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David G. Schatz - One of the best experts on this subject based on the ideXlab platform.
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Direct observation of RAG recombinase recruitment to chromatin and the IgH locus in live pro-B cells
2020Co-Authors: Geoffrey A. Lovely, David G. Schatz, Fatima-zohra Braikia, Amit Singh, Cornelis Murre, Zhe Liu, Ranjan SenAbstract:The RAG1 and RAG2 proteins introduce double-strand DNA breaks at antigen-receptor loci in developing lymphocytes to initiate V(D)J recombination. How RAG proteins find the correct target locus in a vast excess of non-specific chromatin is not known. Here we measured dynamics of RAG1/RAG2 interactions with chromatin in living pro-B cells. We found that the majority of RAG1 or RAG1/RAG2 complex is in a fast 3D diffusive state, and the residual slow diffusive (bound) fraction was determined by a non-core portion of RAG1, and the PHD domain of RAG2. The RAG proteins exhibited distinct dynamics at the IgH locus. In particular, RAG2 increased the probability of RAG1 binding to IgH, a property that likely explains its non-catalytic role in V(D)J recombination. Our observations reveal how RAG finds its targets in developing B cells. One Sentence SummarySingle-molecule imaging of the RAG recombinase reveals its search strategy for chromatin, H3K4me3 and antibody gene loci in living cells.
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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.
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New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination
The FEBS journal, 2017Co-Authors: Lina Marcela Carmona, David G. SchatzAbstract:The adaptive immune system of jawed vertebrates relies on V(D)J recombination as one of the main processes to generate the diverse array of receptors necessary for the recognition of a wide range of pathogens. The DNA cleavage reaction necessary for the assembly of the antigen receptor genes from an array of potential gene segments is mediated by the recombination-activating gene proteins RAG1 and RAG2. The RAG proteins have been proposed to originate from a transposable element (TE) as they share mechanistic and structural similarities with several families of transposases and are themselves capable of mediating transposition. A number of RAG-like proteins and TEs with sequence similarity to RAG1 and RAG2 have been identified, but only recently has their function begun to be characterized, revealing mechanistic links to the vertebrate RAGs. Of particular significance is the discovery of ProtoRAG, a transposon superfamily found in the genome of the basal chordate amphioxus. ProtoRAG has many of the sequence and mechanistic features predicted for the ancestral RAG transposon and is likely to be an evolutionary relative of RAG1 and RAG2. In addition, early observations suggesting that RAG1 is able to mediate V(D)J recombination in the absence of RAG2 have been confirmed, implying independent evolutionary origins for the two RAG genes. Here, recent progress in identifying and characterizing RAG-like proteins and the TEs that encode them is summarized and a refined model for the evolution of V(D)J recombination and the RAG proteins is presented.
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RAG1 targeting in the genome is dominated by chromatin interactions mediated by the non-core regions of RAG1 and RAG2.
Nucleic acids research, 2016Co-Authors: Yaakov Maman, Grace Teng, Rashu B. Seth, Steven H. Kleinstein, David G. SchatzAbstract:The RAG1/RAG2 endonuclease initiates V(D)J recombination at antigen receptor loci but also binds to thousands of places outside of these loci. RAG2 localizes directly to lysine 4 trimethylated histone 3 (H3K4me3) through a plant homeodomain (PHD) finger. The relative contribution of RAG2-dependent and RAG1-intrinsic mechanisms in determining RAG1 binding patterns is not known. Through analysis of deep RAG1 ChIP-seq data, we provide a quantitative description of the forces underlying genome-wide targeting of RAG1. Surprisingly, sequence-specific DNA binding contributes minimally to RAG1 targeting outside of antigen receptor loci. Instead, RAG1 binding is driven by two distinct modes of interaction with chromatin: the first is driven by H3K4me3, promoter-focused and dependent on the RAG2 PHD, and the second is defined by H3K27Ac, enhancer-focused and dependent on 'non-core' portions of RAG1. Based on this and additional chromatin and genomic features, we formulated a predictive model of RAG1 targeting to the genome. RAG1 binding sites predicted by our model correlate well with observed patterns of RAG1-mediated breaks in human pro-B acute lymphoblastic leukemia. Overall, this study provides an integrative model for RAG1 genome-wide binding and off-target activity and reveals a novel role for the RAG1 non-core region in RAG1 targeting.
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Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination.
