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David Baltimore - One of the best experts on this subject based on the ideXlab platform.
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the v d j recombination activating gene Rag 1
Journal of Immunology, 2008Co-Authors: David G. Schatz, Marjorie A. Oettinger, David BaltimoreAbstract:The Rag-1 (recombination activating gene-1) genomic locus, which activates V(D)J recombination when Introduced Into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a Rag-1 genomic probe that detects a single 6.6-7.0 kb mRNA species In transfectants and pre-B and pre-T cells. Rag-1 genomic and cDNA clones were biologically active when introduced Into NIH 3T3 cells. Nucleotide sequencing of human and mouse Rag-1 cDNA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence Identity. Rag-1 has been conserved between species that carry out V(D)J recombination, and its pattern of expression correlates exactly with the pattern of expression of V(D)J recombinase activity. Rag-1 may activate V(D)J recombination indirectly, or It may encode the V(D)J recombinase itself.
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Complementation of V(D)J Recombination Deficiency in Rag-1−/− B Cells Reveals a Requirement for Novel Elements in the N-Terminus of Rag-1
Immunity, 1997Co-Authors: Christopher A. J. Roman, Sara Cherry, David BaltimoreAbstract:Abstract Rag-1 is an essential component of the site-specific V(D)J recombinase. A new assay system has revealed a significant contribution of the catalytically dispensible N-terminal region of Rag-1 to recombination activity. The foundation for this system is an Abelson virus–transformed cell line derived from Rag-1 −/− mice that is dependent on the introduction of exogenous Rag-1 for rearrangement of either plasmid substrates or the endogenous immunoglobulin loci. Use of this line demonstrates that conserved and novel cysteine-containing elements in the N-terminal region are required for full Rag-1 activity when recombination activity is in a Rag-1 dose-responsive range. Our data suggest that the Rag-1 N-terminus enhances the formation of an active recombination complex that facilitates the rearrangement process.
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the homeodomain region of Rag 1 reveals the parallel mechanisms of bacterial and v d j recombination
Cell, 1996Co-Authors: Eugenia Spanopoulou, Florina Zaitseva, Fuhou Wang, Sandro Santagata, David Baltimore, George PanayotouAbstract:The V(D)J recombinase subunits Rag-1 and Rag-2 mediate assembly of antigen receptor gene segments. We studied the mechanisms of DNA recognition by Rag-1/Rag-2 using surface plasmon resonance. The critical step for signal recognition is binding of Rag-1 to the nonamer. This is achieved by a region of Rag-1 homologous to the DNA-binding domain of the Hin family of bacterial invertases and to homeodomain proteins. Strikingly, the Hin homeodomain can functionally substitute for the Rag-1 homologous region. Rag-1 also interacts with the heptamer but with low affinity. Rag-2 shows no direct binding to DNA. Once the Rag-1/Rag-2 complex is engaged on the DNA, subsequent cleavage is directed by the heptamer sequence. This order of events remarkably parallels mechanisms that mediate transposition in bacteria and nematodes.
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Rag 1 interacts with the repeated amino acid motif of the human homologue of the yeast protein srp1
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Patricia Cortes, Zhengsheng Ye, David BaltimoreAbstract:Genes for immunoglobulins and T-cell receptor are generated by a process known as V(D)J recombination. This process is highly regulated and mediated by the recombination activating proteins Rag-1 and Rag-2. By the use of the two-hybrid protein interaction system, we isolated a human protein that specifically interacts with Rag-1. This protein is the human homologue of the yeast SRP1 (suppressor of a temperature-sensitive RNA polymerase I mutation). The SRP1-1 mutation is an allele-specific dominant suppressor of a temperature-sensitive mutation in the zinc binding domain of the 190-kDa subunit of Saccharomyces cerevisiae RNA polymerase I. The human SRP cDNA clone was used to screen a mouse cDNA library. We obtained a 3.9-kbp cDNA clone encoding the mouse SRP1. The open reading frame of this cDNA encodes a 538-amino acid protein with eight degenerate repeats of 40-45 amino acids each. The mouse and human SRP1 are 98% identical, while the mouse and yeast SRP1 have 48% identity. After cotransfection of the genes encoding Rag-1 and human SRP1 into 293T cells, a stable complex was evident. Deletion analysis indicated that the region of the SRP1 protein interacting with Rag-1 involved four repeats. The domain of Rag-1 that associates with SRP1 mapped N-terminal to the zinc finger domain. Because this region of Rag-1 is not required for recombination and SRP1 appears to be bound to the nuclear envelope, we suggest that this interaction helps to localize Rag-1.
