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David G. Schatz - One of the best experts on this subject based on the ideXlab platform.
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Nucleolar localization of RAG1 modulates V(D)J recombination activity.
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Ryan M. Brecht, Catherine C. Liu, Helen A. Beilinson, Alexandra Khitun, Sarah A. Slavoff, David G. SchatzAbstract:V(D)J recombination assembles and diversifies Ig and T cell receptor genes in developing B and T lymphocytes. The reaction is initiated by the RAG1-RAG2 Protein complex which binds and cleaves at discrete gene segments in the antigen receptor loci. To identify mechanisms that regulate V(D)J recombination, we used proximity-dependent biotin identification to analyze the interactomes of full-length and truncated forms of RAG1 in pre-B cells. This revealed an association of RAG1 with numerous nucleolar Proteins in a manner dependent on amino acids 216 to 383 and allowed identification of a motif required for nucleolar localization. Experiments in transformed pre-B cell lines and cultured primary pre-B cells reveal a strong correlation between disruption of nucleoli, reduced association of RAG1 with a nucleolar marker, and increased V(D)J recombination activity. Mutation of the RAG1 nucleolar localization motif boosts recombination while removal of the first 215 amino acids of RAG1, required for efficient egress from nucleoli, reduces recombination activity. Our findings indicate that nucleolar sequestration of RAG1 is a negative regulatory mechanism in V(D)J recombination and identify regions of the RAG1 N-terminal region that control nucleolar association and egress.
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Peripheral subnuclear positioning suppresses Tcrb recombination and segregates Tcrb alleles from RAG2
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Elizabeth A. W. Chan, Craig H. Bassing, David G. Schatz, Grace Teng, Elizabeth L. Corbett, Kingshuk Roy Choudhury, Michael S. KrangelAbstract:Allelic exclusion requires that the two alleles at antigen-receptor loci attempt to recombine variable (V), diversity (D), and joining (J) gene segments [V(D)J recombination] asynchronously in nuclei of developing lymphocytes. It previously was shown that T-cell receptor β (Tcrb) alleles frequently and stochastically associate with the nuclear lamina and pericentromeric heterochromatin in CD4−CD8− thymocytes. Moreover, rearranged alleles were underrepresented at these locations. Here we used 3D immunofluorescence in situ hybridization to identify recently rearranged Tcrb alleles based on the accumulation of the DNA-repair Protein 53BP1. We found that Tcrb alleles recombine asynchronously in double-negative thymocytes and that V(D)J recombination is suppressed on peripheral as compared with central Tcrb alleles. Moreover, the recombination events that did take place at the nuclear periphery preferentially occurred on Tcrb alleles that were partially dissociated from the nuclear lamina. To understand better the mechanism by which V(D)J recombination is suppressed at the nuclear periphery, we evaluated the subnuclear distribution of recombination-activating gene 2 (RAG2) Protein. We found that RAG2 abundance was reduced at the nuclear periphery. Moreover, RAG2 was distributed differently from RNA polymerase II and histone H3K4 trimethylation. Our data suggest that the nuclear periphery suppresses V(D)J recombination, at least in part, by segregating Tcrb alleles from RAG Proteins.
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Regulation of RAG1/RAG2‐mediated transposition by GTP and the C‐terminal region of RAG2
The EMBO journal, 2003Co-Authors: Chia-lun Tsai, David G. SchatzAbstract:The RAG1 and RAG2 Proteins perform critical DNA recognition and cleavage functions in V(D)J recombination, and also catalyze efficient DNA transposition in vitro. No transposition in vivo by the RAG Proteins has been reported, suggesting regulation of the reaction by as yet unknown mechanisms. Here we report that RAG-mediated transposition is suppressed by physiological concentrations of the guanine nucleotide GTP, and by the full-length RAG2 Protein. Both GTP and full-length RAG2 inhibit transposition by blocking the non-covalent ‘capture’ of target DNA, and both are capable of inhibiting RAG-mediated hybrid joint formation in vitro. We also observe that another intracellular signaling molecule, Ca2+, stimulates RAG-mediated transposition and is capable of activating transposition even in reactions containing full-length RAG2 and GTP. RAG-mediated transposition has been proposed to contribute to the chromosomal translocations that underlie the development of lymphoid malignancies, and our findings highlight regulatory mechanisms that might prevent such occurrences, and circumstances in which these regulatory mechanisms could be overcome.
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Identification of Two Catalytic Residues in RAG1 that Define a Single Active Site within the RAG1/RAG2 Protein Complex
Molecular cell, 2000Co-Authors: Sebastian D. Fugmann, Isabelle J. Villey, Leon M. Ptaszek, David G. SchatzAbstract:During V(D)J recombination, the RAG1 and RAG2 Proteins cooperate to catalyze a series of DNA bond breakage and strand transfer reactions. The structure, location, and number of active sites involved in RAG-mediated catalysis have as yet not been determined. Using Protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in RAG1 (D600 and D708) that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal ion binding active site structurally related to the active sites of transposases/integrases and responsible for all catalytic functions of the RAG Protein complex.
