathology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Biochemistry & Molecular Biology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Molecular Microbiology & Immunology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Section of Molecular & Computational Biology, Department of Biological Sciences, University of Southern California (USC), Los Angeles, CA 90089 eli.rothenberg@nyumc.org lieber@usc.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Lieber
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016; eli.rothenberg@nyumc.org lieber@usc.edu.
authors.forename: Eli
authors.initials: E
authors.lastname: Rothenberg
mesh_terms: D000818:Animals; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D024243:HMGB1 Protein / Q000737:chemistry / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008390:Markov Chains; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D055550:Protein Stability; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D060152:V(D)J Recombination
chemical_list: D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D018398:Homeodomain Proteins; D046912:Multiprotein Complexes; C499308:Rag2 protein, mouse; C064658:RAG-1 protein; D004247:DNA
keywords: V(D)J recombination; antigen receptor gene; immunoglobulin; recombination-activating gene; single-molecule FRET