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functional immunoglobulin transgenes guide ordered b cell differentiation in Rag 1 deficient mice
Genes & Development, 1994Co-Authors: E Spanopoulou, Mark S Schlissel, Michel C. Nussenzweig, Daniel P. Silver, C A J Roman, Lynn M Corcoran, David Nemazee, Susan A Shinton, Richard R Hardy, David BaltimoreAbstract:We have examined the regulatory role of the individual components of the immunoglobulin antigen receptor in B-cell development by transgenic complementation of Rag-1 deficient (Rag-1 - ) mice. Complementation with a membrane μ heavy chain (μHC) gene allows progression of developmentally arrested Rag-1 - pro-B-cells to the small pre-B cell stage, whereas the introduction of independently integrated μHC and κ light chain (κLC) transgenes promotes the appearance of peripheral lymphocytes which, however, remain unresponsive to external stimuli. Complete reconstitution of the B-cell lineage and the emergence of functionally mature Rag-1 - peripheral B cells is achieved by the introduction of cointegrated heavy and light chain transgenes encoding an anti-H-2 k antibody
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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Regulation of Rag Transposition
Advances in Experimental Medicine and Biology, 2009Co-Authors: Adam G. W. Matthews, Marjorie A. OettingerAbstract:V(D)J recombination is initiated by the lymphoid specific proteins Rag1 and Rag2, which together constitute the V(D)J recombinase. However, the Rag1/2 complex can also act as a transposase, inserting the broken DNA molecules generated during V(D)J recombination into an unrelated piece of DNA. This process, termed Rag transposition, can potentially cause insertional mutagenesis, chromosomal translocations and genomic instability. This review focuses on the mechanism and regulation of Rag transposition. We first provide a brief overview of the biochemistry of V(D)J recombination. We then discuss the discovery of Rag transposition and present an overview of the Rag transposition pathway. Using this pathway as a framework, we discuss the factors and forces that regulate Rag transposition.
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the v d j recombination activating gene Rag 1
Journal of Immunology, 2008Co-Authors: David G. Schatz, Marjorie A. Oettinger, David BaltimoreAbstract:The Rag-1 (recombination activating gene-1) genomic locus, which activates V(D)J recombination when Introduced Into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a Rag-1 genomic probe that detects a single 6.6-7.0 kb mRNA species In transfectants and pre-B and pre-T cells. Rag-1 genomic and cDNA clones were biologically active when introduced Into NIH 3T3 cells. Nucleotide sequencing of human and mouse Rag-1 cDNA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence Identity. Rag-1 has been conserved between species that carry out V(D)J recombination, and its pattern of expression correlates exactly with the pattern of expression of V(D)J recombinase activity. Rag-1 may activate V(D)J recombination indirectly, or It may encode the V(D)J recombinase itself.
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Roles of the “Dispensable” Portions of Rag-1 and Rag-2 in V(D)J Recombination
Molecular and Cellular Biology, 1999Co-Authors: Sharri Bockheim Steen, Cynthia L. Mundy, Marjorie A. Oettinger, David RothAbstract:Immunoglobulin and T-cell receptor gene segments are rearranged by V(D)J recombination to generate a diverse repertoire of antigen binding domains. The recombinase binds recombination signal sequences (hereafter termed signals) which flank the gene segments and introduces a double-stranded break (DSB) precisely between each signal and gene segment. This cleavage event produces two types of DNA termini, signal ends that terminate in signals and coding ends that contain the gene segment. Signal ends join to form a signal joint, whereas coding ends join to form a coding joint encoding the antigen binding domain (Fig. (Fig.1A)1A) (24, 27, 28, 33, 38, 40). FIG. 1 (A) Schematic diagram of V(D)J recombination intermediates (coding ends and signal ends) and products (coding joints and signal joints) generated from the plasmid substrate pJH290. Signals are represented by triangles, and coding segments are represented ... The V(D)J recombinase minimally consists of the highly conserved, lymphoid-cell-specific proteins Rag-1 and Rag-2 (22, 32). Transfection of the genes encoding Rag-1 and Rag-2 into cultured fibroblasts renders these cells