Genes & development, 2016Co-Authors: Lina Marcela Carmona, Sebastian D. Fugmann, David G. SchatzAbstract:The recombination-activating gene 1 (RAG1) and RAG2 proteins initiate V(D)J recombination, the process that assembles the B- and T-lymphocyte antigen receptor genes of jawed vertebrates. RAG1 and RAG2 are thought to have arisen from a transposable element, but the origins of this element are not understood. We show that two ancestral RAG1 proteins, Transib transposase and purple sea urchin RAG1-like, have a latent ability to initiate V(D)J recombination when coexpressed with RAG2 and that in vitro transposition by Transib transposase is stimulated by RAG2. Conversely, we report low levels of V(D)J recombination by RAG1 in the absence of RAG2. Recombination by RAG1 alone differs from canonical V(D)J recombination in having lost the requirement for asymmetric DNA substrates, implicating RAG2 in the origins of the "12/23 rule," a fundamental regulatory feature of the reaction. We propose that evolution of RAG1/RAG2 began with a Transib transposon whose intrinsic recombination activity was enhanced by capture of an ancestral RAG2, allowing for the development of adaptive immunity.
Glen L Hartman - One of the best experts on this subject based on the ideXlab platform.
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Characterization and genetics of multiple soybean aphid biotype resistance in five soybean plant introductions
Theoretical and Applied Genetics, 2017Co-Authors: Curtis B Hill, Derek Shiao, Glen L HartmanAbstract:Key message Five soybean plant introductions expressed antibiosis resistance to multiple soybean aphid biotypes. Two introductions had resistance genes located in the RAG1, Rag2 , and Rag3 regions; one introduction had resistance genes located in the RAG1, Rag2 , and rag4 regions; one introduction had resistance genes located in the RAG1 and Rag2 regions; and one introduction had a resistance gene located in the Rag2 region. Abstract Soybean aphid ( Aphis glycines Matsumura) is the most important soybean [ Glycine max (L.) Merr.] insect pest in the USA. The objectives of this study were to characterize the resistance expressed in five plant introductions (PIs) to four soybean aphid biotypes, determine the mode of resistance inheritance, and identify markers associated with genes controlling resistance in these accessions. Five soybean PIs, from an initial set of 3000 PIs, were tested for resistance against soybean aphid biotypes 1, 2, 3, and 4 in choice and no-choice tests. Of these five PIs, PI 587663, PI 587677, and PI 587685 expressed antibiosis against all four biotypes, while PI 587972 and PI 594592 expressed antibiosis against biotypes 1, 2, and 3. F_2 populations derived from PI 587663 and PI 587972 were evaluated for resistance against soybean aphid biotype 1, and populations derived from PIs 587677, 587685, and 594592 were tested against biotype 3. In addition, F_2:3 plants were tested against biotypes 2 and 3. Genomic DNA from F_2 plants was screened with markers linked to RAG1, Rag2, Rag3 , and rag4 soybean aphid-resistance genes. Results showed that PI 587663 and PI 594592 each had three genes with variable gene action located in the RAG1, Rag2 , and Rag3 regions. PI 587677 had three genes with variable gene action located in the RAG1, Rag2 and rag4 regions. PI 587685 had one dominant gene located in the RAG1 region and an additive gene in the Rag2 region. PI 587972 had one dominant gene located in the Rag2 region controlling antixenosis- or antibiosis-type resistance to soybean aphid biotypes 1, 2, or 3. PIs 587663, 587677, and 587685 also showed antibiosis-type resistance against biotype 4. Information on multi-biotype aphid resistance and resistance gene markers will be useful for improving soybean aphid resistance in commercial soybean cultivars.
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characterization and genetics of multiple soybean aphid biotype resistance in five soybean plant introductions
Theoretical and Applied Genetics, 2017Co-Authors: Curtis B Hill, Derek Shiao, Glen L HartmanAbstract:Key message Five soybean plant introductions expressed antibiosis resistance to multiple soybean aphid biotypes. Two introductions had resistance genes located in the RAG1, Rag2, and Rag3 regions; one introduction had resistance genes located in the RAG1, Rag2, and rag4 regions; one introduction had resistance genes located in the RAG1 and Rag2 regions; and one introduction had a resistance gene located in the Rag2 region.
