RESUMEN
Immunofluorescent staining is commonly used to generate images to characterize cytological phenotypes. The manual quantification of DNA double-strand breaks and their repair intermediates during meiosis using image data requires a series of subjective steps, from image selection to the counting of particular events per nucleus. Here we describe "synapsis," a bioconductor package, which includes a set of functions to automate the process of identifying meiotic nuclei and quantifying key double-strand break formation and repair events in a rapid, scalable, and reproducible workflow, and compare it to manual user quantification. The software can be extended for other applications in meiosis research, such as incorporating machine learning approaches to categorize meiotic substages.
Asunto(s)
Emparejamiento Cromosómico , Roturas del ADN de Doble Cadena , Reparación del ADN , Meiosis , Programas Informáticos , Intercambio Genético , Humanos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, Fanconi anemia complementation group A (FANCA), is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous FANCA variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic FANCA variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel FANCA variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in FANCA, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes.
Asunto(s)
Alelos , Proteína del Grupo de Complementación A de la Anemia de Fanconi , Insuficiencia Ovárica Primaria , Humanos , Insuficiencia Ovárica Primaria/genética , Femenino , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Adulto , Anemia de Fanconi/genética , Anemia de Fanconi/diagnóstico , Hermanos , Heterocigoto , Predisposición Genética a la Enfermedad , Linaje , Mutación/genéticaRESUMEN
Meiotic crossovers are required for accurate chromosome segregation and producing new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we reveal the tumor suppressor FANCM as a meiotic anti-crossover factor in mammals. We use unique large-scale crossover analyses with both single-gamete sequencing and pedigree-based bulk-sequencing datasets to identify a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss-of-function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway after birth. Despite the gametogenesis phenotypes in Fancm mutants, both sexes are capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.
RESUMEN
At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1 and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.
The Fanconi Anemia (FA) pathway is the subject of intense interest owing to the role of FA as a tumor suppressor. Three FA complex proteins, FANCM, MHF1 and MHF2, were identified as factors that suppress crossover during meiosis in the model plant Arabidopsis thaliana. Here, the authors extended these findings and identified a novel anti-crossover factor and showed that it encodes the plant FANCC homolog, which was previously thought to be vertebrate-specific. They further showed that FANCC regulates meiotic crossover together with two other FA proteins, FANCE and FANCF. This suggests that the FANCCEF subcomplex was already regulating DNA repair in the common ancestor of all living eukaryotes.
Asunto(s)
Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteína del Grupo de Complementación F de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Meiosis , Humanos , Arabidopsis/genética , Arabidopsis/metabolismo , ADN/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación F de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación F de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Recombinación HomólogaRESUMEN
We developed a simple and reliable method for the isolation of haploid nuclei from fresh and frozen testes. The described protocol uses readily available reagents in combination with flow cytometry to separate haploid and diploid nuclei. The protocol can be completed within 1 hour and the resulting individual haploid nuclei have intact morphology. The isolated nuclei are suitable for library preparation for high-throughput DNA and RNA sequencing using bulk or single nuclei. The protocol was optimised with mouse testes and we anticipate that it can be applied for the isolation of mature sperm from other mammals including humans.