competent to rearrange extrachromosomal recombination substrates, indicating that the Rag proteins are the only lymphoid-cell-specific factors necessary for recombination (22). DSBs with the same characteristics as in vivo intermediates (27, 28, 33, 35, 40) are generated in cell-free reactions containing purified, truncated Rag-1 and Rag-2 and the appropriate divalent metal ion (20). After cleavage, the Rag proteins remain associated with the broken DNA ends. Stable complexes have been isolated that contain the Rag proteins and a pair of cleaved signal ends (1). More recently, complexes containing the Rag proteins and all four DNA ends (two signal ends and two coding ends) have been isolated (9). We and others have suggested that disassembly or remodeling of these postcleavage complexes may be necessary to allow the joining machinery to complete formation of coding or signal joints (1, 39). Mutational analyses revealed that Rag-1 and Rag-2 proteins truncated by 30 and 25%, respectively, are still capable of recombining plasmid substrates in fibroblasts, although generally with lower efficiency than full-length Rag proteins (3, 12, 21, 26, 30, 31, 34). These truncated proteins, which contain residues 384 to 1008 of 1,040 amino acids (Rag-1) and 1 to 387 of 527 amino acids (Rag-2) (Fig. (Fig.1B),1B), are more soluble than their full-length counterparts and are, therefore, the forms of the Rag proteins used in cell-free systems (4, 13, 20, 23, 37, 38). Sequence analysis of the portions of Rag-1 and Rag-2 that have been considered dispensable for recombination (amino acids 1 to 383 and 1009 to 1040 of Rag-1 and 388 to 527 of Rag-2) reveals many amino acid residues that are conserved across diverse species (Fig. (Fig.1B),1B), suggesting that they may play an important role(s) in recombination. This proposal is supported by recent work demonstrating that regions within the N terminus of Rag-1 enhance signal joint formation (21, 26). However, these studies did not address which step of recombination, cleavage or joining, is affected by truncation of Rag-1 and Rag-2. Specifically, the effects of the truncations on recombination intermediates were not examined. Here, we analyze the levels of both recombination intermediates (signal ends and coding ends) and products (signal joints and coding joints). In agreement with previous work (3, 12, 21, 26, 31, 34), we found that the levels of V(D)J recombination products, coding and signal joints, are reduced when truncated Rag proteins are used. We expected that the levels of recombination intermediates would be similarly reduced, since the characterized functions of the Rag proteins affect the cleavage step. However, the truncated Rag proteins consistently produced levels of signal ends that were 10-fold-higher than those produced by the full-length Rag proteins. In contrast, levels of coding ends were generally not affected. These observations suggest that the truncated Rag proteins do not increase cleavage but, rather, stabilize the signal end intermediates. Analysis of additional Rag deletion mutants allowed us to separate the effects on intermediates and products, showing that these effects map to distinct regions of the Rag proteins. Thus, these studies have uncovered several unexpected functions of the “dispensable” regions of the Rag proteins, suggesting that they have roles both in the processing of postcleavage complexes and in product formation.
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The recombination activating genes, Rag 1 and Rag 2, are on chromosome 11p in humans and chromosome 2p in mice
Immunogenetics, 1992Co-Authors: Marjorie A. Oettinger, David G. Schatz, Ben Z. Stanger, Thomas M Glaser, Kathy Call, David E. Housman, David BaltimoreAbstract:The recombination activating genes Rag-1 and Rag-2 are adjacent genes that act synergistically to activate variable-diversity-joining (V(D)J) recombination. Southern analysis of hybrid cell lines derived from patients with the Wilms tumor-aniridia-genitourinary defects-mental retardation (WAGR) syndrome and from mutagenized cell hybrids selected for deletions in chromosome 11 has allowed us to map the chromosomal location of the human Rag locus. The Rag locus defines a new interval of human chromosome 11p, but is not associated with any genetically mapped human disease. Guided by the chromosomal localization of the human recombination activating genes, we have also mapped the location of the mouse Rag locus.
David G. Schatz - One of the best experts on this subject based on the ideXlab platform.
