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1.
Cell ; 152(3): 620-32, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23352430

RESUMO

DNA double-strand breaks (DSBs) in B lymphocytes arise stochastically during replication or as a result of targeted DNA damage by activation-induced cytidine deaminase (AID). Here we identify recurrent, early replicating, and AID-independent DNA lesions, termed early replication fragile sites (ERFSs), by genome-wide localization of DNA repair proteins in B cells subjected to replication stress. ERFSs colocalize with highly expressed gene clusters and are enriched for repetitive elements and CpG dinucleotides. Although distinct from late-replicating common fragile sites (CFS), the stability of ERFSs and CFSs is similarly dependent on the replication-stress response kinase ATR. ERFSs break spontaneously during replication, but their fragility is increased by hydroxyurea, ATR inhibition, or deregulated c-Myc expression. Moreover, greater than 50% of recurrent amplifications/deletions in human diffuse large B cell lymphoma map to ERFSs. In summary, we have identified a source of spontaneous DNA lesions that drives instability at preferred genomic sites.


Assuntos
Sítios Frágeis do Cromossomo , Replicação do DNA , Eucariotos/genética , Instabilidade Genômica , Células Procarióticas/fisiologia , Animais , Fenômenos Biomecânicos , Reparo do DNA , Humanos
2.
Cell ; 153(6): 1266-80, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23727112

RESUMO

The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Linfócitos B/metabolismo , Proteína BRCA1/metabolismo , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Instabilidade Genômica , Camundongos , Mutação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
3.
Cell ; 141(2): 243-54, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20362325

RESUMO

Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here, we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.


Assuntos
Proteína BRCA1/genética , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Linfócitos B/metabolismo , Proteínas Cromossômicas não Histona , Quebras de DNA , Proteínas de Ligação a DNA , Feminino , Instabilidade Genômica , Humanos , Camundongos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
4.
Cell ; 135(6): 1028-38, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-19070574

RESUMO

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.


Assuntos
Citidina Desaminase/metabolismo , Genes de Cadeia Pesada de Imunoglobulina , Genes myc , Translocação Genética , Animais , Linfócitos B/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Quebras de DNA de Cadeia Dupla , Células-Tronco Embrionárias , Humanos , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasmocitoma/genética , Plasmocitoma/metabolismo
5.
Nature ; 514(7520): 107-11, 2014 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-25079327

RESUMO

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.


Assuntos
Dano ao DNA , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Mielopoese , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genes BRCA1 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Masculino , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Espécies Reativas de Oxigênio/metabolismo
6.
Mol Cell ; 46(2): 125-35, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22445484

RESUMO

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.


Assuntos
Proteína BRCA1/fisiologia , Reparo do DNA , Recombinação Homóloga/fisiologia , Animais , Antígenos Nucleares/fisiologia , Proteína BRCA1/genética , Proteínas de Ligação a DNA/fisiologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Autoantígeno Ku , Camundongos , Deleção de Sequência
7.
Mol Cell ; 42(3): 319-29, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21549309

RESUMO

53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.


Assuntos
Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Switching de Imunoglobulina/genética , Animais , Linfócitos B/metabolismo , Sítios de Ligação , Western Blotting , Células Cultivadas , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Fosforilação , Multimerização Proteica , Recombinação Genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
8.
Mol Cell ; 34(3): 285-97, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19450527

RESUMO

The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.


Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Linfócitos/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Instabilidade Genômica , Switching de Imunoglobulina , Linfócitos/citologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Timo/citologia , Proteína 28 com Motivo Tripartido , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética
9.
Nature ; 456(7221): 529-33, 2008 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-18931658

RESUMO

Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.