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differential reactions of soybean isolines with combinations of aphid resistance genes RAG1 rag2 and rag3 to four soybean aphid biotypes
Journal of Economic Entomology, 2016Co-Authors: Olutoyosi O Ajayioyetunde, Curtis B Hill, Ursula Reutercarlson, Doris Lagoskutz, Carl A. Bradley, Brian W Diers, Glen L HartmanAbstract:With the discovery of the soybean aphid ( Aphis glycines Matsumura) as a devastating insect pest of soybean ( Glycine max (L.) Merr.) in the United States, host resistance was recognized as an important management option. However, the identification of soybean aphid isolates exhibiting strong virulence against aphid resistance genes ( Rag genes) has highlighted the need for pyramiding genes to help ensure the durability of host resistance as a control strategy. In this study, soybean isolines with all possible combinations of the resistance and susceptibility alleles at RAG1 , Rag2 , and Rag3 were evaluated for their effectiveness against the four characterized soybean aphid biotypes. All soybean isolines, including the susceptible check carrying none of the resistance alleles (S1/S2/S3), were infested with each biotype in no-choice greenhouse tests, and the aphid populations developed on each isoline were enumerated 14 d after infestation. All gene combinations, with the exception of Rag3 alone, provided excellent protection against biotype 1. Isolines with Rag2 alone or in combination with RAG1 and Rag3 had greater levels of resistance to biotype 2 than those with either RAG1 alone, Rag3 alone, or the RAG1/3 pyramid. For biotype 3, the RAG1/3 and RAG1/2/3 pyramided lines significantly reduced aphid populations compared with all other gene combinations, while the RAG1/2/3 pyramid provided the greatest protection against biotype 4. Overall, the RAG1/2/3 pyramided line conferred the greatest protection against all four biotypes.
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identification and molecular mapping of two soybean aphid resistance genes in soybean pi 587732
Theoretical and Applied Genetics, 2014Co-Authors: Anitha Chirumamilla, Curtis B Hill, Glen L Hartman, Brian W DiersAbstract:Soybean [Glycine max (L.) Merr.] continues to be plagued by the soybean aphid (Aphis glycines Matsumura: SA) in North America. New soybean resistance sources are needed to combat the four identified SA biotypes. The objectives of this study were to determine the inheritance of SA resistance in PI 587732 and to map resistance gene(s). For this study, 323 F2 and 214 F3 plants developed from crossing PI 587732 to two susceptible genotypes were challenged with three SA biotypes and evaluated with genetic markers. Choice tests showed that resistance to SA Biotype 1 in the first F2 population was controlled by a gene in the RAG1 region on chromosome 7, while resistance to SA Biotype 2 in the second population was controlled by a gene in the Rag2 region on chromosome 13. When 134 F3 plants segregating in both the RAG1 and Rag2 regions were tested with a 1:1 mixture of SA Biotypes 1 and 2, the Rag2 region and an interaction between the RAG1 and Rag2 regions were significantly associated with the resistance. Based on the results of the non-choice tests, the resistance gene in the RAG1 region in PI 587732 may be a different allele or gene from RAG1 from Dowling because the PI 587732 gene showed antibiosis type resistance to SA Biotype 2 while RAG1 from Dowling did not. The two SA resistance loci and genetic marker information from this study will be useful in increasing diversity of SA resistance sources and marker-assisted selection for soybean breeding programs.
Marjorie A. Oettinger - One of the best experts on this subject based on the ideXlab platform.
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rag a recombinase diversified
Nature Immunology, 2009Co-Authors: Adam G. W. Matthews, Marjorie A. OettingerAbstract:To generate a diverse repertoire of antigen receptors, developing B cells and T cells undergo a complex series of DNA rearrangements collectively termed V(D)J recombination. This process is initiated by the lymphoid-specific proteins RAG1 and RAG2, which function together to generate site-specific DNA double-strand breaks that are then repaired via the classical non-homologous end-joining (NHEJ) pathway. While it is well established that RAG1-RAG2 can function as a recombinase, several recent studies have revealed that RAG1-RAG2 is actually a surprisingly multifaceted enzyme complex that plays an important role in ensuring that V(D)J recombination is faithfully executed and properly regulated in the cell. In this Review, we discuss the role of the RAG1-RAG2 complex in binding to accessible chromatin, mediating allelic pairing during V(D)J recombination, and channeling RAG-generated double-strand breaks towards the classical non-homologous end-joining (NHEJ) pathway (Fig. 1). We conclude by proposing a speculative model in which the RAG1-RAG2 recombinase functions within a specialized subnuclear compartment that we term the V(D)J recombination factory. Figure 1 Multilayered regulation of V(D)J recombination
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rag a recombinase diversified
