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1.
Proc Natl Acad Sci U S A ; 117(40): 24947-24956, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968016

RESUMO

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA , Diploide , Feminino , Endonucleases Flap/genética , Genoma Fúngico/genética , Humanos , Perda de Heterozigosidade/genética , Proteínas de Membrana/genética , Peroxirredoxinas/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
2.
Mol Biol Evol ; 36(12): 2861-2877, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31397846

RESUMO

Mutations, recombinations, and genome duplications may promote genetic diversity and trigger evolutionary processes. However, quantifying these events in diploid hybrid genomes is challenging. Here, we present an integrated experimental and computational workflow to accurately track the mutational landscape of yeast diploid hybrids (MuLoYDH) in terms of single-nucleotide variants, small insertions/deletions, copy-number variants, aneuploidies, and loss-of-heterozygosity. Pairs of haploid Saccharomyces parents were combined to generate ancestor hybrids with phased genomes and varying levels of heterozygosity. These diploids were evolved under different laboratory protocols, in particular mutation accumulation experiments. Variant simulations enabled the efficient integration of competitive and standard mapping of short reads, depending on local levels of heterozygosity. Experimental validations proved the high accuracy and resolution of our computational approach. Finally, applying MuLoYDH to four different diploids revealed striking genetic background effects. Homozygous Saccharomyces cerevisiae showed a ∼4-fold higher mutation rate compared with its closely related species S. paradoxus. Intraspecies hybrids unveiled that a substantial fraction of the genome (∼250 bp per generation) was shaped by loss-of-heterozygosity, a process strongly inhibited in interspecies hybrids by high levels of sequence divergence between homologous chromosomes. In contrast, interspecies hybrids exhibited higher single-nucleotide mutation rates compared with intraspecies hybrids. MuLoYDH provided an unprecedented quantitative insight into the evolutionary processes that mold diploid yeast genomes and can be generalized to other genetic systems.


Assuntos
Evolução Molecular , Técnicas Genéticas , Hibridização Genética , Mutação , Polimorfismo Genético , Diploide , Genoma Fúngico , Saccharomyces cerevisiae
3.
DNA Repair (Amst) ; 76: 40-49, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30818168

RESUMO

DNA polymerases influence genome stability through their involvement in DNA replication, response to DNA damage, and DNA repair processes. Saccharomyces cerevisiae possess four non-essential DNA polymerases, Pol λ, Pol η, Pol ζ, and Rev1, which have varying roles in genome stability. In order to assess the contribution of the non-essential DNA polymerases in genome stability, we analyzed the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant in microhomology mediated repair, due to recent studies linking some of these DNA polymerases to this repair pathway. Our results suggest that the length and quality of microhomology influence both the overall efficiency of repair and the involvement of DNA polymerases. Furthermore, the non-essential DNA polymerases demonstrate overlapping and redundant functions when repairing double-strand breaks using short microhomologies containing mismatches. Then, we examined genome-wide mutation accumulation in the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant compared to wild type cells. We found a significant decrease in the overall rate of mutation accumulation in the quadruple mutant cells compared to wildtype, but an increase in frameshift mutations and a shift towards transversion base-substitution with a preference for G:C to T:A or C:G. Thus, the non-essential DNA polymerases have an impact on the nature of the mutational spectrum. The sequence and functional homology shared between human and S. cerevisiae non-essential DNA polymerases suggest these DNA polymerases may have a similar role in human cells.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Mutação
4.
J Cell Sci ; 129(17): 3332-41, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27466378

RESUMO

The diversity of microtubule functions is dependent on the status of tubulin C-termini. To address the physiological role of the C-terminal aromatic residue of α-tubulin, a tub1-Glu yeast strain expressing an α-tubulin devoid of its C-terminal amino acid was used to perform a genome-wide-lethality screen. The identified synthetic lethal genes suggested links with endocytosis and related processes. In the tub1-Glu strain, the routing of the v-SNARE Snc1 was strongly impaired, with a loss of its polarized distribution in the bud, and Abp1, an actin patch or endocytic marker, developed comet-tail structures. Snc1 trafficking required dynamic microtubules but not dynein and kinesin motors. Interestingly, deletion of the microtubule plus-end-tracking protein Bik1 (a CLIP170 ortholog), which is preferentially recruited to the C-terminal residue of α-tubulin, similarly resulted in Snc1 trafficking defects. Finally, constitutively active Rho1 rescued both Bik1 localization at the microtubule plus-ends in tub1-Glu strain and a correct Snc1 trafficking in a Bik1-dependent manner. Our results provide the first evidence for a role of microtubule plus-ends in membrane cargo trafficking in yeast, through Rho1- and Bik1-dependent mechanisms, and highlight the importance of the C-terminal α-tubulin amino acid in this process.


Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/química , Proteínas R-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas rho de Ligação ao GTP/metabolismo , Temperatura Baixa , Testes Genéticos , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microtúbulos/metabolismo , Transporte Proteico , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
5.
PLoS Genet ; 12(2): e1005781, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26828862

RESUMO

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.


Assuntos
Diploide , Hibridização Genética , Meiose/genética , Recombinação Genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Separação Celular , Mapeamento Cromossômico , Troca Genética , Conversão Gênica/genética , Variação Genética , Genoma Fúngico , Haplótipos/genética , Homozigoto , Fenótipo , Locos de Características Quantitativas/genética
6.
G3 (Bethesda) ; 4(4): 707-15, 2014 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-24558267

RESUMO

Advances in high-throughput sequencing (HTS) technologies have accelerated our knowledge of genomes in hundreds of organisms, but the presence of repetitions found in every genome raises challenges to unambiguously map short reads. In particular, short polymorphic reads that are multialigned hinder our capacity to detect mutations. Here, we present two complementary bioinformatics strategies to perform more robust analyses of genome content and sequencing data, validated by use of the Saccharomyces cerevisiae fully sequenced genome. First, we created an annotated HTS profile for the reference genome, based on the production of virtual HTS reads. Using variable read lengths and different numbers of mismatches, we found that 35 nt-reads, with a maximum of 6 mismatches, targets 89.5% of the genome to unique (U) regions. Longer reads consisting of 50-100 nt provided little additional benefits on the U regions extent. Second, to analyze the remaining multialigned (M) regions, we identified the intragenomic single-nucleotide variants and thus defined the unique (MU) and multialigned (MM) subregions, as exemplified for the polymorphic copies of the six flocculation genes and the 50 Ty retrotransposons. As a resource, the coordinates of the U and M regions of the yeast genome have been added to the Saccharomyces Genome Database (www.yeastgenome.org). The benefit of this advanced method of genome annotation was confirmed by our ability to identify acquired single nucleotide polymorphisms in the U and M regions of an experimentally sequenced variant wild-type yeast strain.


Assuntos
Genoma Fúngico , Saccharomyces cerevisiae/genética , Pareamento Incorreto de Bases , Mapeamento Cromossômico , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Retroelementos/genética , Análise de Sequência de DNA
7.
Proc Natl Acad Sci U S A ; 111(5): 1897-902, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24449905

RESUMO

The acquisition of mutations is relevant to every aspect of genetics, including cancer and evolution of species on Darwinian selection. Genome variations arise from rare stochastic imperfections of cellular metabolism and deficiencies in maintenance genes. Here, we established the genome-wide spectrum of mutations that accumulate in a WT and in nine Saccharomyces cerevisiae mutator strains deficient for distinct genome maintenance processes: pol32Δ and rad27Δ (replication), msh2Δ (mismatch repair), tsa1Δ (oxidative stress), mre11Δ (recombination), mec1Δ tel1Δ (DNA damage/S-phase checkpoints), pif1Δ (maintenance of mitochondrial genome and telomere length), cac1Δ cac3Δ (nucleosome deposition), and clb5Δ (cell cycle progression). This study reveals the diversity, complexity, and ultimate unique nature of each mutational spectrum, composed of punctual mutations, chromosomal structural variations, and/or aneuploidies. The mutations produced in clb5Δ/CCNB1, mec1Δ/ATR, tel1Δ/ATM, and rad27Δ/FEN1 strains extensively reshape the genome, following a trajectory dependent on previous events. It comprises the transmission of unstable genomes that lead to colony mosaicisms. This comprehensive analytical approach of mutator defects provides a model to understand how genome variations might accumulate during clonal evolution of somatic cell populations, including tumor cells.


Assuntos
Mutação/genética , Saccharomyces cerevisiae/genética , Aneuploidia , Cromossomos Fúngicos/genética , Rearranjo Gênico/genética , Genes Fúngicos/genética , Haploidia , Análise de Sequência de DNA
8.
Bioinformatics ; 26(15): 1895-6, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20639544

RESUMO

SUMMARY: We present SVDetect, a program designed to identify genomic structural variations from paired-end and mate-pair next-generation sequencing data produced by the Illumina GA and ABI SOLiD platforms. Applying both sliding-window and clustering strategies, we use anomalously mapped read pairs provided by current short read aligners to localize genomic rearrangements and classify them according to their type, e.g. large insertions-deletions, inversions, duplications and balanced or unbalanced inter-chromosomal translocations. SVDetect outputs predicted structural variants in various file formats for appropriate graphical visualization. AVAILABILITY: Source code and sample data are available at http://svdetect.sourceforge.net/


Assuntos
Genoma/genética , Variação Estrutural do Genoma , Genômica/métodos , Software
9.
Mol Biol Cell ; 16(11): 5258-68, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16162818

RESUMO

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60delta mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39delta partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export.


Assuntos
Proteínas Nucleares/fisiologia , Transporte de RNA , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Núcleo Celular/metabolismo , Deleção de Genes , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Íntrons , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
DNA Repair (Amst) ; 4(4): 459-68, 2005 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-15725626

RESUMO

The yeast RAD27 gene encodes a functional homolog of the mammalian FEN1 protein, a structure-specific endo/exonuclease which plays an important role in DNA replication and repair. Previous genetic interaction studies, including a synthetic genetic array (SGA) analysis, showed that the survival of rad27Delta cells requires several DNA metabolic processes, in particular those mediated by all members of the Rad52-dependent recombinational repair pathway. Here, we report the results of our SGA analysis of the collection of non-essential yeast genes against the rad27Delta mutation, which resulted in the identification of a novel synthetic lethal interaction conferred by mutations affecting the Nup84 nuclear pore subcomplex (nup133Delta, nup120Delta and nup84Delta). Additional screens showed that all Rad52 group genes are required for the survival of the nup133Delta and nup120Delta mutants, which are defective in nuclear pore distribution and mRNA export, but not of the nup133DeltaN mutant, which is solely defective in pore distribution. This requirement for the DNA double-strand break (DSB) repair pathway is consistent with the observation that, like rad27Delta, the nup133Delta, nup120Delta and nup84Delta mutants are sensitive to methyl methanesulfonate (MMS). Furthermore, nup133Delta cells exhibit an increased number of spontaneous DNA repair foci containing Rad52. Altogether, these data suggest that the pathological interactions between the rad27Delta and specific nupDelta mutations result from the accumulation of unrepaired DNA damages.


Assuntos
Reparo do DNA/genética , Replicação do DNA/genética , Endonucleases Flap/genética , Poro Nuclear/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Bleomicina/farmacologia , Replicação do DNA/efeitos dos fármacos , Genótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Deleção de Sequência
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