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David G Schatz - One of the best experts on this subject based on the ideXlab platform.
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The Role of RAG in V(D)J Recombination
Encyclopedia of Immunobiology, 2016Co-Authors: Lina Marcela Carmona, David G SchatzAbstract:V(D)J Recombination is initiated by the lymphoid-specific proteins, RAG1 and RAG2. The Recombination activating genes (RAGs) are found primarily in jawed vertebrates and are highly conserved in these species. Their transcription is tightly regulated throughout lymphocyte development with a number of enhancer elements essential for their expression. The posttranslational modification of RAG2 in a cell cycle–dependent manner also links RAG activity to the cell cycle. RAG cleavage occurs at conserved sequence elements known as Recombination Signal Sequences (RSSs) which demarcate the antigen receptor gene segments for Recombination. The mechanism of DNA cleavage occurs in a two-step direct transesterification reaction which yields a pair of hairpin and blunt ends which are repaired by the nonhomologous end joining pathway. This mechanism shares similarities with the transposition mechanism used by families of cut-and-paste transposable elements (TEs). This and other similarities support the hypothesis that the RAGs and V(D)J Recombination evolved from the integration of a TE. This article will review the mechanism of RAG-mediated DNA cleavage and transposition and provide an overview of the structure of the RAG proteins and locus and their transcriptional and translational regulation.
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single molecule analysis of rag mediated v d j dna cleavage
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Geoffrey Lovely, David G Schatz, David Baltimore, Robert C Brewster, Rob PhillipsAbstract:The Recombination-activating gene products, RAG1 and RAG2, initiate V(D)J Recombination during lymphocyte development by cleaving DNA adjacent to conserved Recombination Signal Sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites.
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synapsis alters rag mediated nicking at tcrb Recombination Signal Sequences implications for the beyond 12 23 rule
Molecular and Cellular Biology, 2014Co-Authors: Joydeep K Banerjee, David G SchatzAbstract:At the Tcrb locus, Vβ-to-Jβ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the “beyond 12/23” rule (B12/23 rule). Previous work showed that Vβ Recombination Signal Sequences (RSSs) do not recombine with Jβ RSSs because Jβ RSSs are crippled for either nicking or synapsis. This result raised the following question: how can crippled Jβ RSSs recombine with Dβ RSSs? We report here that the nicking of some Jβ RSSs can be substantially stimulated by synapsis with a 3′Dβ1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of a partner. Slow-nicking RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial RAG binding can occur on RSSs with either 12- or 23-bp spacers (12- or 23-RSSs, respectively).
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Synapsis alters RAG-mediated nicking at Tcrb Recombination Signal Sequences: implications for the “beyond 12/23” rule.
Molecular and Cellular Biology, 2014Co-Authors: Joydeep K Banerjee, David G SchatzAbstract:At the Tcrb locus, Vβ-to-Jβ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the “beyond 12/23” rule (B12/23 rule). Previous work showed that Vβ Recombination Signal Sequences (RSSs) do not recombine with Jβ RSSs because Jβ RSSs are crippled for either nicking or synapsis. This result raised the following question: how can crippled Jβ RSSs recombine with Dβ RSSs? We report here that the nicking of some Jβ RSSs can be substantially stimulated by synapsis with a 3′Dβ1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of a partner. Slow-nicking RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial RAG binding can occur on RSSs with either 12- or 23-bp spacers (12- or 23-RSSs, respectively).
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Single Molecule Dynamics Governing the Initiation of V(D)J Recombination
Biophysical Journal, 2014Co-Authors: Geoffrey Lovely, David G Schatz, Martin Lindén, Pradeep Ramesh, David Baltimore, Rob PhillipsAbstract:The Recombination activating genes (RAG)1 and RAG2 perform V(D)J Recombination by rearranging conserved Recombination Signal Sequences (RSSs) to generate antigen-receptors during lymphopoiesis. However the orchestration of V(D)J Recombination on biologically relevant (long) length scales has resisted experimental investigation. Here we develop single-molecule assays to watch in real time as RAG1/2 and its co-factor HMGB1 carry out V(D)J Recombination from start (RSS binding) to finish (hairpin formation) on long DNA molecules. We capture various intermediate states preceding hairpin formation, show how RAG1/2 and HMGB1 form bends on the DNA, demonstrate how the identity of the Recombination Signal sequence modulates bending with single bp resolution and show HMGB1 must compact DNA flanking RSSs to form hairpins. Our results provide single-molecule mechanistic insight into the orchestration of V(D)J Recombination.