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the v d j recombination activating gene Rag 1
Journal of Immunology, 2008Co-Authors: David G. Schatz, Marjorie A. Oettinger, David BaltimoreAbstract:The Rag-1 (recombination activating gene-1) genomic locus, which activates V(D)J recombination when Introduced Into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a Rag-1 genomic probe that detects a single 6.6-7.0 kb mRNA species In transfectants and pre-B and pre-T cells. Rag-1 genomic and cDNA clones were biologically active when introduced Into NIH 3T3 cells. Nucleotide sequencing of human and mouse Rag-1 cDNA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence Identity. Rag-1 has been conserved between species that carry out V(D)J recombination, and its pattern of expression correlates exactly with the pattern of expression of V(D)J recombinase activity. Rag-1 may activate V(D)J recombination indirectly, or It may encode the V(D)J recombinase itself.
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The half-life of Rag-1 protein in precursor B cells is increased in the absence of Rag-2 expression.
Journal of Experimental Medicine, 1996Co-Authors: Ulf Grawunder, David G. Schatz, Antonius G. Rolink, Fritz MelchersAbstract:Site-specific recombination of immunoglobulin and T cell receptor gene segments in B and T lymphocytes is dependent on the expression of two recombinant activation genes, Rag-1 and Rag-2. Here, we show that Rag-1 protein turnover in pre-B cells depends on the expression of Rag-2. The apparent half-life of Rag-1 protein is increased when Rag-2 is not expressed in differentiating pre-B cells.
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Rag-1 and Rag-2: Biochemistry and Protein Interactions
Current Topics in Microbiology and Immunology, 1996Co-Authors: David G. SchatzAbstract:The process of V(D)J recombination is the defining characteristic of lymphocyte development. This site-specific recombination reaction assembles the genes that encode immunoglobulin (lg) and T cell receptor proteins, and in many species is the source of much of the diversity in these gene products. The reaction is complex and almost certainly requires the coordinated activity of a large number of proteins. Most components of the V(D)J recombination enzymatic machinery (hereafter referred to as the V(D)J recombinase) are ubiquitously expressed, with only three lymphocyte-specific factors thus far identified: the products of the recombination activating genes (Rag-1 and Rag-2), and terminal deoxynucleotidyl transferase (TdT). TdT is not required for V(D)J recombination, but when present it functions to add nongermline-encoded nucleotides (N regions) to coding junctions and thereby greatly increases the diversity of the products of the reaction. The Rag-1 and Rag-2 proteins are central to the process of V(D)J recombination, and their biochemical properties and protein-protein interactions are the focus of this chapter.
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Rag-1 and Rag-2 are components of a high-molecular-weight complex, and association of Rag-2 with this complex is Rag-1 dependent.
Molecular and Cellular Biology, 1995Co-Authors: David G. SchatzAbstract:Despite the essential and synergistic functions of the Rag-1 and Rag-2 proteins in V(D)J recombination and lymphocyte development, little is known about the biochemical properties of the two proteins. We have developed cell lines expressing high levels of the Rag proteins and specific, sensitive immunological reagents for their detection, and we have examined the physical properties of the Rag proteins in vitro and their subcellular localizations in vivo. Rag-1 is tightly associated with nuclear structures, requires a high salt concentration to maintain its solubility, and is a component of large, heterogeneously sized complexes. Furthermore, the presence of Rag-1 alters the behavior of Rag-2, conferring on it properties similar to those of Rag-1 and changing its distribution in the nucleus. We demonstrate that Rag-1 and Rag-2 are present in the same complex by coimmunoprecipitation, and we provide evidence that these complexes contain more molecules of Rag-2 than of Rag-1. The demonstration of intracellular complexes containing Rag-1 and Rag-2 raises the possibility that interaction between these proteins is necessary for their biological function.
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The recombination activating genes, Rag 1 and Rag 2, are on chromosome 11p in humans and chromosome 2p in mice
Immunogenetics, 1992Co-Authors: Marjorie A. Oettinger, David G. Schatz, Ben Z. Stanger, Thomas M Glaser, Kathy Call, David E. Housman, David BaltimoreAbstract:The recombination activating genes Rag-1 and Rag-2 are adjacent genes that act synergistically to activate variable-diversity-joining (V(D)J) recombination. Southern analysis of hybrid cell lines derived from patients with the Wilms tumor-aniridia-genitourinary defects-mental retardation (WAGR) syndrome and from mutagenized cell hybrids selected for deletions in chromosome 11 has allowed us to map the chromosomal location of the human Rag locus. The Rag locus defines a new interval of human chromosome 11p, but is not associated with any genetically mapped human disease. Guided by the chromosomal localization of the human recombination activating genes, we have also mapped the location of the mouse Rag locus.