Assuntos
DNA/metabolismo , Rearranjo Gênico do Linfócito T/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Recombinação Genética , Animais , Apoptose , Proteínas Cromossômicas não Histona , DNA/genética , Quebras de DNA , Proteínas de Ligação a DNA , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfopenia/genética , Linfopenia/patologia , Camundongos , Modelos Genéticos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Homologia de Sequência , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/citologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
10.
Nat Cell Biol ; 7(7): 675-85, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15965469

RESUMO

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Hidrolases Anidrido Ácido , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/imunologia , Linfócitos B/patologia , Sítios de Ligação/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Aberrações Cromossômicas , Quebra Cromossômica , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/metabolismo , Transtornos Cromossômicos/patologia , Dano ao DNA/genética , Dano ao DNA/fisiologia , Enzimas Reparadoras do DNA , Replicação do DNA/genética , Feminino , Predisposição Genética para Doença/genética , Gônadas/anormalidades , Humanos , Switching de Imunoglobulina/genética , Linfoma não Hodgkin/etiologia , Linfoma não Hodgkin/genética , Proteína Homóloga a MRE11 , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Síndrome , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
11.
Nature ; 440(7080): 105-9, 2006 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-16400328

RESUMO

Chromosomal translocations involving the immunoglobulin switch region are a hallmark feature of B-cell malignancies. However, little is known about the molecular mechanism by which primary B cells acquire or guard against these lesions. Here we find that translocations between c-myc and the IgH locus (Igh) are induced in primary B cells within hours of expression of the catalytically active form of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosine to produce uracil in DNA. Translocation also requires uracil DNA glycosylase (UNG), which removes uracil from DNA to create abasic sites that are then processed to double-strand breaks. The pathway that mediates aberrant joining of c-myc and Igh differs from intrachromosomal repair during immunoglobulin class switch recombination in that it does not require histone H2AX, p53 binding protein 1 (53BP1) or the non-homologous end-joining protein Ku80. In addition, translocations are inhibited by the tumour suppressors ATM, Nbs1, p19 (Arf) and p53, which is consistent with activation of DNA damage- and oncogenic stress-induced checkpoints during physiological class switching. Finally, we demonstrate that accumulation of AID-dependent, IgH-associated chromosomal lesions is not sufficient to enhance c-myc-Igh translocations. Our findings reveal a pathway for surveillance and protection against AID-dependent DNA damage, leading to chromosomal translocations.


Assuntos
Citidina Desaminase/metabolismo , Genes de Cadeia Pesada de Imunoglobulina/genética , Genes myc/genética , Instabilidade Genômica/genética , Modelos Genéticos , Translocação Genética/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular , Citidina Desaminase/genética , Dano ao DNA/genética , Expressão Gênica , Switching de Imunoglobulina/genética , Camundongos , Mutação/genética , Proteína Supressora de Tumor p53/genética
12.
Nat Cell Biol ; 4(12): 993-7, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12447390

RESUMO

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.


Assuntos
Proteínas de Transporte/genética , Dano ao DNA/genética , Fase G2/genética , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular , Mitose/genética , Fosfoproteínas , Proteínas Serina-Treonina Quinases , Animais , Linhagem Celular , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Fase G2/fisiologia , Regulação da Expressão Gênica , Camundongos , Mitose/fisiologia , Fosforilação , Proteínas Quinases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
13.
J Exp Med ; 200(9): 1103-10, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15520243

RESUMO

Ataxia telangiectasia mutated (ATM) kinase is critical for initiating the signaling pathways that lead to cell cycle checkpoints and DNA double strand break repair. In the absence of ATM, humans and mice show a primary immunodeficiency that includes low serum antibody titers, but the role of ATM in antigen-driven immunoglobulin gene diversification has not been defined. Here, we show that although ATM is dispensable for somatic hypermutation, it is required for efficient class switch recombination (CSR). The defect in CSR is not due to alterations in switch region transcription, accessibility, DNA damage checkpoint protein recruitment, or short-range intra-switch region recombination. Only long-range inter-switch recombination is defective, indicating an unexpected role for ATM in switch region synapsis during CSR.


