RESUMO
The genes that encode immunoglobulin and T cell receptor proteins are assembled from component gene segments in a reaction known as V(D)J recombination. The reaction, and its crucial mediators RAG1 and RAG2, are essential for lymphocyte development and hence for adaptive immunity. Here we consider the biochemistry of this reaction, focusing on the DNA transactions and the proteins involved. We discuss how the RAG proteins interact with DNA and how coordinate cleavage of the DNA at two sites might be achieved. Finally, we consider the RAG proteins and V(D)J recombination from an evolutionary point of view.
Assuntos
Linfócitos B/fisiologia , Genes de Imunoglobulinas , Genes Codificadores dos Receptores de Linfócitos T , Recombinação Genética , Linfócitos T/fisiologia , VDJ Recombinases , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , VDJ Recombinases/genética , VDJ Recombinases/metabolismoRESUMO
In V(D)J recombination, the RAG1 and RAG2 proteins are the essential components of the complex that catalyzes DNA cleavage. RAG1 has been shown to play a central role in DNA binding and catalysis. In contrast, the molecular roles of RAG2 in V(D)J recombination are unknown. To address this, we individually mutated 36 evolutionarily conserved basic and hydroxy group containing residues within RAG2. Biochemical analysis of the recombinant RAG2 proteins led to the identification of a number of basic residue mutants defective in catalysis in vitro and V(D)J recombination in vivo. Five of these were deficient in binding of the RAG1-RAG2 complex to its cognate DNA target sequence while interacting normally with RAG1. Our findings provide support for the direct involvement of RAG2 in DNA binding during all steps of the cleavage reaction.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Ligação a DNA/química , Genes Reporter/genética , Proteínas de Homeodomínio/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Técnicas do Sistema de Duplo-HíbridoAssuntos
Genes de Imunoglobulinas , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Recombinação Genética , Animais , Cromatina/química , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Modelos Genéticos , Proteínas Nucleares , Nucleossomos/fisiologia , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Targeted hypermutation of immunoglobulin variable region genes occurs in B cells during an immune response, and gives rise to families of related mutant antibodies which are then selected for their binding affinity to the immunizing antigen. Somatic hypermutation predominantly generates point mutations, many of which occur at specific residues (hotspots). The reaction has been linked to transcription and requires the presence of immunoglobulin enhancers, but replacement of the variable gene by heterologous sequences, or the variable region promoter by a heterologous promoter, does not interfere with the mutation process. Here we show the existence of abundant DNA double-strand breaks (DSBs) in hypermutating sequences. Generation of the DSBs is coupled to transcription, enhancer-dependent, and correlates with the appearance of nearby mutations. Furthermore, the DSBs are cell-cycle restricted, being found almost exclusively in cells that have completed, or nearly completed, DNA replication. We propose a model for somatic hypermutation in which mutations are introduced into the DNA during repair of DSBs by homologous recombination. The finding of DSBs during somatic hypermutation may help to explain the chromosomal translocations found in some B-cell tumours.
Assuntos
Ciclo Celular/fisiologia , Dano ao DNA , DNA/metabolismo , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Mutação , Animais , Linhagem Celular , Clonagem Molecular , Reparo do DNA , Elementos Facilitadores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
Lymphocyte antigen receptors are not encoded by germline genes, but rather are produced by combinatorial joining between clusters of gene segments in somatic cells. Within a given cluster, gene segment usage during recombination is thought to be largely random, with biased representation in mature T lymphocytes resulting from protein-mediated selection of a subset of the total repertoire. Here we show that T cell receptor D beta and J beta gene segment usage is not random, but is patterned at the time of recombination. The hierarchy of gene segment usage is independent of gene segment proximity, but rather is influenced by the ability of the flanking recombination signal sequences (RSS) to bind the recombinase and/or to form a paired synaptic complex. Importantly, the relative frequency of gene segment usage established during recombination is very similar to that found after protein-mediated selection, suggesting that in addition to targeting recombinase activity, the RSS may have evolved to bias the naive repertoire in favor of useful gene products.