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identification of two catalytic residues in rag1 that define a single active site within the rag1 RAG2 Protein complex
Molecular Cell, 2000Co-Authors: Sebastian D. Fugmann, Isabelle J. Villey, Leon M. Ptaszek, David G. SchatzAbstract:During V(D)J recombination, the RAG1 and RAG2 Proteins cooperate to catalyze a series of DNA bond breakage and strand transfer reactions. The structure, location, and number of active sites involved in RAG-mediated catalysis have as yet not been determined. Using Protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in RAG1 (D600 and D708) that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal ion binding active site structurally related to the active sites of transposases/integrases and responsible for all catalytic functions of the RAG Protein complex.
Martin Gellert - One of the best experts on this subject based on the ideXlab platform.
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Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes
The Journal of biological chemistry, 2015Co-Authors: Mikalai Lapkouski, Martin Gellert, Min Sung Kim, Watchalee Chuenchor, Wei YangAbstract:Abstract Mammalian immune-receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 Protein complex (RAG1/2) initiates this process by binding to two types of Recombination Signal Sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12 and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex (PC) that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 PC is active in the presence of Mg2+, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex (SEC). We report here the purification and initial crystallization of the RAG1/2 SEC complex for atomic-resolution structure elucidation. Strict pairing of the 12 and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.
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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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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 Yang, Martin GellertAbstract: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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V(D)J Recombination
Mobile DNA II, 2002Co-Authors: Martin GellertAbstract:This chapter focuses on V(D)J recombination, but other mechanisms also contribute to antigen receptor diversity. Although in mice and humans V(D)J recombination is the major source of diversity, this is not true of all vertebrates. Terminal deoxynucleotidyltransferase (TdT) is normally expressed only in early lymphoid cells, so these insertions are relatively specific to V(D)J recombination (compared with other types of double-strand break repair). Work of the past several years has shown that V(D)J recombination has two distinct stages. In the first stage, the RAG1 Protein and RAG2 Protein act together to recognize the RSSs and their correct 12/23 pairing, and make double-strand breaks at the border between each heptamer and the neighboring coding sequence. In the second stage, an array of factors also used in other types of ‘‘nonhomologous end joining’’ acts to assemble the coding joints and signal joints. The RAG1 and RAG2 Proteins are the only lymphoid-specific factors needed for V(D)J recombination. RAG1 and RAG2 are normally coexpressed only in early lymphoid cells, where V(D)J recombination takes place. Transcription of the two neighboring RAG genes is convergent, and it has been shown that the control region of both genes is located upstream of RAG2. As for the regulation of V(D)J recombination, several new experimental systems should soon lead to a better understanding of locus accessibility, and make experimental modification of rearrangement possible.
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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.
Marjorie A. Oettinger - One of the best experts on this subject based on the ideXlab platform.
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A PHD finger motif in the C terminus of RAG2 modulates recombination activity.
The Journal of biological chemistry, 2005Co-Authors: Sheryl K. Elkin, Dmitri N. Ivanov, Mark D. Ewalt, Colin G. Ferguson, Sven G. Hyberts, Zhen-yu J. Sun, Glenn D. Prestwich, Junying Yuan, Gerhard Wagner, Marjorie A. OettingerAbstract:Abstract The RAG1 and RAG2 Proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a “core” domain that is required for the recombination reaction and a C-terminal nonessential or “non-core” region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 Protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an α-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.
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ATP-dependent remodeling by SWI/SNF and ISWI Proteins stimulates V(D)J cleavage of 5 S arrays.
The Journal of biological chemistry, 2004Co-Authors: Nadja Patenge, Sheryl K. Elkin, Marjorie A. OettingerAbstract:Abstract Control of V(D)J recombination is critical for the generation of a fully developed immune repertoire. The molecular mechanisms underlying the regulation of antigen receptor gene assembly are beginning to be revealed. Here we studied the influence of chromatin modifications on V(D)J cleavage of a polynucleosomal substrate, in which V(D)J cleavage is greatly reduced compared with naked DNA. ATP-dependent remodeling by human SWI/SNF (hSWI/SNF) in the presence of HMG1 led to a substantial increase of cleavage by the recombination activation gene (RAG) Proteins. Either BRG1, the ATPase subunit of hSWI/SNF, or SNF2h, the ATPase of human ISWI complexes, was capable of stimulating V(D)J cleavage of the array, although these remodelers act by different mechanisms. No effect of histone hyperacetylation was detectable in this system. As is observed on naked DNA, in the presence of core RAG1, the full-length RAG2 Protein proved to be more active than core RAG2 on these polynucleosomal arrays, reinforcing the importance of the RAG2 C-terminal domain for efficient recombination. Comparison of 5 S array cleavage by the RAG Proteins or by the restriction enzyme HhaI after remodeling by hSWI/SNF suggested that RAG Proteins and HhaI might have different requirements for maximal accessibility of the substrate.
