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1.
Mol Cell ; 39(1): 25-35, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20598602

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 Fisiológico
2.
PLoS Genet ; 9(3): e1003335, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23505384

RESUMO

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-Like
3.
Proc Natl Acad Sci U S A ; 109(9): 3440-5, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22331911

RESUMO

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/fisiologia
4.
Biology (Basel) ; 12(11)2023 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-37997977

RESUMO

We investigated the effects of dietary delivered self-DNA in the model insect Drosophila melanogaster. Self-DNA administration resulted in low but significant lethality in Drosophila larvae and considerably extended the fly developmental time. This was characterized by the abnormal persistence of the larvae in the L2 and L3 stages, which largely accounted for the average 72 h delay observed in pupariation, as compared to controls. In addition, self-DNA exposure affected adult reproduction by markedly reducing both female fecundity and fertility, further demonstrating its impact on Drosophila developmental processes. The effects on the metabolites of D. melanogaster larvae after exposure to self-DNA were studied by NMR, LC-MS, and molecular networking. The results showed that self-DNA feeding reduces the amounts of all metabolites, particularly amino acids and N-acyl amino acids, which are known to act as lipid signal mediators. An increasing amount of phloroglucinol was found after self-DNA exposure and correlated to developmental delay and egg-laying suppression. Pidolate, a known intermediate in the γ-glutamyl cycle, also increased after exposure to self-DNA and correlated to the block of insect oogenesis.

5.
Biology (Basel) ; 11(2)2022 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-35205128

RESUMO

All organisms, from bacteria to mammals, sense and respond to foreign nucleic acids to fight infections in order to survive and preserve genome integrity across generations. The innate immune system is an evolutionarily conserved defence strategy. Complex organisms have developed various cellular processes to respond to and recognise not only infections, i.e., pathogen-associated molecular patterns (PAMPs), but also to sense injury and tissue dysfunctions, i.e., damage-associated molecular patterns (DAMPs). Mis-localized self-DNA can be sensed as DAMP by specific DNA-sensing pathways, and self-DNA chronic exposure can be detrimental to the organisms. Here, we investigate the effects of dietary delivered self-DNA in the nematode Caenorhabditis elegans. The hermaphrodite worms were fed on Escherichia coli genomic libraries: a C. elegans library (self) and a legume (Medicago truncatula) library (non-self). We show that the self-library diet affects embryogenesis, larval development and gametogenesis. DNA damage and activation of p53/CEP-1-dependent apoptosis occur in gonadal germ cells. Studies of self-DNA exposure in this model organism were not pursued up to now. The genetic tractability of C. elegans will help to identify the basic molecular pathways involved in such mechanisms. The specificity of the adverse effects associated with a self-DNA enriched diet suggests applications in biological pest control approaches.

6.
Sci Rep ; 10(1): 20913, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262405

RESUMO

Cystathionine ß-synthase (CBS) is a eukaryotic enzyme that maintains the cellular homocysteine homeostasis and catalyzes the conversion of homocysteine to L-cystathionine and Hydrogen sulfide, via the trans-sulfuration pathway. In Caenorhabditis elegans, two cbs genes are present: cbs-1 functions similarly as to human CBS, and cbs-2, whose roles are instead unknown. In the present study we performed a phenotypic characterization of the cbs-2 mutant. The null cbs-2 mutant is viable, fertile and shows the wild-type complement of six bivalents in most oocyte nuclei, which is indicative of a correct formation of crossover recombination. In absence of synaptonemal complex formation (syp-2 mutant), loss of cbs-2 leads to chromosome fragmentation, suggesting that cbs-2 is essential during inter-sister repair. Interestingly, although proficient in the activation of the DNA damage checkpoint after exposure to genotoxic stress, the cbs-2 mutant is defective in DNA damage-induced apoptosis in meiotic germ cells. These results suggest possible functions for CBS-2 in meiosis, distinct from a role in the trans-sulfuration pathway. We propose that the C. elegans CBS-2 protein is required for both inter-sister repair and execution of DNA damage-induced apoptosis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Meiose/genética , Animais , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/metabolismo , Dano ao DNA/genética , Reparo do DNA , Genes Letais
7.
Sci Rep ; 10(1): 103, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919410

RESUMO

Fanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Meiose , Recombinação Genética , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética
8.
Dev Cell ; 5(3): 463-74, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12967565

RESUMO

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 Tempo
9.
Sci Rep ; 9(1): 6889, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053748

RESUMO

DNA alkylguanine DNA alkyltransferases (AGTs) are evolutionary conserved proteins that repair alkylation damage in DNA, counteracting the effects of agents inducing such lesions. Over the last years AGTs have raised considerable interest for both the peculiarity of their molecular mechanism and their relevance in cancer biology. AGT knock out mice show increased tumour incidence in response to alkylating agents, and over-expression of the human AGT protein in cancer cells is frequently associated with resistance to alkylating chemotherapy. While all data available point to a function of AGT proteins in the cell response to alkylation lesions, we report for the first time that one of the two AGT paralogs of the model organism C. elegans, called AGT-2, also plays unexpected roles in meiosis and early development under physiological conditions. Our data suggest a role for AGT-2 in conversion of homologous recombination intermediates into post-strand exchange products in meiosis, and show that agt-2 gene down-regulation, or treatment of animals with an AGT inhibitor results in increased number of germ cells that are incompatible with producing viable offspring and are eliminated by apoptosis. These results suggest possible functions for AGTs in cell processes distinct from repair of alkylating damage.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Meiose , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Reparo do DNA/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Meiose/genética , O(6)-Metilguanina-DNA Metiltransferase/genética
10.
Genetics ; 209(4): 1017-1028, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884745

RESUMO

The evolutionarily conserved RAD-51 protein is essential for homologous recombination in the germ line as well as homologous repair of DNA double-strand breaks in all eukaryotic cells. In the nematode Caenorhabditis elegans, the rad-51 gene is transcribed into messenger RNAs potentially coding three alternative protein isoforms. Null rad-51 alleles display embryonic lethality, severe defects in chromosome structure, and high levels of germ line apoptosis. To dissect its functions, we genetically modified the C. elegans rad-51 gene by clustered regularly interspaced short palindromic repeats/Cas9 genome-editing technology, obtaining a separation-of-function (sfi-) mutant allele that only disrupts the long-transcript isoform. This mutant shows no defects in an otherwise wild-type meiosis and is able to activate physiological germ cell death, which occurs at the late pachytene stage. However, although the mutant is competent in DNA damage checkpoint activation after exposure to ionizing radiation, it is defective for induction of DNA damage-induced apoptosis in meiotic germ cells. These results suggest that RAD-51 plays a novel role in germ line apoptosis independent of RAD-51-mediated strand invasion for homologous recombination.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Germinativas/citologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Animais , Apoptose , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Dano ao DNA , Feminino , Células Germinativas/metabolismo , Masculino , Mutação
11.
Worm ; 1(4): 212-5, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24058851

RESUMO

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.

12.
EMBO Rep ; 9(3): 287-92, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18219312

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.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Troca Genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Meiose , Animais , Apoptose , Caenorhabditis elegans/enzimologia , Perda do Embrião , Endodesoxirribonucleases , Esterases/metabolismo , Indóis , Prófase Meiótica I , Mutação/genética , Rad51 Recombinase/metabolismo
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