Mark S Schlissel - One of the best experts on this subject based on the ideXlab platform.
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gfi1b negatively regulates Rag expression directly and via the repression of foxo1
Journal of Experimental Medicine, 2012Co-Authors: Danae Schulz, Lothar Vassen, Kwan T Chow, Sarah M Mcwhirter, Rupesh H Amin, Tarik Moroy, Mark S SchlisselAbstract:Precise regulation of Rag ( recombination-activating gene ) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5′ of the B cell–specific ERag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1 . Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase.
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a conserved transcriptional enhancer regulates Rag gene expression in developing b cells
Immunity, 2003Co-Authors: Josh Lauring, Hong Erh Liang, Stephen Greenbaum, Dragana Cado, Yuan Zhuang, Mark S SchlisselAbstract:Although expression of the Rag1 and Rag2 genes is essential for lymphocyte development, the mechanisms responsible for the lymphoid- and developmental stage-specific regulation of these genes are poorly understood. We have identified a novel, evolutionarily conserved transcriptional enhancer in the Rag locus, called ERag, which was essential for the expression of a chromosomal reporter gene driven by either Rag promoter. Targeted deletion of ERag in the mouse germline results in a partial block in B cell development associated with deficient V(D)J recombination, whereas T cell development appears unaffected. We found that E2A transcription factors bind to ERag in vivo and can transactivate ERag-dependent reporter constructs in cotransfected cell lines. These findings lead us to conclude that Rag transcription is regulated by distinct elements in developing B and T cells and that ERag is required for optimal levels of Rag expression in early B cell precursors but not in T cells.
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functional immunoglobulin transgenes guide ordered b cell differentiation in Rag 1 deficient mice
Genes & Development, 1994Co-Authors: E Spanopoulou, Mark S Schlissel, Michel C. Nussenzweig, Daniel P. Silver, C A J Roman, Lynn M Corcoran, David Nemazee, Susan A Shinton, Richard R Hardy, David BaltimoreAbstract:We have examined the regulatory role of the individual components of the immunoglobulin antigen receptor in B-cell development by transgenic complementation of Rag-1 deficient (Rag-1 - ) mice. Complementation with a membrane μ heavy chain (μHC) gene allows progression of developmentally arrested Rag-1 - pro-B-cells to the small pre-B cell stage, whereas the introduction of independently integrated μHC and κ light chain (κLC) transgenes promotes the appearance of peripheral lymphocytes which, however, remain unresponsive to external stimuli. Complete reconstitution of the B-cell lineage and the emergence of functionally mature Rag-1 - peripheral B cells is achieved by the introduction of cointegrated heavy and light chain transgenes encoding an anti-H-2 k antibody
Stephen Desiderio - One of the best experts on this subject based on the ideXlab platform.
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a plant homeodomain in Rag 2 that binds hypermethylated lysine 4 of histone h3 is necessary for efficient antigen receptor gene rearrangement
Immunity, 2007Co-Authors: Ramesh Subrahmanyam, Tirtha Chakraborty, Stephen DesiderioAbstract:Summary V(D)J recombination is initiated by the recombination activating gene (Rag) proteins Rag-1 and Rag-2. The ability of antigen-receptor-gene segments to undergo V(D)J recombination is correlated with spatially- and temporally-restricted chromatin modifications. We have found that Rag-2 bound specifically to histone H3 and that this binding was absolutely dependent on dimethylation or trimethylation at lysine 4 (H3K4me2 or H3K4me3). The interaction required a noncanonical plant homeodomain (PHD) that had previously been described within the noncore region of Rag-2. Binding of the Rag-2 PHD finger to chromatin across the IgH D-J H -C locus showed a strong correlation with the distribution of trimethylated histone H3 K4. Mutation of a conserved tryptophan residue in the Rag-2 PHD finger abolished binding to H3K4me3 and greatly impaired recombination of extrachromosomal and endogenous immunoglobulin gene segments. Together, these findings are consistent with the interpretation that recognition of hypermethylated histone H3 K4 promotes efficient V(D)J recombination in vivo.