Asunto(s)
Ácidos Nucleicos , Animales , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mamíferos , Ratones , Semen , EspermatozoidesRESUMEN
Profiling gametes of an individual enables the construction of personalised haplotypes and meiotic crossover landscapes, now achievable at larger scale than ever through the availability of high-throughput single-cell sequencing technologies. However, high-throughput single-gamete data commonly have low depth of coverage per gamete, which challenges existing gamete-based haplotype phasing methods. In addition, haplotyping a large number of single gametes from high-throughput single-cell DNA sequencing data and constructing meiotic crossover profiles using existing methods requires intensive processing. Here, we introduce efficient software tools for the essential tasks of generating personalised haplotypes and calling crossovers in gametes from single-gamete DNA sequencing data (sgcocaller), and constructing, visualising, and comparing individualised crossover landscapes from single gametes (comapr). With additional data pre-possessing, the tools can also be applied to bulk-sequenced samples. We demonstrate that sgcocaller is able to generate impeccable phasing results for high-coverage datasets, on which it is more accurate and stable than existing methods, and also performs well on low-coverage single-gamete sequencing datasets for which current methods fail. Our tools achieve highly accurate results with user-friendly installation, comprehensive documentation, efficient computation times and minimal memory usage.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Algoritmos , Células Germinativas , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Análisis de Expresión Génica de una Sola Célula , Programas Informáticos , Intercambio GenéticoRESUMEN
Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Reparación del ADN/efectos de los fármacos , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Anemia de Fanconi/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteína BRCA1/deficiencia , Proteína BRCA2/deficiencia , Daño del ADN , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/uso terapéutico , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/antagonistas & inhibidores , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Terapia Molecular Dirigida/métodos , Morfolinas/uso terapéutico , Pironas/uso terapéutico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Mutaciones Letales Sintéticas , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/antagonistas & inhibidores , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Genetic maps have been fundamental to building our understanding of disease genetics and evolutionary processes. The gametes of an individual contain all of the information required to perform a de novo chromosome-scale assembly of an individual's genome, which historically has been performed with populations and pedigrees. Here, we discuss how single-cell gamete sequencing offers the potential to merge the advantages of short-read sequencing with the ability to build personalized genetic maps and open up an entirely new space in personalized genetics.
Asunto(s)
Genoma , Genómica/métodos , Células Germinativas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Medicina de Precisión/métodos , Análisis de la Célula Individual/métodos , Animales , Mapeo Cromosómico , Biología Computacional/métodos , Biología Computacional/normas , Interpretación Estadística de Datos , Heterogeneidad Genética , Genómica/normas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Medicina de Precisión/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de la Célula Individual/normas , Secuenciación Completa del GenomaRESUMEN
DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular Tumoral , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Humanos , Ubiquitinación/efectos de los fármacosRESUMEN
Germline mutations in Fanconi anemia (FA) genes predispose to chromosome instability syndromes, such as FA and cancers. FA gene products have traditionally been studied for their role in interstrand cross link (ICL) repair. A fraction of FA gene products are classical homologous recombination (HR) factors that are involved in repairing DNA double-strand breaks (DSBs) in an error-free manner. Emerging evidence suggests that, independent of ICL and HR repair, FA genes protect DNA replication forks in the presence of replication stress. Therefore, understanding the precise function of FA genes and their role in promoting genome stability in response to DNA replication stress is crucial for diagnosing FA and FA-associated cancers. Moreover, molecular understanding of the FA pathway will greatly help to establish proper functional assays for variants of unknown significance (VUS), often encountered in clinics. In this short review, we discuss the recently uncovered molecular details of FA genes in replication fork protection pathways. Finally, we examine how novel FA variants predispose to FA and cancer, due to defective replication fork protection activity.
Asunto(s)
Reparación del ADN , Replicación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Anemia de Fanconi/patología , Inestabilidad Genómica , Recombinación Homóloga , Anemia de Fanconi/genética , HumanosRESUMEN
Among the hundreds of recombination events initiated at meiosis, only a subset is selected to become crossovers. In this issue of Developmental Cell, Sandhu et al. (2020) reveal that budding yeast Mph1/FANCM dismantles recombination events between sister chromatids at early meiosis, thus favoring recombination with homologs.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Saccharomyces cerevisiae , ADN Helicasas , Reparación del ADN/genética , Meiosis/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.