Assuntos
Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Genes Codificadores dos Receptores de Linfócitos T , Recombinação Genética , Linfócitos T/imunologia , Animais , Sequência de Bases , Sequência Consenso , Primers do DNA , DNA Ribossômico/genética , Rim/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da PolimeraseRESUMO
V(D)J recombination proceeds through a series of protein:DNA complexes mediated in part by the RAG1 and RAG2 proteins. These proteins are responsible for sequence-specific DNA recognition and DNA cleavage, and they appear to perform multiple postcleavage roles in the reaction as well. Here we review the interaction of the RAG proteins with DNA, the chemistry of the cleavage reaction, and the higher order complexes in which these events take place. We also discuss postcleavage functions of the RAG proteins, including recent evidence indicating that they initiate the process of coding end processing by nicking hairpin DNA termini. Finally, we discuss the evolutionary and functional implications of the finding that RAG1 and RAG2 constitute a transposase, and we consider RAG protein biochemistry in the context of several bacterial transposition systems. This suggests a model of the RAG protein active site in which two divalent metal ions serve alternating and opposite roles as activators of attacking hydroxyl groups and stabilizers of oxyanion leaving groups.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação Genética , Animais , Elementos de DNA Transponíveis , Humanos , Proteínas Nucleares , Sinais Direcionadores de Proteínas , VDJ RecombinasesRESUMO
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.
Assuntos
Ácido Aspártico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , DNA/química , DNA/metabolismo , DNA Nucleotidiltransferases/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transposases/química , Transposases/metabolismo , VDJ RecombinasesRESUMO
V(D)J recombination plays a prominent role in the generation of the antigen receptor repertoires of B and T lymphocytes. It is also likely to be involved in the formation of chromosomal translocations, some of which may result from interchromosomal recombination. We have investigated the potential of the V(D)J recombination machinery to perform intermolecular recombination between two plasmids, either unlinked or linked by catenation. In either case, recombination occurs in trans to yield signal and coding joints, and the results do not support the existence of a mechanistic block to the formation of coding joints in trans. Instead, we observe that linearization of the substrate, which does not alter the cis or trans status of the recombination signals, causes a specific and dramatic reduction in coding joint formation. This unexpected result leads us to propose a "release and recapture" model for V(D)J recombination in which coding ends are frequently released from the postcleavage complex and the efficiency of coding joint formation is influenced by the efficiency with which such ends are recaptured by the complex. This implies the existence of mechanisms, operative during recombination of chromosomal substrates, that act to prevent coding end release or to facilitate coding end recapture.
Assuntos
Rearranjo Gênico , Modelos Genéticos , Plasmídeos/genética , Receptores de Antígenos/genética , Recombinação Genética , DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Homeodomínio/metabolismo , Conformação de Ácido Nucleico , Plasmídeos/química , Recombinases , Especificidade por Substrato , Transposases/metabolismoRESUMO
The RAG1 and RAG2 proteins together initiate V(D)J recombination by performing cleavage of chromosomal DNA adjacent to antigen receptor gene segments. Like the adaptive immune system itself, RAG1 and RAG2 are found only in jawed vertebrates. The hypothesis that RAG1 and RAG2 arose in evolution as components of a transposable element has received dramatic support from our recent finding that the RAG proteins are a fully functional transposase in vitro. This result strongly suggests that antigen receptor genes acquired their unusual structure as a consequence of the insertion of a transposable element into an ancestral receptor gene by RAG1 and RAG2 approx 450 million years ago.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Sistema Imunitário/fisiologia , Recombinação Genética , Animais , Evolução Molecular , Humanos , Proteínas NuclearesAssuntos
Linfócitos B/fisiologia , Leucopoese/fisiologia , Animais , Linfócitos B/imunologia , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Rearranjo Gênico do Linfócito B , Rearranjo Gênico do Linfócito T , Genes RAG-1 , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Camundongos , Modelos Imunológicos , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.