Nature Immunology, 2009Co-Authors: Adam G. W. Matthews, Marjorie A. OettingerAbstract:To generate a diverse repertoire of antigen receptors, developing B cells and T cells undergo a complex series of DNA rearrangements collectively termed V(D)J recombination. This process is initiated by the lymphoid-specific proteins RAG1 and RAG2, which function together to generate site-specific DNA double-strand breaks that are then repaired via the classical non-homologous end-joining (NHEJ) pathway. While it is well established that RAG1-RAG2 can function as a recombinase, several recent studies have revealed that RAG1-RAG2 is actually a surprisingly multifaceted enzyme complex that plays an important role in ensuring that V(D)J recombination is faithfully executed and properly regulated in the cell. In this Review, we discuss the role of the RAG1-RAG2 complex in binding to accessible chromatin, mediating allelic pairing during V(D)J recombination, and channeling RAG-generated double-strand breaks towards the classical non-homologous end-joining (NHEJ) pathway (Fig. 1). We conclude by proposing a speculative model in which the RAG1-RAG2 recombinase functions within a specialized subnuclear compartment that we term the V(D)J recombination factory. Figure 1 Multilayered regulation of V(D)J recombination
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The C-terminal portion of RAG2 protects against transposition in vitro
The EMBO journal, 2003Co-Authors: Sheryl K. Elkin, Adam G. W. Matthews, Marjorie A. OettingerAbstract:The assembly of antigen receptor genes by V(D)J recombination is initiated by the RAG1/RAG2 protein complex, which introduces double-strand breaks between recombination signal sequences and their coding DNA. Truncated forms of RAG1 and RAG2 are functional in vivo and have been used to study V(D)J cleavage, hybrid joint formation and transposition in vitro. Here we have characterized the activities of the full-length proteins. Unlike core RAG2, which supports robust transposition in vitro, full-length RAG2 blocks transposition of signal ends following V(D)J cleavage. Thus, one role of this non-catalytic domain may be to prevent transposition in developing lymphoid cells. Although full-length RAG1 and RAG2 proteins rarely form hybrid joints in vivo in the absence of non-homologous end-joining factors, we show that the full-length proteins alone can catalyze this reaction in vitro.
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Assembly of the RAG1/RAG2 Synaptic Complex
Molecular and cellular biology, 2002Co-Authors: Cynthia L. Mundy, Adam G. W. Matthews, Nadja Patenge, Marjorie A. OettingerAbstract:Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.
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assembly of the RAG1 rag2 synaptic complex
Molecular and Cellular Biology, 2002Co-Authors: Cynthia L. Mundy, Adam G. W. Matthews, Nadja Patenge, Marjorie A. OettingerAbstract:Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.
Martin Gellert - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the v d j recombinase RAG1 rag2
Nature, 2015Co-Authors: Min Sung Kim, Mikalai Lapkouski, Wei Yang, Martin GellertAbstract:V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2 A resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.
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Organization of RAG1/2 and RSS DNA in the Post-Cleavage Complex
Biophysical Journal, 2010Co-Authors: Svetlana L. Kotova, Martin Gellert, Santiago Ramón-maiques, Emilios K. Dimitriadis, J. Bernard Heymann, Alasdair C. Steven, Gabriel J. Grundy, Wei YangAbstract:V(D)J recombination is central to establishing a functional adaptive immune system. The large repertoire of immunoglobulins and T-cell receptors is generated by combinatorial rearrangement of an extensive array of variable (V), diversity (D), and joining (J) gene segments that are joined to encode the variable domains of the protein chains. The recombination signal sequences (RSS) that flank these gene segments are recognized, paired in a synaptic complex, and cleaved by collaboration of the lymphoid-specific proteins RAG1 and RAG2. After cleavage, the signal ends remain tightly bound to the RAG proteins in a particularly stable Signal-End Complex (SEC).To obtain 3D structural information about RAG1/2 bound to RSS DNA, isolated and purified SEC were visualized by AFM. To better define the arrangement of the RAG proteins and RSS DNA in the complex, we used RAG1 and RAG2 fused with maltose binding protein (MBP). A wide variety of complex shapes was recorded, however, it was clear that the two DNA chains predominantly exited the SEC complex from adjacent points. The volume of the protein core was consistent with the expected mass of 500 kDa corresponding to (RAG1)2-(RAG2)2 composition. MBP protrusions could be observed on the protein particles marking the N-termini of RAG1 and RAG2. To make their appearance more noticeable, we used selective antibody labeling. Fab-labeled MBPs were clearly identified peripheral to the recombinase core. When only the RAG2 MBPs were labeled, the two DNAs most often exited together from the SEC on the opposite side to the Fabs. Consistent with this observation, when only the RAG1 MBPs were labeled, they were situated closer to the exiting DNAs.The parallel arrangement of DNA and protein subunits found by AFM is in an excellent agreement with the 3D model based on EM data.