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ubiquitylation of Rag 2 by skp2 scf links destruction of the v d j recombinase to the cell cycle
Molecular Cell, 2005Co-Authors: Hao Jiang, Fu Chung Chang, Ashley E Ross, Keiichi I Nakayama, Keiko Nakayama, Stephen DesiderioAbstract:Summary The periodic destruction of Rag-2 at the G1-to-S transition couples V(D)J recombination to the G0 and G1 cell cycle phases and coordinates Rag-mediated DNA cleavage with DNA repair by nonhomologous end joining. To define the mechanism by which this occurs, we reproduced cell cycle-dependent regulation of the V(D)J recombinase in a cell-free system. The ubiquitin-proteasomal pathway carries out destruction of Rag-2 in lysates of S phase cells and during S phase in vivo. Remarkably, the Skp2-SCF ubiquitin ligase, which plays a central role in cell cycle regulation through the destruction of p27, mediates ubiquitylation of Rag-2 in vitro and degradation of Rag-2 in vivo. The regulation of antigen receptor gene assembly by Skp2-SCF provides an unexpected and direct mechanistic link between DNA recombination and the cell cycle.
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Rag-2 promotes heptamer occupancy by Rag-1 in the assembly of a V(D)J initiation complex.
Molecular and Cellular Biology, 1999Co-Authors: Patrick C. Swanson, Stephen DesiderioAbstract:V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. Rag-1 and Rag-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. Rag-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. Rag-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of Rag-1; how Rag-2 collaborates with Rag-1 has been poorly understood. Here we examine the composition of Rag-RSS complexes and the relative contributions of Rag-1 and Rag-2 to heptamer binding. Rag-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether Rag-2 is present; only a single subunit of Rag-1, however, participates in nonamer binding. In contrast, multimeric Rag-2 is not detectable by electrophoretic mobility shift assays in complexes containing both Rag proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by Rag-2, are mediated primarily by Rag-1. Rag-2 cross-linking, while less efficient than that of Rag-1, is detectable near the heptamer-coding junction. These observations provide evidence that Rag-2 alters the conformation or orientation of Rag-1, thereby stabilizing interactions of Rag-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.
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Rag-1 and Rag-2-dependent assembly of functional complexes with V(D)J recombination substrates in solution.
Molecular and Cellular Biology, 1997Co-Authors: Wenhui Li, Patrick C. Swanson, Stephen DesiderioAbstract:V(D)J recombination is initiated by Rag-1 and Rag-2, which introduce double-strand DNA breaks at recombination signal sequences (RSSs) of antigen receptor gene segments to produce signal ends, terminating in blunt, double-strand breaks, and coding ends, terminating in DNA hairpins. While the formation of Rag-RSS complexes has been documented, observations regarding the individual contributions of Rag-1 and Rag-2 to RSS recognition are in conflict. Here we describe an assay for formation and maintenance of functional Rag-RSS complexes in the course of the DNA cleavage reaction. Under conditions of in vitro cleavage, the Rag proteins sequester intact substrate DNA in a stable complex which is formed prior to strand scission. The cleavage reaction subsequently proceeds through nicking and hairpin formation without dissociation of substrate. Notably, the presence of both Rag-1 and Rag-2 is essential for formation of stable, functional complexes with substrate DNA under conditions of the sequestration assay. Two classes of substrate mutation are distinguished by their effects on Rag-mediated DNA cleavage in vitro. A mutation of the first class, residing within the RSS nonamer and associated with coordinate impairment of nicking and hairpin formation, greatly reduces the stability of Rag association with intact substrate DNA. In contrast, a mutation of the second class, lying within the RSS heptamer and associated with selective abolition of hairpin formation, has little or no effect on the half-life of the Rag-substrate complex.
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regulation of v d j recombination activator protein Rag 2 by phosphorylation
Science, 1993Co-Authors: Stephen DesiderioAbstract:Antigen receptor genes are assembled by site-specific DNA rearrangement. The recombination activator genes Rag-1 and Rag-2 are essential for this process, termed V(D)J rearrangement. The activity and stability of the Rag-2 protein have now been shown to be regulated by phosphorylation. In fibroblasts Rag-2 was phosphorylated predominantly at two serine residues, one of which affected Rag-2 activity in vivo. The threonine at residue 490 was phosphorylated by p34cdc2 kinase in vitro; phosphorylation at this site in vivo was associated with rapid degradation of Rag-2. Instability was transferred to chimeric proteins by a 90-residue portion of Rag-2. Mutation of the p34cdc2 phosphorylation site of the tumor suppressor protein p53 conferred a similar phenotype, suggesting that this association between phosphorylation and degradation is a general mechanism.
