RESUMO
Fanconi anemia (FA) is a complex cancer susceptibility disorder associated with DNA repair defects and infertility, yet the precise function of the FA proteins in genome maintenance remains unclear. Here we report that C. elegans FANCD2 (fcd-2) is dispensable for normal meiotic recombination but is required in crossover defective mutants to prevent illegitimate repair of meiotic breaks by nonhomologous end joining (NHEJ). In mitotic cells, we show that DNA repair defects of C. elegans fcd-2 mutants and FA-deficient human cells are significantly suppressed by eliminating NHEJ. Moreover, NHEJ factors are inappropriately recruited to sites of replication stress in the absence of FANCD2. Our findings are consistent with the interpretation that FA results from the promiscuous action of NHEJ during DNA repair. We propose that a critical function of the FA pathway is to channel lesions into accurate, as opposed to error-prone, repair pathways.
Assuntos
Reparo do DNA/genética , Anemia de Fanconi/genética , Recombinação Genética , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Troca Genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Proteína Quinase Ativada por DNA/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Meiose/genética , Mutação/genética , Rad51 Recombinase/metabolismo , Estresse FisiológicoRESUMO
Faithful chromosome segregation during meiosis I depends on the establishment of a crossover between homologous chromosomes. This requires induction of DNA double-strand breaks (DSBs), alignment of homologs, homolog association by synapsis, and repair of DSBs via homologous recombination. The success of these events requires coordination between chromosomal events and meiotic progression. The conserved SUN/KASH nuclear envelope bridge establishes transient linkages between chromosome ends and cytoskeletal forces during meiosis. In Caenorhabditis elegans, this bridge is essential for bringing homologs together and preventing nonhomologous synapsis. Chromosome movement takes place during synapsis and recombination. Concomitant with the onset of chromosome movement, SUN-1 clusters at chromosome ends associated with the nuclear envelope, and it is phosphorylated in a chk-2- and plk-2-dependent manner. Identification of all SUN-1 phosphomodifications at its nuclear N terminus allowed us to address their role in prophase I. Failures in recombination and synapsis led to persistent phosphorylations, which are required to elicit a delay in progression. Unfinished meiotic tasks elicited sustained recruitment of PLK-2 to chromosome ends in a SUN-1 phosphorylation-dependent manner that is required for continued chromosome movement and characteristic of a zygotene arrest. Furthermore, SUN-1 phosphorylation supported efficient synapsis. We propose that signals emanating from a failure to successfully finish meiotic tasks are integrated at the nuclear periphery to regulate chromosome end-led movement and meiotic progression. The single unsynapsed X chromosome in male meiosis is precluded from inducing a progression delay, and we found it was devoid of a population of phosphorylated SUN-1. This suggests that SUN-1 phosphorylation is critical to delaying meiosis in response to perturbed synapsis. SUN-1 may be an integral part of a checkpoint system to monitor establishment of the obligate crossover, inducible only in leptotene/zygotene. Unrepaired DSBs and unsynapsed chromosomes maintain this checkpoint, but a crossover intermediate is necessary to shut it down.
Assuntos
Proteínas de Caenorhabditis elegans , Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Meiose/genética , Receptores Citoplasmáticos e Nucleares , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Quebras de DNA de Cadeia Dupla , Masculino , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Recombinação Genética/genética , Cromossomo X/genética , Quinase 1 Polo-LikeRESUMO
Introduction of multiple copies of a germ-line-expressed gene elicits silencing of the corresponding endogenous gene during Caenorhabditis elegans oogenesis; this process is referred to as germ-line cosuppression. Transformed plasmids assemble into extrachromosomal arrays resembling extra minichromosomes with repetitive structures. Loss of the transgene extrachromosomal array leads to reversion of the silencing phenomenon. Cosuppression and RNAi depend upon some of the same genes. In the C. elegans germ line, about half the cells undergo a physiological programmed cell death that shares most genetic requirements with somatic apoptosis. In addition, apoptosis is stimulated by DNA damage and synaptic failure mediated through different apoptotic checkpoints. We found that both germ-line cosuppression and RNAi of germ-line-expressed genes enhance apoptosis during C. elegans oogenesis. In contrast, apoptosis is not enhanced by extrachromosomal arrays carrying genes not driven by germ-line-specific promoters that thus do not elicit transgene-mediated cosuppression/silencing. Similarly, introduction of doubled-stranded RNA that shares no homology with endogenous genes has no effect on apoptosis. "Silencing-induced apoptosis" is dependent upon sir-2.1 and cep-1 (the worm p53 ortholog), and is accompanied by a rise in RAD-51 foci, a marker for ongoing DNA repair, indicating induction of DNA double-strand breaks. This finding suggests that the DNA damage-response pathway is involved. RNAi and cosuppression have been postulated as defense mechanisms against genomic intruders. We speculate that the mechanism here described may trigger the elimination of germ cells that have undergone viral infection or transposon activation.
Assuntos
Apoptose/genética , Caenorhabditis elegans/genética , Interferência de RNA , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/fisiologia , Reparo do DNA , Herança Extracromossômica , Dosagem de Genes , Células Germinativas/patologia , Mutação em Linhagem Germinativa , Meiose/genética , Mutagênese Insercional , Plasmídeos/genética , RNA de Cadeia Dupla/genética , Rad51 Recombinase/fisiologia , Sirtuínas/fisiologia , Transgenes , Proteína Supressora de Tumor p53/fisiologiaRESUMO
In most cells, the DNA damage checkpoint delays cell division when replication is stalled by DNA damage. In early Caenorhabditis elegans embryos, however, the checkpoint responds to developmental signals that control the timing of cell division, and checkpoint activation by nondevelopmental inputs disrupts cell cycle timing and causes embryonic lethality. Given this sensitivity to inappropriate checkpoint activation, we were interested in how embryos respond to DNA damage. We demonstrate that the checkpoint response to DNA damage is actively silenced in embryos but not in the germ line. Silencing requires rad-2, gei-17, and the polh-1 translesion DNA polymerase, which suppress replication fork stalling and thereby eliminate the checkpoint-activating signal. These results explain how checkpoint activation is restricted to developmental signals during embryogenesis and insulated from DNA damage. They also show that checkpoint activation is not an obligatory response to DNA damage and that pathways exist to bypass the checkpoint when survival depends on uninterrupted progression through the cell cycle.
Assuntos
Caenorhabditis elegans/embriologia , Proteínas de Ciclo Celular/fisiologia , Dano ao DNA , Embrião não Mamífero/embriologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem , Citocalasina B/farmacologia , Reparo do DNA , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Regulação para Baixo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/efeitos da radiação , Endodesoxirribonucleases/genética , Genes cdc/fisiologia , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Células Germinativas/efeitos da radiação , Hidroxiureia/farmacologia , Ligases/genética , Metanossulfonato de Metila/farmacologia , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Modelos Biológicos , Mutação , Fosfotransferases/genética , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinases/genética , Enzimas de Conjugação de Ubiquitina/genética , Raios UltravioletaRESUMO
Here we probe the relationships between assembly of the synaptonemal complex (SC) and progression of recombination between homologous chromosomes during Caenorhabditis elegans meiosis. We identify SYP-2 as a structural component of the SC central region and show that central region assembly depends on proper morphogenesis of chromosome axes. We find that the SC central region is dispensable for initiation of recombination and for loading of DNA strand-exchange protein RAD-51, despite the fact that extensive RAD-51 loading normally occurs in the context of assembled SC. Further, persistence of RAD-51 foci and absence of crossover products in meiotic mutants suggests that SC central region components and recombination proteins MSH-4 and MSH-5 are required to promote conversion of resected double-strand breaks into stable post-strand exchange intermediates. Our data also suggest that early prophase barriers to utilization of sister chromatids as repair templates do not depend on central region assembly.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Meiose , Proteínas do Tecido Nervoso/fisiologia , Recombinação Genética/fisiologia , Complexo Sinaptonêmico/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/isolamento & purificação , Proteínas de Caenorhabditis elegans/metabolismo , Pareamento Cromossômico , Cromossomos/metabolismo , Troca Genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Esterases/metabolismo , Imuno-Histoquímica , Indóis/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Complexo Sinaptonêmico/ultraestrutura , Fatores de TempoAssuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Eucariotos/genética , Meiose/genética , Reprodução/fisiologia , Animais , Reparo do DNA/genética , Eucariotos/fisiologia , Feminino , Humanos , Masculino , Modelos Biológicos , Reprodução/genética , Sexo , Especificidade da EspécieRESUMO
We have investigated the role of Caenorhabditis elegans RAD-51 during meiotic prophase and embryogenesis, making use of the silencing effect of RNA interference (RNAi). rad-51 RNAi leads to severe defects in chromosome morphology in diakinesis oocytes. We have explored the effect of rad-51 RNAi in mutants lacking fundamental components of the recombination machinery. If double-strand breaks are prevented by spo-11 mutation, rad-51 RNAi does not affect chromosome appearance. This is consistent with a role for RAD-51 downstream of the initiation of recombination. In the absence of MRE-11, as in the absence of SPO-11, RAD-51 depletion has no effect on the chromosomes, which appear intact, thus indicating a role for MRE-11 in DSB induction. Intriguingly, rad-51 silencing in oocytes that lack MSH-5 leads to chromosome fragmentation, a novel trait that is distinct from that seen in msh-5 mutants and in rad-51 RNAi oocytes, suggesting new potential roles for the msh-5 gene. Silencing of the rad-51 gene also causes a reduction in fecundity, which is suppressed by mutation in the DNA damage checkpoint gene rad-5, but not in the cell death effector gene ced-3. Finally, RAD-51 depletion is also seen to affect the soma, resulting in hypersensitivity to ionizing radiation in late embryogenesis.
Assuntos
Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Proteínas de Ligação a DNA/fisiologia , Meiose/fisiologia , Tolerância a Radiação/genética , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Aberrações Cromossômicas/embriologia , Aberrações Cromossômicas/efeitos da radiação , Embrião não Mamífero/embriologia , Embrião não Mamífero/efeitos da radiação , Raios gama , Meiose/efeitos da radiação , RNA Mensageiro/metabolismo , Rad51 RecombinaseRESUMO
RNA interference and transgene-mediated cosuppression are trans-generational silencing mechanisms acting both at a post-transcriptional and epigenetic level. We have recently shown that both these procedures, which share several common factors and are commonly used to phenocopy gene deletions, also induce germ-line DNA damage and apoptosis. These observations shed new light on the cross-talk between different pathways devoted to the protection of genome stability in germ cells.
RESUMO
The breast and ovarian cancer susceptibility protein BRCA1 is evolutionarily conserved and functions in DNA double-strand break (DSB) repair through homologous recombination, but its role in meiosis is poorly understood. By using genetic analysis, we investigated the role of the Caenorhabditis elegans BRCA1 orthologue (brc-1) during meiotic prophase. The null mutant in the brc-1 gene is viable, fertile and shows the wild-type complement of six bivalents in most diakinetic nuclei, which is indicative of successful crossover recombination. However, brc-1 mutants show an abnormal increase in apoptosis and RAD-51 foci at pachytene that are abolished by loss of spo-11 function, suggesting a defect in meiosis rather than during premeiotic DNA replication. In genetic backgrounds in which chiasma formation is abrogated, such as him-14/MSH4 and syp-2, loss of brc-1 leads to chromosome fragmentation suggesting that brc-1 is dispensable for crossing over but essential for DSB repair through inter-sister recombination.