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Martin Gellert - One of the best experts on this subject based on the ideXlab platform.
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metabolic sensor ampk directly phosphorylates RAG1 Protein and regulates v d j recombination
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Alexandra L Brown, Samarendra K Singh, Yong Chen, Marjan Gucek, Baeckseung Lee, Megan A Luckey, Myung K Kim, Junghyun Park, Barry P Sleckman, Martin GellertAbstract:The ability to sense metabolic stress is critical for successful cellular adaptation. In eukaryotes, the AMP-activated Protein kinase (AMPK), a highly conserved serine/threonine kinase, functions as a critical metabolic sensor. AMPK is activated by the rising ADP/ATP and AMP/ATP ratios during conditions of energy depletion and also by increasing intracellular Ca2+. In response to metabolic stress, AMPK maintains energy homeostasis by phosphorylating and regulating Proteins that are involved in many physiological processes including glucose and fatty acid metabolism, transcription, cell growth, mitochondrial biogenesis, and autophagy. Evidence is mounting that AMPK also plays a role in a number of pathways unrelated to energy metabolism. Here, we identify the recombination-activating gene 1 Protein (RAG1) as a substrate of AMPK. The RAG1/RAG2 complex is a lymphoid-specific endonuclease that catalyzes specific DNA cleavage during V(D)J recombination, which is required for the assembly of the Ig and T-cell receptor genes of the immune system. AMPK directly phosphorylates RAG1 at serine 528, and the phosphorylation enhances the catalytic activity of the RAG complex, resulting in increased cleavage of oligonucleotide substrates in vitro, or increased recombination of an extrachromosomal substrate in a cellular assay. Our results suggest that V(D)J recombination can be regulated by AMPK activation, providing a potential new link between metabolic stress and development of B and T lymphocytes.
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Initiation of V(D)J recombination in a cell-free system
Cell, 1995Co-Authors: Dik C. Van Gent, Dale A. Ramsden, Joanne E Hesse, J. Fraser Mcblane, Moshe J. Sadofsky, Martin GellertAbstract:Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-length, blunt, 5'-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 Protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 Protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.
Ester Mejstrikova - One of the best experts on this subject based on the ideXlab platform.
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similar recombination activating gene rag mutations result in similar immunobiological effects but in different clinical phenotypes
The Journal of Allergy and Clinical Immunology, 2014Co-Authors: Hanna Ijspeert, Gertjan J Driessen, Michael Moorhouse, Nico G Hartwig, Beata Wolskakusnierz, Krzysztof Kalwak, Anna Pituchnoworolska, Irina Kondratenko, Joris M Van Montfrans, Ester MejstrikovaAbstract:Background V(D)J recombination takes place during lymphocyte development to generate a large repertoire of T- and B-cell receptors. Mutations in recombination-activating gene 1 (RAG1) and RAG2 result in loss or reduction of V(D)J recombination. It is known that different mutations in RAG genes vary in residual recombinase activity and give rise to a broad spectrum of clinical phenotypes. Objective We sought to study the immunologic mechanisms causing the clinical spectrum of RAG deficiency. Methods We included 22 patients with similar RAG1 mutations (c.519delT or c.368_369delAA) resulting in N-terminal truncated RAG1 Protein with residual recombination activity but presenting with different clinical phenotypes. We studied precursor B-cell development, immunoglobulin and T-cell receptor repertoire formation, receptor editing, and B- and T-cell numbers. Results Clinically, patients were divided into 3 main categories: T − B − severe combined immunodeficiency, Omenn syndrome, and combined immunodeficiency. All patients showed a block in the precursor B-cell development, low B- and T-cell numbers, normal immunoglobulin gene use, limited B- and T-cell repertoires, and slightly impaired receptor editing. Conclusion This study demonstrates that similar RAG mutations can result in similar immunobiological effects but different clinical phenotypes, indicating that the level of residual recombinase activity is not the only determinant for clinical outcome. We postulate a model in which the type and moment of antigenic pressure affect the clinical phenotypes of these patients.
Marjorie A. Oettinger - One of the best experts on this subject based on the ideXlab platform.
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Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase
Genes & development, 1999Co-Authors: Deok Ryong Kim, Yan Dai, Cynthia L. Mundy, Wei Yang, Marjorie A. OettingerAbstract:The RAG1 and RAG2 Proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG Proteins requires a divalent metal ion but does not involve a covalent Protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two chemical steps of V(D)J cleavage (nicking and hairpin formation) is severely impaired. Because both chemical steps are affected by the same mutations, a single active site appears responsible for both reactions. Two independent lines of evidence demonstrate that at least two of these acidic residues are directly involved in coordinating a divalent metal ion: The substitution of Cys for Asp allows rescue of some catalytic function, whereas an alanine substitution is no longer subject to iron-induced hydroxyl radical cleavage. Our results support a model in which the RAG1 Protein contains the active site of the V(D)J recombinase and are interpreted in light of predictions about the structure of RAG1.