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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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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, Emilios K. Dimitriadis, Svetlana Kotova, Christian Biertümpfel, Alasdair C. Steven, Santiago Ramonmaiques, Bernard J Heymann, 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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A Stable RAG1–RAG2–DNA Complex That Is Active in V(D)J Cleavage
Cell, 1997Co-Authors: Kevin Hiom, Martin GellertAbstract:Abstract The RAG1 and RAG2 proteins initiate V(D)J recombination by making specific double-strand DNA breaks at recombination signal sequences. We show here that RAG1 and RAG2 bind specifically to this sequence, forming a stable protein–DNA complex. The complex requires the conserved heptamer and nonamer motifs of the recombination signal as well as both the RAG1 and RAG2 proteins. This complex is able to either nick or form hairpins at the V(D)J signal sequence, depending on the divalent cation present. A complex trapped using Ca 2+ is subsequently active when transferred to Mg 2+ or Mn 2+ . After cleavage, the complex is destabilized and the RAG proteins dissociate. We term this early precursor in the V(D)J recombination reaction a "stable cleavage complex."
Wei Yang - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the v d j recombinase RAG1 rag2
Nature, 2015Co-Authors: Min Sung Kim, Mikalai Lapkouski, Wei Yang, Martin GellertAbstract:V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2 A resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.
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Crystal structure of the V(D)J recombinase RAG1–RAG2
Nature, 2015Co-Authors: Min Sung Kim, Mikalai Lapkouski, Wei YangAbstract:V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2 A resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.
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Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Gabrielle J. Grundy, Wei YangAbstract:Gene assembly of the variable domain of antigen receptors is initiated by DNA cleavage by the RAG1–RAG2 protein complex at sites flanking V, D, and J gene segments. Double-strand breaks are produced via a single-strand nick that is converted to a hairpin end on coding DNA and a blunt end on the neighboring recombination signal sequence. We demonstrate that the C-terminal regions of purified murine RAG1 (aa 1009–1040) and RAG2 (aa 388–520, including a plant homeodomain [PHD domain]) collaborate to inhibit the hairpinning stage of DNA cleavage. The C-terminal region of RAG2 stabilizes the RAG1/2 heterotetramer but destabilizes the RAG–DNA precleavage complex. This destabilization is reversed by binding of the PHD domain to a histone H3 peptide trimethylated on lysine 4 (H3K4me3). The addition of H3K4me3 likewise alleviates the RAG1/RAG2 C-terminus-mediated inhibition of hairpinning and the PHD-mediated inhibition of transposition activity. Thus a negative regulatory function of the noncore regions of RAG1/2 limits the RAG endonuclease activity in the absence of an activating methylated histone tail bound to the complex.
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Organization of RAG1/2 and RSS DNA in the Post-Cleavage Complex
Biophysical Journal, 2010Co-Authors: Svetlana L. Kotova, Martin Gellert, Santiago Ramón-maiques, Emilios K. Dimitriadis, J. Bernard Heymann, Alasdair C. Steven, Gabriel J. Grundy, Wei YangAbstract:V(D)J recombination is central to establishing a functional adaptive immune system. The large repertoire of immunoglobulins and T-cell receptors is generated by combinatorial rearrangement of an extensive array of variable (V), diversity (D), and joining (J) gene segments that are joined to encode the variable domains of the protein chains. The recombination signal sequences (RSS) that flank these gene segments are recognized, paired in a synaptic complex, and cleaved by collaboration of the lymphoid-specific proteins RAG1 and RAG2. After cleavage, the signal ends remain tightly bound to the RAG proteins in a particularly stable Signal-End Complex (SEC).To obtain 3D structural information about RAG1/2 bound to RSS DNA, isolated and purified SEC were visualized by AFM. To better define the arrangement of the RAG proteins and RSS DNA in the complex, we used RAG1 and RAG2 fused with maltose binding protein (MBP). A wide variety of complex shapes was recorded, however, it was clear that the two DNA chains predominantly exited the SEC complex from adjacent points. The volume of the protein core was consistent with the expected mass of 500 kDa corresponding to (RAG1)2-(RAG2)2 composition. MBP protrusions could be observed on the protein particles marking the N-termini of RAG1 and RAG2. To make their appearance more noticeable, we used selective antibody labeling. Fab-labeled MBPs were clearly identified peripheral to the recombinase core. When only the RAG2 MBPs were labeled, the two DNAs most often exited together from the SEC on the opposite side to the Fabs. Consistent with this observation, when only the RAG1 MBPs were labeled, they were situated closer to the exiting DNAs.The parallel arrangement of DNA and protein subunits found by AFM is in an excellent agreement with the 3D model based on EM data.
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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.