Craig H. Bassing - One of the best experts on this subject based on the ideXlab platform.
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Poor quality Vβ Recombination Signal Sequences stochastically enforce TCRβ allelic exclusion.
Journal of Experimental Medicine, 2020Co-Authors: Katherine S. Yang-iott, Morgann A. Klink, Katharina E. Hayer, Kyutae D. Lee, Craig H. BassingAbstract:The monoallelic expression of antigen receptor (AgR) genes, called allelic exclusion, is fundamental for highly specific immune responses to pathogens. This cardinal feature of adaptive immunity is achieved by the assembly of a functional AgR gene on one allele, with subsequent feedback inhibition of V(D)J Recombination on the other allele. A range of epigenetic mechanisms have been implicated in sequential Recombination of AgR alleles; however, we now demonstrate that a genetic mechanism controls this process for Tcrb. Replacement of V(D)J recombinase targets at two different mouse Vβ gene segments with a higher quality target elevates Vβ rearrangement frequency before feedback inhibition, dramatically increasing the frequency of T cells with TCRβ chains derived from both Tcrb alleles. Thus, TCRβ allelic exclusion is enforced genetically by the low quality of Vβ recombinase targets that stochastically restrict the production of two functional rearrangements before feedback inhibition silences one allele.
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Poor Quality Vβ Recombination Signal Sequences Enforce TCRβ Allelic Exclusion by Limiting the Frequency of Vβ Recombination
bioRxiv, 2020Co-Authors: Katherine S. Yang-iott, Craig H. Bassing, Katharina E. Hayer, Kyutae D. Lee, Morgann A. ReedAbstract:Monoallelic expression (allelic exclusion) of T and B lymphocyte antigen receptor genes is achieved by the assembly of a functional gene through V(D)J Recombination on one allele and subsequent feedback inhibition of Recombination on the other allele. There has been no validated mechanism for how only one allele of any antigen receptor locus assembles a functional gene prior to feedback inhibition. Here, we demonstrate that replacement of a single V{beta} Recombination Signal sequence (RSS) with a better RSS increases V{beta} rearrangement, reveals Tcrb alleles compete for utilization in the {beta} T cell receptor (TCR) repertoire, and elevates the fraction of {beta} T cells expressing TCR{beta} protein from both alleles. The data indicate that poor qualities of V{beta} RSSs for Recombination with D{beta} and J{beta} RSSs enforces allelic exclusion by stochastically limiting the incidence of functional V{beta} rearrangements on both alleles before feedback inhibition terminates V{beta} Recombination.
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Noncore RAG1 Regions Promote Vβ Rearrangements and αβ T Cell Development by Overcoming Inherent Inefficiency of Vβ Recombination Signal Sequences
Journal of Immunology, 2014Co-Authors: Julie Horowitz, Craig H. BassingAbstract:The RAG proteins are comprised of core endonuclease domains and noncore regions that modulate endonuclease activity. Mutation or deletion of noncore RAG regions in humans causes immunodeficiency and altered TCR repertoire, and mice expressing core but not full-length Rag1 (Rag1C/C) or Rag2 (Rag2C/C) exhibit lymphopenia, reflecting impaired V(D)J Recombination and lymphocyte development. Rag1C/C mice display reduced D-to-J and V-to-DJ rearrangements of TCRβ and IgH loci, whereas Rag2C/C mice show decreased V-to-DJ rearrangements and altered Vβ/VH repertoire. Because Vβs/VHs only recombine to DJ complexes, the Rag1C/C phenotype could reflect roles for noncore RAG1 regions in promoting Recombination during only the D-to-J step or during both steps. In this study, we demonstrate that a preassembled TCRβ gene, but not a preassembled DβJβ complex or the prosurvival BCL2 protein, completely rescues αβ T cell development in Rag1C/C mice. We find that Rag1C/C mice exhibit altered Vβ utilization in Vβ-to-DJβ rearrangements, increased usage of 3′Jα gene segments in Vα-to-Jα rearrangements, and abnormal changes in Vβ repertoire during αβ TCR selection. Inefficient Vβ/VH Recombination Signal Sequences (RSSs) have been hypothesized to cause impaired V-to-DJ Recombination on the background of a defective recombinase as in core-Rag mice. We show that replacement of the Vβ14 RSS with a more efficient RSS increases Vβ14 Recombination and rescues αβ T cell development in Rag1C/C mice. Our data indicate that noncore RAG1 regions establish a diverse TCR repertoire by overcoming Vβ RSS inefficiency to promote Vβ Recombination and αβ T cell development, and by modulating TCRβ and TCRα gene segment utilization.