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Regions of RAG1 Protein critical for V(D)J recombination.
European journal of immunology, 1996Co-Authors: Susan A. Kirch, Priya Sudarsanam, Marjorie A. OettingerAbstract:The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutations for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conserved sequence. We show here that this “dispensable” region of RAG1 is not necessary for coding joint formation or for recombination of an integrated substrate, and that this portion is not functionally redundant with the “dispensable” region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.
Moshe J. Sadofsky - One of the best experts on this subject based on the ideXlab platform.
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a ww like module in the RAG1 n terminal domain contributes to previously unidentified Protein Protein interactions
Nucleic Acids Research, 2009Co-Authors: Radhashree Maitra, Moshe J. SadofskyAbstract:More than one-third of the RAG1 Protein can be truncated from the N-terminus with only subtle effects on the products of V(D)J recombination in vitro or in a mouse. What, then, is the function of the N-terminal domain? We believe it to be regulatory. We determined, several years ago, that an included RING motif could function as an ubiquitin E3 ligase. Whether this activity is limited to automodification, or may alter other Proteins in the cell, remains an open question. We revisited the issue of additional Protein-Protein interactions between RAG1 and other Proteins by means of the yeast two-hybrid assay. We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66. A luciferase reporter assay demonstrates that a Protein complex containing RAG Proteins and the transcription factor can assemble in cells. Further mapping identified a region within the N-terminal domain resembling a WW motif. Point mutation directed at residues conserved in WW motifs eliminated binding to one of the partners. Phylogenetic analysis shows the WW-like module to be highly conserved. The module contributes to Protein-Protein interactions that may also influence how RAG1 binds DNA targets.
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Initiation of V(D)J recombination in a cell-free system
Cell, 1995Co-Authors: Dik C. Van Gent, Dale A. Ramsden, Joanne E Hesse, J. Fraser Mcblane, Moshe J. Sadofsky, Martin GellertAbstract:Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-length, blunt, 5'-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 Protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 Protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.
Craig H. Bassing - One of the best experts on this subject based on the ideXlab platform.
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the RAG1 ubiquitin ligase domain enhances t cell receptor gene assembly and thymic selection
bioRxiv, 2021Co-Authors: Craig H. Bassing, Jessica M. Jones, Thomas N Burn, Charline Miot, Portia A Kreiger, Katharina E Hayer, Anamika Bhattacharyya, Edward M BehrensAbstract:RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte antigen receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. However, this process creates highly self-reactive cells that must be negatively selected to suppress autoimmunity. The RAG1 Protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity. We report here that mice lacking RAG1 ubiquitin ligase activity exhibit diminished recombination of T cell receptor (TCR) {beta} and loci, and impaired thymocyte developmental transitions that require the assembly of these genes and signaling by their Proteins. The mice also have reduced expression of TCR signaling Proteins within thymocytes, less efficient negative selection of highly self-reactive thymocytes, and mature {beta} T cells of elevated autoimmune potential. Thus, we propose that the RAG1 ubiquitin ligase domain provides {beta} T cell developmental stage-specific means to augment TCR signaling and thereby enhance selection for beneficial TCR genes and against {beta} TCRs possessing high autoimmune potential.
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immature lymphocytes inhibit RAG1 and rag2 transcription and v d j recombination in response to dna double strand breaks
Journal of Immunology, 2017Co-Authors: David G. Schatz, Megan R Fisher, Adrian Riverareyes, Noah B Bloch, Craig H. BassingAbstract:Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double-strand breaks (DSBs) induced at Igκ loci by the RAG1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one Igκ allele signal through the ataxia telangiectasia mutated (ATM) kinase to feedback-inhibit RAG expression and RAG cleavage of the other Igκ allele. In this article, we show that DSBs induced by ionizing radiation, etoposide, or bleomycin suppress RAG1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of RAG1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of RAG1 and Rag2 , causing downregulation of both RAG1 and Rag2 mRNA, but only RAG1 Protein. This transcriptional inhibition requires the ATM kinase and the NF-κB essential modulator Protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igκ loci and a chromosomally integrated substrate. Our data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic Ag receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of monospecific lymphocytes while suppressing oncogenic Ag receptor locus translocations.