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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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Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Yoshiko Akamatsu, Robert J. Monroe, Darryll D. Dudley, Sheryl K. Elkin, Frank Gärtner, Sadiqur R. Talukder, Yousuke Takahama, Frederick W. Alt, Craig H. Bassing, Marjorie A. OettingerAbstract:The recombination-activating gene (RAG)1 and RAG2 Proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 Protein (“core” RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombination in vitro and in transfected cell lines. Here we examine the effect of replacing the endogenous RAG2 locus in mice with core RAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 Protein retains significant in vivo function. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.
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Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly
The EMBO journal, 1998Co-Authors: Susan A. Kirch, Gary A. Rathbun, Marjorie A. OettingerAbstract:Immunoglobulin genes are assembled during lymphoid development by a series of site-specific rearrangements that are tightly regulated to ensure that functional antibodies are generated in B (but not T) cells and that a unique receptor is present on each cell. Because a common V(D)J recombinase comprising RAG1 and RAG2 Proteins is used for both B- and T-cell antigen receptor assembly, lineage-specific rearrangement must be modulated through differential access to sites of recombination. We show here that the C-terminus of the RAG2 Protein, although dispensable for the basic recombination reaction and for Ig heavy chain DH to JH joining, is essential for efficient VH to DJH rearrangement at the IgH locus. Thus, the RAG2 Protein plays a dual role in V(D)J recombination, acting both in catalysis of the reaction and in governing access to particular loci.
Sheryl K. Elkin - One of the best experts on this subject based on the ideXlab platform.
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A PHD finger motif in the C terminus of RAG2 modulates recombination activity.
The Journal of biological chemistry, 2005Co-Authors: Sheryl K. Elkin, Dmitri N. Ivanov, Mark D. Ewalt, Colin G. Ferguson, Sven G. Hyberts, Zhen-yu J. Sun, Glenn D. Prestwich, Junying Yuan, Gerhard Wagner, Marjorie A. OettingerAbstract:Abstract The RAG1 and RAG2 Proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a “core” domain that is required for the recombination reaction and a C-terminal nonessential or “non-core” region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 Protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an α-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.
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ATP-dependent remodeling by SWI/SNF and ISWI Proteins stimulates V(D)J cleavage of 5 S arrays.
The Journal of biological chemistry, 2004Co-Authors: Nadja Patenge, Sheryl K. Elkin, Marjorie A. OettingerAbstract:Abstract Control of V(D)J recombination is critical for the generation of a fully developed immune repertoire. The molecular mechanisms underlying the regulation of antigen receptor gene assembly are beginning to be revealed. Here we studied the influence of chromatin modifications on V(D)J cleavage of a polynucleosomal substrate, in which V(D)J cleavage is greatly reduced compared with naked DNA. ATP-dependent remodeling by human SWI/SNF (hSWI/SNF) in the presence of HMG1 led to a substantial increase of cleavage by the recombination activation gene (RAG) Proteins. Either BRG1, the ATPase subunit of hSWI/SNF, or SNF2h, the ATPase of human ISWI complexes, was capable of stimulating V(D)J cleavage of the array, although these remodelers act by different mechanisms. No effect of histone hyperacetylation was detectable in this system. As is observed on naked DNA, in the presence of core RAG1, the full-length RAG2 Protein proved to be more active than core RAG2 on these polynucleosomal arrays, reinforcing the importance of the RAG2 C-terminal domain for efficient recombination. Comparison of 5 S array cleavage by the RAG Proteins or by the restriction enzyme HhaI after remodeling by hSWI/SNF suggested that RAG Proteins and HhaI might have different requirements for maximal accessibility of the substrate.
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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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Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Yoshiko Akamatsu, Robert J. Monroe, Darryll D. Dudley, Sheryl K. Elkin, Frank Gärtner, Sadiqur R. Talukder, Yousuke Takahama, Frederick W. Alt, Craig H. Bassing, Marjorie A. OettingerAbstract:The recombination-activating gene (RAG)1 and RAG2 Proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 Protein (“core” RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombination in vitro and in transfected cell lines. Here we examine the effect of replacing the endogenous RAG2 locus in mice with core RAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 Protein retains significant in vivo function. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.
Wei Yang - One of the best experts on this subject based on the ideXlab platform.
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Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes
The Journal of biological chemistry, 2015Co-Authors: Mikalai Lapkouski, Martin Gellert, Min Sung Kim, Watchalee Chuenchor, Wei YangAbstract:Abstract Mammalian immune-receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 Protein complex (RAG1/2) initiates this process by binding to two types of Recombination Signal Sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12 and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex (PC) that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 PC is active in the presence of Mg2+, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex (SEC). We report here the purification and initial crystallization of the RAG1/2 SEC complex for atomic-resolution structure elucidation. Strict pairing of the 12 and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.
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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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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 Yang, Martin GellertAbstract: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.