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Recombination Signal Sequences restrict chromosomal V(D)J Recombination beyond the 12/23 rule.
Nature, 2000Co-Authors: Craig H. Bassing, Frederick W. Alt, Maureen M. Hughes, Margaux D'auteuil, Tara D. Wehrly, Barbara B. Woodman, Frank Gärtner, J. Michael White, Laurie Davidson, Barry P. SleckmanAbstract:The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments1. V(D)J Recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking Recombination Signal Sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction2,3. The conserved heptamer and nonamer Sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting4,5, specifies that V(D)J Recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS1. Vβ segments are appended to DJβ rearrangements, with little or no direct Vβ to Jβ joining, despite 12/23 compatibility of Vβ 23-RSSs and Jβ12-RSSs6,7. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)β locus containing only one Dβ (Dβ1) gene segment and one Jβ (Jβ1) gene cluster to show that the 5′ Dβ1 12-RSS, but not the Jβ1 12-RSSs, targets rearrangement of a diverse Vβ repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J Recombination has important implications for the regulation of variable region gene assembly and repertoire development.
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Recombination Signal Sequences restrict chromosomal v d j Recombination beyond the 12 23 rule
Nature, 2000Co-Authors: Craig H. Bassing, Frederick W. Alt, Maureen M. Hughes, Tara D. Wehrly, Barbara B. Woodman, Frank Gärtner, Laurie Davidson, Margaux Dauteuil, Michael J White, Barry P. SleckmanAbstract:The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments1. V(D)J Recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking Recombination Signal Sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction2,3. The conserved heptamer and nonamer Sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting4,5, specifies that V(D)J Recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS1. Vβ segments are appended to DJβ rearrangements, with little or no direct Vβ to Jβ joining, despite 12/23 compatibility of Vβ 23-RSSs and Jβ12-RSSs6,7. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)β locus containing only one Dβ (Dβ1) gene segment and one Jβ (Jβ1) gene cluster to show that the 5′ Dβ1 12-RSS, but not the Jβ1 12-RSSs, targets rearrangement of a diverse Vβ repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J Recombination has important implications for the regulation of variable region gene assembly and repertoire development.
Michael S. Krangel - One of the best experts on this subject based on the ideXlab platform.
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RSSs set the odds for exclusion.
Journal of Experimental Medicine, 2020Co-Authors: Michael S. KrangelAbstract:In this issue of JEM, Wu et al. (https://doi.org/10.1084/jem.20200412) provide new insights into allelic exclusion. They demonstrate that Vβ-to-DβJβ rearrangement occurs stochastically on two competing Tcrb alleles, with suboptimal Vβ Recombination Signal Sequences limiting synchronous rearrangements and essential for allelic exclusion.
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Orchestrating T-cell receptor α gene assembly through changes in chromatin structure and organization
Immunologic Research, 2011Co-Authors: Han-yu Shih, Bingtao Hao, Michael S. KrangelAbstract:V(D)J Recombination is regulated through changes in chromatin structure that allow recombinase proteins access to Recombination Signal Sequences and through changes in three-dimensional chromatin organization that bring pairs of distant Recombination Signal Sequences into proximity. The Tcra/Tcrd locus is complex and undergoes distinct Recombination programs in double negative and double positive thymocytes that lead to the assembly of Tcrd and Tcra genes, respectively. Our studies provide insights into how locus chromatin structure is regulated and how changes in locus chromatin structure can target and then retarget the recombinase to create developmental progressions of Recombination events. Our studies also reveal distinct locus conformations in double negative and double positive thymocytes and suggest how these conformations may support the distinct Recombination programs in the two compartments.
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Transcription-Dependent Mobilization of Nucleosomes at Accessible TCR Gene Segments In Vivo
Journal of Immunology, 2010Co-Authors: Hrisavgi D. Kondilis-mangum, Robin Milley Cobb, Oleg Osipovich, Sruti Srivatsan, Eugene M. Oltz, Michael S. KrangelAbstract:Accessibility of chromosomal Recombination Signal Sequences to the RAG protein complex is known to be essential for V(D)J Recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis -acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible Recombination Signal Sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at D β , J β , and J α segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to Recombination Signal Sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at J α 61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified.
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Mechanics of T cell receptor gene rearrangement.
Current Opinion in Immunology, 2009Co-Authors: Michael S. KrangelAbstract:The four T cell receptor genes (Tcra, Tcrb, Tcrg, Tcrd) are assembled by V(D)J Recombination according to distinct programs during intrathymic T cell development. These programs depend on genetic factors, including gene segment order and Recombination Signal Sequences. They also depend on epigenetic factors. Regulated changes in chromatin structure, directed by enhancers and promoter, can modify the availability of Recombination Signal Sequences to the RAG recombinase. Regulated changes in locus conformation may control the synapsis of distant Recombination Signal Sequences, and regulated changes in subnuclear positioning may influence locus Recombination events by unknown mechanisms. Together these influences may explain the ordered activation and inactivation of T cell receptor locus Recombination events and the phenomenon of Tcrb allelic exclusion.
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Gene segment selection in V(D)J Recombination: accessibility and beyond
Nature Immunology, 2003Co-Authors: Michael S. KrangelAbstract:V(D)J Recombination assembles genes encoding antigen receptors according to defined developmental programs in immature B and T lymphocytes. The 'accessibility hypothesis' was initially invoked to explain how a single recombinase complex could control the locus and allele specificity of V(D)J Recombination. It has been since shown that Recombination Signal Sequences themselves influence Recombination efficiency and specificity in ways that had not been previously appreciated. Recent developments have increased our understanding of how the chromatin barrier to V(D)J Recombination is regulated, and how chromatin control and the properties of the underlying Recombination Signal Sequences may cooperate to create diverse, lineage-restricted and allelically excluded repertoires of antigen receptors.
Martin Gellert - One of the best experts on this subject based on the ideXlab platform.
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Initial Stages of V(D)J Recombination: The Organization of RAG1/2 and RSS DNA in the Postcleavage Complex
Molecular cell, 2009Co-Authors: Gabrielle J. Grundy, Martin Gellert, Santiago Ramón-maiques, Emilios K. Dimitriadis, Svetlana Kotova, Christian Biertümpfel, J. Bernard Heymann, Alasdair C. Steven, Wei YangAbstract:To obtain structural information on the early stages of V(D)J Recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved Recombination Signal Sequences (RSS). Stoichiometric and molecular mass analysis established that this Signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.
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An Activation-Induced Cytidine Deaminase-Independent Mechanism of Secondary VH Gene Rearrangement in Preimmune Human B Cells
Journal of Immunology, 2008Co-Authors: Nancy S. Longo, Gabrielle J. Grundy, Jisoo Lee, Martin Gellert, Peter E. LipskyAbstract:VH replacement is a form of IgH chain receptor editing that is believed to be mediated by recombinase cleavage at cryptic Recombination Signal Sequences (cRSS) embedded in VH genes. Whereas there are several reports of VH replacement in primary and transformed human B cells and murine models, it remains unclear whether VH replacement contributes to the normal human B cell repertoire. We identified VH→VH(D)JH compound rearrangements from fetal liver, fetal bone marrow, and naive peripheral blood, all of which involved invading and recipient VH4 genes that contain a cryptic heptamer, a 13-bp spacer, and nonamer in the 5′ portion of framework region 3. Surprisingly, all pseudohybrid joins lacked the molecular processing associated with typical VH(D)JH Recombination or nonhomologous end joining. Although inefficient compared with a canonical Recombination Signal Sequences, the VH4 cRSS was a significantly better substrate for in vitro RAG-mediated cleavage than the VH3 cRSS. It has been suggested that activation-induced cytidine deamination (AICDA) may contribute to VH replacement. However, we found similar secondary rearrangements using VH4 genes in AICDA-deficient human B cells. The data suggest that VH4 replacement in preimmune human B cells is mediated by an AICDA-independent mechanism resulting from inefficient but selective RAG activity.