Assuntos
DNA/metabolismo , Rearranjo Gênico/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Ligação Competitiva , Catálise , Linhagem Celular , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Dimerização , Escherichia coli/genética , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/isolamento & purificação , Camundongos , Mutação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Titulometria , Transfecção , Zinco/análiseAssuntos
DNA Complementar/análise , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Mapeamento por Restrição/métodos , Animais , Sequência de Bases , Indicadores e Reagentes , Hibridização de Ácido Nucleico/métodos , Oligodesoxirribonucleotídeos , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Sensibilidade e Especificidade , Leveduras/genéticaRESUMO
The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn2+, a synthetic hairpin is nicked 5 nucleotides (nt) 5' of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg2+, hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5' of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination.
Assuntos
Reparo do DNA/fisiologia , Proteínas de Homeodomínio/genética , Recombinação Genética , Southern Blotting , Clonagem Molecular , Primers do DNA , Proteínas de Ligação a DNA , Exonucleases/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Modelos Genéticos , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
Somatic DNA recombination is essential for production of functional antigen receptor genes of T and B lymphocytes, but it is thought to be unique to the immune system. Recent studies have now shown that recombination-related genes are also necessary for normal neuronal development.
Assuntos
Sistema Nervoso Central/embriologia , Animais , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Rearranjo Gênico/genética , Proteínas de Homeodomínio/genética , Sistema Imunitário/citologia , Sistema Imunitário/embriologia , Sistema Imunitário/metabolismo , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/metabolismo , Neurônios/citologia , Neurônios/metabolismoRESUMO
V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.
Assuntos
DNA/metabolismo , Genes de Imunoglobulinas/genética , Proteínas de Homeodomínio/genética , Recombinação Genética/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Modelos Moleculares , Mutação/genética , Raios UltravioletaRESUMO
Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.
Assuntos
Rearranjo Gênico do Linfócito T , Linfócitos T/fisiologia , Animais , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Receptores de Hialuronatos/análise , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Receptores de Interleucina-2/análiseRESUMO
Janus kinase 3 (Jak3) plays a central role in the transduction of signals mediated by the IL-2 family of cytokine receptors. Targeted deletion of the murine Jak3 gene results in severe reduction of alphabeta and complete elimination of gammadelta lineage thymocytes and NK cells. The developmental blockade appears to be imposed on early thymocyte differentiation and/or expansion. In this study, we show that bcl-2 expression and in vivo survival of immature thymocytes are greatly compromised in Jak3-/- mice. There is no gross deficiency in rearrangements of the TCRdelta and certain gamma loci in pre-T cells, and a functional gammadelta TCR transgene cannot rescue gammadelta lineage differentiation in Jak3-/- mice. In contrast, a TCRbeta transgene is partially able to restore alphabeta thymocyte development. These data suggest that the signals mediated by Jak3 are critical for survival of all thymocyte precursors particularly during TCRbeta-chain gene rearrangement, and are continuously required in the gammadelta lineage. The results also emphasize the fundamentally different requirements for differentiation of the alphabeta and gammadelta T cell lineages.
Assuntos
Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Animais , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Expressão Gênica , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Genes bcl-2 , Janus Quinase 3 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de Sinais , Subpopulações de Linfócitos T/citologiaRESUMO
Immunoglobulin and T-cell-receptor genes are assembled from component gene segments in developing lymphocytes by a site-specific recombination reaction, V(D)J recombination. The proteins encoded by the recombination-activating genes, RAG1 and RAG2, are essential in this reaction, mediating sequence-specific DNA recognition of well-defined recombination signals and DNA cleavage next to these signals. Here we show that RAG1 and RAG2 together form a transposase capable of excising a piece of DNA containing recombination signals from a donor site and inserting it into a target DNA molecule. The products formed contain a short duplication of target DNA immediately flanking the transposed fragment, a structure like that created by retroviral integration and all known transposition reactions. The results support the theory that RAG1 and RAG2 were once components of a transposable element, and that the split nature of immunoglobulin and T-cell-receptor genes derives from germline insertion of this element into an ancestral receptor gene soon after the evolutionary divergence of jawed and jawless vertebrates.