Assuntos
Linfócitos B/imunologia , Switching de Imunoglobulina/genética , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Southern Blotting , Proteínas de Ciclo Celular , Análise Mutacional de DNA , Proteínas de Ligação a DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/imunologia , Proteínas Supressoras de Tumor
14.
J Exp Med ; 196(4): 469-80, 2002 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-12186839

RESUMO

Nonreciprocal translocations and gene amplifications are commonly found in human tumors. Although little is known about the mechanisms leading to such aberrations, tissue culture models predict that they can arise from DNA breakage, followed by cycles of chromatid fusion, asymmetric mitotic breakage, and replication. Mice deficient in both a nonhomologous end joining (NHEJ) DNA repair protein and the p53 tumor suppressor develop lymphomas at an early age harboring amplification of an IgH/c-myc fusion. Here we report that these chromosomal rearrangements are initiated by a recombination activating gene (RAG)-induced DNA cleavage. Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway. Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture. Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.


Assuntos
Antígenos Nucleares , Dano ao DNA , DNA Helicases , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-myc/genética , Neoplasias do Timo/genética , Translocação Genética , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Amplificação de Genes , Rearranjo Gênico , Autoantígeno Ku , Camundongos , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Oncogenes , Telômero , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia
15.
Sci Signal ; 9(445): ra91, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27625305

RESUMO

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Neoplasias Experimentais , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Nat Med ; 21(10): 1199-208, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26366710

RESUMO

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Linfoma de Células B/etiologia , Proteínas de Neoplasias/fisiologia , Animais , Linfócitos B/patologia , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas de Neoplasias/genética
17.
J Exp Med ; 211(6): 1027-36, 2014 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-24842372

RESUMO

Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3' ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.


Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Instabilidade Genômica , Recombinação Homóloga , Immunoblotting , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
18.
J Cell Biol ; 193(2): 295-305, 2011 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-21482716

RESUMO

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)-dependent protein kinase catalytic subunit (DNA-PKcs)-null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice.


Assuntos
Reparo do DNA/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Mutação , Proteínas Nucleares/genética , Animais , Apoptose , Células Cultivadas , Reagentes de Ligações Cruzadas/toxicidade , Dano ao DNA , Fibroblastos/metabolismo , Técnicas de Introdução de Genes , Hiperpigmentação/genética , Camundongos , Camundongos Knockout , Recombinação Genética , Proteína Supressora de Tumor p53/metabolismo
19.
J Exp Med ; 207(5): 973-81, 2010 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-20385748

RESUMO

53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1's interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase-dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain-associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX(-/-) mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8(-/-) males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage-inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.


Assuntos
Switching de Imunoglobulina/fisiologia , Meiose/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Switching de Imunoglobulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfopenia/genética , Linfopenia/fisiopatologia , Meiose/genética , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Recombinação Genética , Transcrição Gênica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética
20.
Science ; 329(5994): 917-23, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20671152

RESUMO

Programmed genetic rearrangements in lymphocytes require transcription at antigen receptor genes to promote accessibility for initiating double-strand break (DSB) formation critical for DNA recombination and repair. Here, we showed that activated B cells deficient in the PTIP component of the MLL3 (mixed-lineage leukemia 3)-MLL4 complex display impaired trimethylation of histone 3 at lysine 4 (H3K4me3) and transcription initiation of downstream switch regions at the immunoglobulin heavy-chain (Igh) locus, leading to defective immunoglobulin class switching. We also showed that PTIP accumulation at DSBs contributes to class switch recombination (CSR) and genome stability independently of Igh switch transcription. These results demonstrate that PTIP promotes specific chromatin changes that control the accessibility of the Igh locus to CSR and suggest a nonredundant role for the MLL3-MLL4 complex in altering antibody effector function.


Assuntos
Proteínas de Transporte/fisiologia , Switching de Imunoglobulina/fisiologia , Proteínas Nucleares/fisiologia , Animais , Especificidade de Anticorpos/genética , Proteínas de Transporte/genética , Citidina Desaminase/metabolismo , DNA , Proteínas de Ligação a DNA , Histonas/metabolismo , Switching de Imunoglobulina/genética , Região de Troca de Imunoglobulinas , Metilação , Camundongos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Recombinação Genética , Ativação Transcricional
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