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1.
Methods Mol Biol ; 2391: 31-43, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34686974

RESUMO

In fungi, karyotyping is fundamental to understanding their genome organization. It is also essential to study various genome- or chromosome-related topics such as karyotype polymorphisms and supernumerary or pathogenicity chromosomes. Here, we describe the protocols of pulsed-field gel electrophoresis and the germ tube burst method for molecular and cytological karyotyping of Fusarium oxysporum. The combined use of the two methods is valuable for determining definitive and comprehensive karyotypes of these fungi.


Assuntos
Fusarium , Cromossomos Fúngicos/genética , Eletroforese em Gel de Campo Pulsado , Fusarium/genética , Cariotipagem
2.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34747981

RESUMO

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.


Assuntos
Antígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Homeostase , Meiose , Ploidias , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Corpos Polares do Fuso/metabolismo , Cromossomos Fúngicos/metabolismo , Esporos Fúngicos/metabolismo , Zigoto/citologia
3.
Cells ; 10(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34440781

RESUMO

Nuclear movements during meiotic prophase, driven by cytoskeleton forces, are a broadly conserved mechanism in opisthokonts and plants to promote pairing between homologous chromosomes. These forces are transmitted to the chromosomes by specific associations between telomeres and the nuclear envelope during meiotic prophase. Defective chromosome movements (CMs) harm pairing and recombination dynamics between homologues, thereby affecting faithful gametogenesis. For this reason, modelling the behaviour of CMs and their possible microvariations as a result of mutations or physico-chemical stress is important to understand this crucial stage of meiosis. Current developments in high-throughput imaging and image processing are yielding large CM datasets that are suitable for data mining approaches. To facilitate adoption of data mining pipelines, we present ChroMo, an interactive, unsupervised cloud application specifically designed for exploring CM datasets from live imaging. ChroMo contains a wide selection of algorithms and visualizations for time-series segmentation, motif discovery, and assessment of causality networks. Using ChroMo to analyse meiotic CMs in fission yeast, we found previously undiscovered features of CMs and causality relationships between chromosome morphology and trajectory. ChroMo will be a useful tool for understanding the behaviour of meiotic CMs in yeast and other model organisms.


Assuntos
Algoritmos , Segregação de Cromossomos , Cromossomos Fúngicos , Interpretação de Imagem Assistida por Computador , Meiose , Microscopia de Fluorescência , Schizosaccharomyces/crescimento & desenvolvimento , Imagem com Lapso de Tempo , Automação Laboratorial , Computação em Nuvem , Ensaios de Triagem em Larga Escala , Schizosaccharomyces/genética , Fatores de Tempo
4.
Sci Rep ; 11(1): 14940, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294749

RESUMO

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a chromosome segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and, probably, anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses, and GFP-tagged DNA damage markers suggest that neither recombination intermediates nor unfinished replication account for the postreplicative PFGE shift, which is further supported by the fact that the shift does not trigger the G2/M checkpoint. We propose that the absence of Top2 activity leads to a general chromosome structural/topological change in mitosis.


Assuntos
Cromossomos Fúngicos/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Ciclo Celular , Segregação de Cromossomos , DNA Topoisomerases Tipo II/deficiência , Eletroforese em Gel de Campo Pulsado , Técnicas de Inativação de Genes , Mitose , Saccharomyces cerevisiae/genética
5.
Nucleic Acids Res ; 49(13): 7537-7553, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197600

RESUMO

The synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. In budding yeast, Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. During SC elongation, the SUMOylated SC component Ecm11 and the Ecm11-interacting protein Gmc2 facilitate the polymerization of Zip1, an SC central region component. Through physical recombination, cytological, and genetic analyses, we found that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies on a short chromosome (chromosome III) were reduced, whereas CO and non-crossover frequencies on a long chromosome (chromosome VII) were elevated. Further, in ecm11 and gmc2 mutants, more double-strand breaks (DSBs) were formed on a long chromosome during late prophase I, implying that the Ecm11-Gmc2 (EG) complex is involved in the homeostatic regulation of DSB formation. The EG complex may participate in joint molecule (JM) processing and/or double-Holliday junction resolution for ZMM-dependent CO-designated recombination. Absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role for the EG complex in suppressing ZMM-independent recombination. Our results suggest that the SC central region functions as a compartment for sequestering recombination-associated proteins to regulate meiosis specificity during recombination.


Assuntos
Proteínas de Ciclo Celular/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexo Sinaptonêmico/metabolismo , Cromossomos Fúngicos , Replicação do DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Retroalimentação Fisiológica , Deleção de Genes , Recombinação Genética , Saccharomyces cerevisiae/genética , Temperatura , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
6.
PLoS Genet ; 17(7): e1009560, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34260586

RESUMO

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.


Assuntos
Citoplasma/genética , Proteínas Nucleares/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Pontos de Checagem do Ciclo Celular , Membrana Celular/metabolismo , Pareamento Cromossômico , Cromossomos Fúngicos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Meiose , Microrganismos Geneticamente Modificados , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Commun Biol ; 4(1): 707, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108627

RESUMO

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis (Arabidopsis thaliana) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.


Assuntos
Cromossomos Fúngicos/genética , Fusarium/genética , Doenças das Plantas/microbiologia , Arabidopsis/imunologia , Arabidopsis/microbiologia , Brassicaceae/imunologia , Brassicaceae/microbiologia , Cromossomos Fúngicos/fisiologia , Fusarium/patogenicidade , Fusarium/fisiologia , Genoma Fúngico/genética , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/imunologia
8.
Nat Commun ; 12(1): 3418, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103502

RESUMO

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.


Assuntos
Cryptococcus/efeitos dos fármacos , Farmacorresistência Fúngica , Flucitosina/farmacologia , Cromossomos Fúngicos/genética , Células Clonais , Cryptococcus/genética , Cryptococcus/crescimento & desenvolvimento , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Duplicação Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Supressores , Variação Genética , Genoma Fúngico , Espaço Intracelular/metabolismo , Testes de Sensibilidade Microbiana , Mutação/genética , Reprodutibilidade dos Testes , Uridina Difosfato Ácido Glucurônico/metabolismo
9.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34081091

RESUMO

The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.


Assuntos
Segregação de Cromossomos , Cromossomos Fúngicos , Endopeptidases/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Endopeptidases/química , Endopeptidases/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação
10.
Nat Commun ; 12(1): 2763, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980827

RESUMO

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Motivos de Aminoácidos , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/química , Exodesoxirribonucleases/química , Modelos Moleculares , Complexos Multiproteicos , Mutação , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética
11.
Fungal Genet Biol ; 153: 103566, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33991664

RESUMO

Fusarium graminearum is one of the most frequent causal agents of the Fusarium Head Blight, a cereal disease spread throughout the world, reducing grain production and quality. F. graminearum isolates are genetically and phenotypically highly diverse. Notably, remarkable variations of aggressiveness between isolates have been observed, which could reflect an adaptive potential of this pathogen. In this study, we aimed to characterize the genetic basis of aggressiveness variation observed in an F1 population (n = 94), for which genome sequences of both parental strains are available. Aggressiveness was assessed by a panel of in planta and in vitro proxies during two phenotyping trials including, among others, disease severity and mycotoxin accumulation in wheat spike. One major and single QTL was mapped for all the traits measured, on chromosome I, that explained up to 90% of the variance for disease severity. The confidence interval at the QTL spanned 1.2 Mb and contained 428 genes on the reference genome. Of these, four candidates were selected based on the postulate that a non-synonymous mutation affecting protein function may be responsible for phenotypic differences. Finally, a new mutation was identified and functionally validated in the gene FgVe1, coding for a velvet protein known to be involved in pathogenicity and secondary metabolism production in several fungi.


Assuntos
Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/patogenicidade , Doenças das Plantas/microbiologia , Triticum/microbiologia , Alelos , Mapeamento Cromossômico , Cromossomos Fúngicos , Genes Fúngicos , Mutação , Fenótipo , Locos de Características Quantitativas , Metabolismo Secundário/genética
13.
FEMS Yeast Res ; 21(4)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33893795

RESUMO

Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.


Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Cromossomos Fúngicos/genética , Microbiologia Industrial , Plasmídeos , RNA Guia
14.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800857

RESUMO

Fusarium wilt of flax is an aggressive disease caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. lini. It is a challenging pathogen presenting a constant threat to flax production industry worldwide. Previously, we reported chromosome-level assemblies of 5 highly pathogenic F. oxysporum f. sp. lini strains. We sought to characterize the genomic architecture of the fungus and outline evolutionary mechanisms shaping the pathogen genome. Here, we reveal the complex multi-compartmentalized genome organization and uncover its diverse evolutionary dynamics, which boosts genetic diversity and facilitates host adaptation. In addition, our results suggest that host of functions implicated in the life cycle of mobile genetic elements are main contributors to dissimilarity between proteomes of different Fusaria. Finally, our experiments demonstrate that mobile genetics elements are expressed in planta upon infection, alluding to their role in pathogenicity. On the whole, these results pave the way for further in-depth studies of evolutionary forces shaping the host-pathogen interaction.


Assuntos
Linho/microbiologia , Fusarium/genética , Genoma Fúngico , Doenças das Plantas/microbiologia , Cromossomos Fúngicos/genética , Evolução Molecular , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno/genética , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Filogenia , Proteoma , Especificidade da Espécie , Virulência/genética
15.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33875604

RESUMO

Meiotic drive elements cause their own preferential transmission following meiosis. In fungi, this phenomenon takes the shape of spore killing, and in the filamentous ascomycete Neurospora sitophila, the Sk-1 spore killer element is found in many natural populations. In this study, we identify the gene responsible for spore killing in Sk-1 by generating both long- and short-read genomic data and by using these data to perform a genome-wide association test. We name this gene Spk-1 Through molecular dissection, we show that a single 405-nt-long open reading frame generates a product that both acts as a poison capable of killing sibling spores and as an antidote that rescues spores that produce it. By phylogenetic analysis, we demonstrate that the gene has likely been introgressed from the closely related species Neurospora hispaniola, and we identify three subclades of N. sitophila, one where Sk-1 is fixed, another where Sk-1 is absent, and a third where both killer and sensitive strain are found. Finally, we show that spore killing can be suppressed through an RNA interference-based genome defense pathway known as meiotic silencing by unpaired DNA. Spk-1 is not related to other known meiotic drive genes, and similar sequences are only found within Neurospora These results shed light on the diversity of genes capable of causing meiotic drive, their origin and evolution, and their interaction with the host genome.


Assuntos
Introgressão Genética , Neurospora/genética , Interferência de RNA , Sequências Repetitivas de Ácido Nucleico , Cromossomos Fúngicos
16.
Biochem Biophys Res Commun ; 556: 179-184, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33839413

RESUMO

Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4-S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT)12 and (ACT)12 act as strong nucleosome-promoting sequences, while (AGG)12 and (CCG)12 act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG)12, is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.


Assuntos
Nucleossomos/química , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Repetições de Trinucleotídeos/genética , Sequência de Bases/genética , Cromossomos Fúngicos/química , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo
17.
PLoS Genet ; 17(3): e1009442, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33735169

RESUMO

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.


Assuntos
Centrômero/genética , Cromossomos Fúngicos , Fenótipo , Saccharomycetales/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Fúngicas , Recombinação Homóloga , Saccharomycetales/metabolismo
18.
Gene ; 784: 145584, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33753149

RESUMO

Sister chromatid cohesion (SCC) is essential for the maintenance of genome integrity. The establishment of SCC is coupled to DNA replication, and this is achieved in budding yeast Saccharomyces cerevisiae by a mechanism that is dependent on the interaction between Eco1 acetyltransferase and PCNA in the DNA replication complex. In vertebrates, the Eco1 homolog ESCO2 has been reported to interact with MCM complex in the DNA replication complex to establish DNA replication-dependent cohesion. Here we show that budding yeast Eco1 is also physically interacted with the MCM complex. We found that Eco1 was specifically bound to Mcm2 subunit in the MCM complex and they interacted via their N-terminal regions, using yeast two-hybrid system. The underlying mechanism of the interaction was different between yeast and vertebrates. Intensive molecular dissection of Eco1 identified residues important for interaction with Mcm2 and/or PCNA. Mutant forms of Eco1 (Eco1mWW and Eco1mGRK), where sets of the identified residues were substituted with alanine, resulted in impaired SCC, decreased level of acetylation of Smc3, and a reduction of Eco1 protein amount in yeast cells. We, hence, suggest that Eco1 is stabilized by its interactions with MCM complex and PCNA, which allows it to promote DNA replication-coupled SCC establishment.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Sítios de Ligação , Cromossomos Fúngicos/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estabilidade Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
19.
Curr Genet ; 67(3): 431-437, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33604699

RESUMO

The programmed formation of DNA double-strand breaks (DSBs) in meiotic prophase I initiates the homologous recombination process that yields crossovers between homologous chromosomes, a prerequisite to accurately segregating chromosomes during meiosis I (MI). In the budding yeast Saccharomyces cerevisiae, proteins required for meiotic DSB formation (DSB proteins) accumulate to higher levels specifically on short chromosomes to ensure that these chromosomes make DSBs. We previously demonstrated that as-yet undefined cis-acting elements preferentially recruit DSB proteins and promote higher levels of DSBs and recombination and that these intrinsic features are subject to selection pressure to maintain the hyperrecombinogenic properties of short chromosomes. Thus, this targeted boosting of DSB protein binding may be an evolutionarily recurrent strategy to mitigate the risk of meiotic mis-segregation caused by karyotypic constraints. However, the underlining mechanisms are still elusive. Here, we discuss possible scenarios in which components of the meiotic chromosome axis (Red1 and Hop1) bind to intrinsic features independent of the meiosis-specific cohesin subunit Rec8 and DNA replication, promoting preferential binding of DSB proteins to short chromosomes. We also propose a model where chromosome position in the nucleus, influenced by centromeres, promotes the short-chromosome boost of DSB proteins.


Assuntos
Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Prófase Meiótica I/genética , Saccharomyces cerevisiae/genética
20.
PLoS Genet ; 17(2): e1009386, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33591993

RESUMO

Supernumerary mini-chromosomes-a unique type of genomic structural variation-have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.


Assuntos
Ascomicetos/genética , Cromossomos Fúngicos/genética , Rearranjo Gênico/genética , Genoma Fúngico/genética , Genômica/métodos , Ascomicetos/patogenicidade , Eleusine/genética , Eleusine/microbiologia , Evolução Molecular , Genes Fúngicos/genética , Variação Genética , Interações Hospedeiro-Patógeno/genética , Milhetes/genética , Milhetes/microbiologia , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Virulência/genética
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