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Inverse transposition by the RAG1 and RAG2 proteins: role reversal of donor and target DNA.
The EMBO journal, 2002Co-Authors: I‐hung Shih, Meni Melek, Nadeesha D. Jayaratne, Martin GellertAbstract:The lymphoid-specific proteins RAG1 and RAG2 initiate V(D)J Recombination by introducing DNA double-strand breaks at the Recombination Signal Sequences (RSSs). In addition to DNA cleavage, the versatile RAG1/2 complex is capable of catalyzing several other reactions, including hybrid joint formation and the transposition of Signal ends into a second DNA. Here we show that the RAG1/2 complex also mediates an unusual strand transfer reaction, inverse transposition, in which non-RSS DNA is cleaved and subsequently transferred to an RSS sequence by direct transesterification. Characterization of the reaction products and requirements suggests that inverse transposition is related to both hybrid joint formation and Signal-end transposition. This aberrant activity provides another possible mechanism for some chromosomal translocations present in lymphoid tumors.
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v d j Recombination rag proteins repair factors and regulation
Annual Review of Biochemistry, 2002Co-Authors: Martin GellertAbstract:▪ Abstract V(D)J Recombination is the specialized DNA rearrangement used by cells of the immune system to assemble immunoglobulin and T-cell receptor genes from the preexisting gene segments. Because there is a large choice of segments to join, this process accounts for much of the diversity of the immune response. Recombination is initiated by the lymphoid-specific RAG1 and RAG2 proteins, which cooperate to make double-strand breaks at specific recognition Sequences (Recombination Signal Sequences, RSSs). The neighboring coding DNA is converted to a hairpin during breakage. Broken ends are then processed and joined with the help of several factors also involved in repair of radiation-damaged DNA, including the DNA-dependent protein kinase (DNA-PK) and the Ku, Artemis, DNA ligase IV, and Xrcc4 proteins, and possibly histone H2AX and the Mre11/Rad50/Nbs1 complex. There may be other factors not yet known. V(D)J Recombination is strongly regulated by limiting access to RSS sites within chromatin, so that par...
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RAG1/2-Mediated Resolution of Transposition Intermediates: Two Pathways and Possible ConSequences
Cell, 2000Co-Authors: Meni Melek, Martin GellertAbstract:During B and T cell development, the RAG1/RAG2 protein complex cleaves DNA at conserved Recombination Signal Sequences (RSS) to initiate V(D)J Recombination. RAG1/2 has also been shown to catalyze transpositional strand transfer of RSS-containing substrates into target DNA to form branched DNA intermediates. We show that RAG1/2 can resolve these intermediates by two pathways. RAG1/2 catalyzes hairpin formation on target DNA adjacent to transposed RSS ends in a manner consistent with a model leading to chromosome translocations. Alternatively, disintegration removes transposed donor DNA from the intermediate. At high magnesium concentrations, such as are present in mammalian cells, disintegration is the favored pathway of resolution. This may explain in part why RAG1/2-mediated transposition does not occur at high frequency in cells.
Hitoshi Sakano - One of the best experts on this subject based on the ideXlab platform.
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RAG-Heptamer Interaction in the Synaptic Complex Is a Crucial Biochemical Checkpoint for the 12/23 Recombination Rule
The Journal of biological chemistry, 2007Co-Authors: Tadashi Nishihara, Fumikiyo Nagawa, Takeshi Imai, Hitoshi SakanoAbstract:In V(D)J Recombination, the RAG1 and RAG2 protein complex cleaves the Recombination Signal Sequences (RSSs), generating a hairpin structure at the coding end. The cleavage occurs only between two RSSs with different spacer lengths of 12 and 23 bp. Here we report that in the synaptic complex, Recombination-activating gene (RAG) proteins interact with the 7-mer and unstack the adjacent base in the coding region. We generated a RAG1 mutant that exhibits reduced RAG-7-mer interaction, unstacking of the coding base, and hairpin formation. Mutation of the 23-RSS at the first position of the 7-mer, which has been reported to impair the cleavage of the partner 12-RSS, demonstrated phenotypes similar to those of the RAG1 mutant; the RAG interaction and base unstacking in the partner 12-RSS are reduced. We propose that the RAG-7-mer interaction is a critical step for coding DNA distortion and hairpin formation in the context of the 12/23 rule.
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Footprint analysis of Recombination Signal Sequences in the 12/23 synaptic complex of V(D)J Recombination.
Molecular and Cellular Biology, 2002Co-Authors: Fumikiyo Nagawa, Masami Kodama, Tadashi Nishihara, Kei-ichiro Ishiguro, Hitoshi SakanoAbstract:In V(D)J joining of antigen receptor genes, two Recombination Signal Sequences (RSSs), 12-RSS and 23-RSS, are paired and complexed with the protein products of Recombination-activating genes RAG1 and RAG2. Using magnetic beads, we purified the pre- and postcleavage complexes of V(D)J joining and analyzed them by DNase I footprinting. In the precleavage synaptic complex, strong protection was seen not only in the 9-mer and spacer regions but also near the coding border of the 7-mer. This is a sharp contrast to the single RSS-RAG complex where the 9-mer plays a major role in the interaction. We also analyzed the postcleavage Signal end complex by footprinting. Unlike what was seen with the precleavage complex, the entire 7-mer and its neighboring spacer regions were protected. The present study indicates that the RAG-RSS interaction in the 7-mer region drastically changes once the synaptic complex is formed for cleavage.
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footprint analysis of Recombination Signal Sequences in the 12 23 synaptic complex of v d j Recombination
Molecular and Cellular Biology, 2002Co-Authors: Fumikiyo Nagawa, Masami Kodama, Tadashi Nishihara, Kei-ichiro Ishiguro, Hitoshi SakanoAbstract:In V(D)J joining of antigen receptor genes, two Recombination Signal Sequences (RSSs), 12-RSS and 23-RSS, are paired and complexed with the protein products of Recombination-activating genes RAG1 and RAG2. Using magnetic beads, we purified the pre- and postcleavage complexes of V(D)J joining and analyzed them by DNase I footprinting. In the precleavage synaptic complex, strong protection was seen not only in the 9-mer and spacer regions but also near the coding border of the 7-mer. This is a sharp contrast to the single RSS-RAG complex where the 9-mer plays a major role in the interaction. We also analyzed the postcleavage Signal end complex by footprinting. Unlike what was seen with the precleavage complex, the entire 7-mer and its neighboring spacer regions were protected. The present study indicates that the RAG-RSS interaction in the 7-mer region drastically changes once the synaptic complex is formed for cleavage.
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The DNA-bending protein, HMG1, is required for correct cleavage of 23 bp Recombination Signal Sequences by Recombination activating gene proteins in vitro.
International Immunology, 2000Co-Authors: Tomoyuki Yoshida, Fumikiyo Nagawa, Kei-ichiro Ishiguro, Akio Tsuboi, Hitoshi SakanoAbstract:DNA-bending proteins are known to facilitate the in vitro V(D)J joining of antigen receptor genes. Here we report that the high-mobility group protein, HMG1, is necessary for the correct nicking of the 23 bp Recombination Signal sequence (23-RSS) by the products of the Recombination activating gene (RAG) proteins, RAG1 and RAG2. Without HMG1, the mouse Jκ1 23-RSS was recognized as if it were the 12-RSS and nicked at a site 12 7 nucleotides away from the 9mer Signal, even though no 7mer-like sequence was evident at the cryptic nicking site. When increased amounts of HMG1 were added, the 23-RSS substrate was nicked correctly at a site 23 7 nucleotides from the 9mer, and nicking at the cryptic site disappeared. Unlike the 23-RSS, the 12-RSS did not require HMG1 for correct nicking, although HMG1 was found to increase the interaction between RSS and RAG proteins. Modification-interference assays demonstrated that HMG1 caused changes in the interaction between the 23-RSS and RAG proteins specifically at the 7mer and the cryptic nicking site.
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FOOTPRINT ANALYSIS OF THE RAG PROTEIN Recombination Signal SEQUENCE COMPLEX FOR V(D)J TYPE Recombination
Molecular and Cellular Biology, 1998Co-Authors: Fumikiyo Nagawa, Kei-ichiro Ishiguro, Tomoyuki Yoshida, Akio Tsuboi, Akiko Ishikawa, Toshitada Takemori, Anthony J. Otsuka, Hitoshi SakanoAbstract:We have studied the interaction between Recombination Signal Sequences (RSSs) and protein products of the truncated forms of Recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.