Bone marrow is the spongy tissue inside bones that produces blood cells. Fanconi anemia is the most common form of inherited bone marrow death and affects children and young adults. In this disease, bone marrow cells cannot attach a protein tag called ubiquitin to another protein called FANCD2. When DNA becomes damaged, FANCD2 helps cells to respond and repair the damage but without ubiquitin it cannot do this correctly. Without ubiquitin linked to FANCD2 bone marrow cells die from damaged DNA. Another protein, called FANCI, works in partnership with FANCD2 and also gets linked to ubiquitin. Tan et al. studied purified proteins in the laboratory to understand how linking ubiquitin changes the behavior of FANCD2 and FANCI. When the proteins have ubiquitin attached, they can form stable attachments to DNA. Without ubiquitin, however, the proteins only attach to DNA for short periods of time. Using electron microscopy, Tan et al. discovered that large numbers of the modified proteins become tightly attached to damaged DNA, helping to protect it and triggering DNA repair processes. Understanding the role of FANCD2 in Fanconi anemia could lead to new treatments. FANCD2 and FANCI have similar roles in other cells too. Stopping them from protecting damaged DNA in cancer cells could be used to enhance the success of chemotherapies and radiotherapies.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Replicación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , UbiquitinaciónRESUMEN
Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.
Asunto(s)
Avidina/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Antígeno Nuclear de Célula en Proliferación , Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/aislamiento & purificación , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/aislamiento & purificación , Humanos , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Células Sf9 , Spodoptera , Ubiquitina/química , Ubiquitina/aislamiento & purificación , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/aislamiento & purificación , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/aislamiento & purificaciónRESUMEN
Fanconi anemia (FA) is a life-threatening syndrome characterized by bone marrow failure and cancer predispositions. The past two decades have seen an explosion of data in the FA field, both in humans and other organisms, following the cloning of 22 FA genes. A common but notably understudied clinical feature of the disease is the reduced fertility of individuals with FA. This review focuses on the known causes of reduced fertility in FA, and integrates them with the emerging role of the FA pathway in double-strand break (DSB) repair at meiosis in a variety of organisms, as well as providing future directions for research and diagnostics.
Asunto(s)
Trastornos de Fallo de la Médula Ósea/genética , Anemia de Fanconi/genética , Fertilidad/genética , Trastornos de Fallo de la Médula Ósea/complicaciones , Trastornos de Fallo de la Médula Ósea/patología , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Anemia de Fanconi/complicaciones , Anemia de Fanconi/patología , Humanos , Meiosis/genéticaRESUMEN
At meiosis, hundreds of programmed DNA double-strand breaks (DSBs) form and are repaired by homologous recombination. From this large number of DSBs, only a subset yields crossovers (COs), with a minimum of one CO per chromosome pair. All DSBs must be repaired and every recombination intermediate must be resolved to avoid subsequent entanglement and chromosome breakage. The conserved BLM-TOP3α-RMI1 (BTR) complex acts on early and late meiotic recombination intermediates to both limit CO outcome and promote chromosome integrity. In Arabidopsis, the BLM homologues RECQ4A and RECQ4B act redundantly to prevent meiotic extra COs, but recombination intermediates are fully resolved in their absence. In contrast, TOP3α is needed for both processes. Here we show through the characterization of specific mutants that RMI1 is a major anti-CO factor, in addition to being essential to prevent chromosome breakage and entanglement. Further, our findings suggest a specific role of the C-terminal domains of RMI1 and TOP3α, that respectively contain an Oligo Binding domain (OB2) and ZINC finger motifs, in preventing extra-CO. We propose that these domains of TOP3α and RMI1 define a sub-domain of the BTR complex which is dispensable for the resolution of recombination intermediates but crucial to limit extra-COs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Intercambio Genético , ADN-Topoisomerasas de Tipo I/metabolismo , Meiosis , Dominios y Motivos de Interacción de Proteínas , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Portadoras/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo I/química , Epistasis Genética , Modelos Biológicos , Mutación , Unión Proteica , Recombinación Genética , Dedos de ZincRESUMEN
Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.
Asunto(s)
Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitinación , Línea Celular , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Factores de Tiempo , Transfección , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Ciclinas/metabolismo , Meiosis , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromosomas de las Plantas/genética , Ciclinas/genética , Epistasis Genética , Genes Dominantes , Pruebas Genéticas , Modelos Biológicos , Mutación/genética , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismo , Tetraploidía , Tubulina (Proteína)/metabolismoRESUMEN
Meiotic crossovers (COs) generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression.