RESUMO
The structure of broken DNA ends is a critical determinant of the pathway used for DNA double-strand break (DSB) repair. Here, we develop an approach involving the hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single-nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1 phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3' overhangs, many of these DNA ends unexpectedly form long 5' single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify features of DNA end processing during DSB repair.
Assuntos
Cromossomos Humanos/genética , Análise de Sequência de DNA/métodos , Sequência de Bases , Células Cultivadas , Quebras de DNA de Cadeia Dupla , HumanosRESUMO
The plasmacytoid dendritic cell (pDC) is vital to the coordinated action of innate and adaptive immunity. pDC development has not been unequivocally traced, nor has its transcriptional regulatory network been fully clarified. Here we confirm an essential requirement for the BCL11A transcription factor in fetal pDC development, and demonstrate this lineage-specific requirement in the adult organism. Furthermore, we identify BCL11A gene targets and provide a molecular mechanism for its action in pDC commitment. Embryonic germ-line deletion of Bcl11a revealed an absolute cellular, molecular, and functional absence of pDCs in fetal mice. In adults, deletion of Bcl11a in hematopoietic stem cells resulted in perturbed yet continued generation of progenitors, loss of downstream pDC and B-cell lineages, and persisting myeloid, conventional dendritic, and T-cell lineages. Challenge with virus resulted in a marked reduction of antiviral response in conditionally deleted adults. Genome-wide analyses of BCL11A DNA binding and expression revealed that BCL11A regulates transcription of E2-2 and other pDC differentiation modulators, including ID2 and MTG16. Our results identify BCL11A as an essential, lineage-specific factor that regulates pDC development, supporting a model wherein differentiation into pDCs represents a primed "default" pathway for common dendritic cell progenitors.
Assuntos
Proteínas de Transporte/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes/imunologia , Proteínas Nucleares/imunologia , Animais , Linfócitos B/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Células Dendríticas/citologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The ability to sense metabolic stress is critical for successful cellular adaptation. In eukaryotes, the AMP-activated protein kinase (AMPK), a highly conserved serine/threonine kinase, functions as a critical metabolic sensor. AMPK is activated by the rising ADP/ATP and AMP/ATP ratios during conditions of energy depletion and also by increasing intracellular Ca(2+). In response to metabolic stress, AMPK maintains energy homeostasis by phosphorylating and regulating proteins that are involved in many physiological processes including glucose and fatty acid metabolism, transcription, cell growth, mitochondrial biogenesis, and autophagy. Evidence is mounting that AMPK also plays a role in a number of pathways unrelated to energy metabolism. Here, we identify the recombination-activating gene 1 protein (RAG1) as a substrate of AMPK. The RAG1/RAG2 complex is a lymphoid-specific endonuclease that catalyzes specific DNA cleavage during V(D)J recombination, which is required for the assembly of the Ig and T-cell receptor genes of the immune system. AMPK directly phosphorylates RAG1 at serine 528, and the phosphorylation enhances the catalytic activity of the RAG complex, resulting in increased cleavage of oligonucleotide substrates in vitro, or increased recombination of an extrachromosomal substrate in a cellular assay. Our results suggest that V(D)J recombination can be regulated by AMPK activation, providing a potential new link between metabolic stress and development of B and T lymphocytes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Homeodomínio/metabolismo , Serina/metabolismo , Recombinação V(D)J , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Fosforilação , Homologia de Sequência de Aminoácidos , Serina/genética , Especificidade por SubstratoRESUMO
The ataxia telangiectasia mutated (ATM) kinase and H2AX histone tumor suppressor proteins are each critical for maintenance of cellular genomic stability and suppression of lymphomas harboring clonal translocations. ATM is the predominant kinase that phosphorylates H2AX in chromatin around DNA double-strand breaks, including along lymphocyte Ag receptor loci cleaved during V(D)J recombination. However, combined germline inactivation of Atm and H2ax in mice causes early embryonic lethality associated with substantial cellular genomic instability, indicating that ATM and H2AX exhibit nonredundant functions in embryonic cells. To evaluate potential nonredundant roles of ATM and H2AX in somatic cells, we generated and analyzed Atm-deficient mice with conditional deletion of H2ax in αß T-lineage lymphocytes. Combined Atm/H2ax inactivation starting in early-stage CD4(-)/CD8(-) thymocytes resulted in lower numbers of later-stage CD4(+)/CD8(+) thymocytes, but led to no discernible V(D)J recombination defect in G1 phase cells beyond that observed in Atm-deficient cells. H2ax deletion in Atm-deficient thymocytes also did not affect the incidence or mortality of mice from thymic lymphomas with clonal chromosome 14 (TCRα/δ) translocations. Yet, in vitro-stimulated Atm/H2ax-deficient splenic αß T cells exhibited a higher frequency of genomic instability, including radial chromosome translocations and TCRß translocations, compared with cells lacking Atm or H2ax. Collectively, our data demonstrate that both redundant and nonredundant functions of ATM and H2AX are required for normal recombination of TCR loci, proliferative expansion of developing thymocytes, and maintenance of genomic stability in cycling αß T-lineage cells.
Assuntos
Ataxia Telangiectasia/imunologia , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Histonas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/imunologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/genética , Ciclo Celular/imunologia , Proteínas de Ciclo Celular/deficiência , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Loci Gênicos/imunologia , Instabilidade Genômica/imunologia , Histonas/deficiência , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Recombinação Genética/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Proteínas Supressoras de Tumor/deficiênciaRESUMO
Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination.
Assuntos
Quebra Cromossômica , Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação Genética/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Fenômenos Imunogenéticos/fisiologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Deleção de Sequência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.
RESUMO
BACKGROUND: Chromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development. RESULTS: Alternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles. CONCLUSION: We propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.
Assuntos
Proteínas de Transporte/metabolismo , Centro Germinativo/metabolismo , Linfoma de Células B/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Processamento Alternativo/genética , Animais , Western Blotting , Células COS , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Perfilação da Expressão Gênica , Centro Germinativo/patologia , Células HeLa , Humanos , Imunoprecipitação , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/análise , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas RepressorasRESUMO
The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G1-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G1-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G1-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G1-phase cells and suggest that there may be species-specific features to this activity.
Assuntos
Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos/metabolismo , Animais , Células Cultivadas , DNA/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/genéticaRESUMO
Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre-B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.
Assuntos
Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico de Cadeia Leve de Linfócito B , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Histonas/deficiência , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Modelos Biológicos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genéticaRESUMO
Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a(lox/lox) deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/metabolismo , Ativação Transcricional , Recombinação V(D)J , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Linhagem Celular , Células Cultivadas , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Células Precursoras de Linfócitos B/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras , Regulação para CimaRESUMO
V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Recombinação V(D)J , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Domínio Catalítico , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Células Precursoras de Linfócitos B/metabolismo , Domínios e Motivos de Interação entre ProteínasRESUMO
Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/enzimologia , Catálise , Deleção de Genes , Genoma , Instabilidade Genômica , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/metabolismo , Fosforilação , Mutação Puntual , Poli(ADP-Ribose) Polimerases/metabolismo , Recombinação GenéticaRESUMO
The Mre11-Rad50-Nbs1 (MRN) complex functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) at postreplicative stages of the cell cycle. During HR, the MRN complex functions directly in the repair of DNA DSBs and in the initiation of DSB responses through activation of the ataxia telangiectasia-mutated (ATM) serine-threonine kinase. Whether MRN functions in DNA damage responses before DNA replication in G0/G1 phase cells has been less clear. In developing G1-phase lymphocytes, DNA DSBs are generated by the Rag endonuclease and repaired during the assembly of antigen receptor genes by the process of V(D)J recombination. Mice and humans deficient in MRN function exhibit lymphoid phenotypes that are suggestive of defects in V(D)J recombination. We show that during V(D)J recombination, MRN deficiency leads to the aberrant joining of Rag DSBs and to the accumulation of unrepaired coding ends, thus establishing a functional role for MRN in the repair of Rag-mediated DNA DSBs. Moreover, these defects in V(D)J recombination are remarkably similar to those observed in ATM-deficient lymphocytes, suggesting that ATM and MRN function in the same DNA DSB response pathways during lymphocyte antigen receptor gene assembly.