Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Nat Genet ; 36(1): 46-54, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14702040

RESUMO

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein involved in DNA replication, recombination and repair. We show here that RPA is present at the telomeres of the budding yeast Saccharomyces cerevisiae, with a maximal association in S phase. A truncation of the N-terminal region of Rfa2p (associated with the rfa2Delta40 mutated allele) results in severe telomere shortening caused by a defect in the in vivo regulation of telomerase activity. Cells carrying rfa2Delta40 show impaired binding of the protein Est1p, which is required for telomerase action. In addition, normal telomere length can be restored by expressing a Cdc13-Est1p hybrid protein. These findings indicate that RPA activates telomerase by loading Est1p onto telomeres during S phase. We propose a model of in vivo telomerase action that involves synergistic action of RPA and Cdc13p at the G-rich 3' overhang of telomeric DNA.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo , Replicação do DNA , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Proteína de Replicação A , Fase S/fisiologia , Telômero/química , Proteínas de Ligação a Telômeros/metabolismo
2.
Nature ; 435(7046): 1275-9, 2005 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-15965464

RESUMO

RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. Here we show that in fission yeast (Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Transcrição Gênica , Proteínas Argonautas , Centrômero/genética , Centrômero/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Polimerase II/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Ribonucleases/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Virais/metabolismo
3.
Curr Opin Genet Dev ; 14(2): 174-80, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15196464

RESUMO

RNA interference (RNAi) is widely used for knocking down expression of genes of interest and in systematic screens for desired phenotypes. In post-transcriptional gene silencing, double-stranded RNA triggers are processed to small interfering RNAs, which act to seek out and destroy homologous transcripts. A variety of organisms utilise the RNAi pathway to silence expression of potentially harmful endogenous mobile elements and to eliminate unnecessary sequences. In plants and fission yeast, RNAi can also mediate chromatin-based silencing resulting in transcriptional shutdown of homologous transcription units (transcriptional gene silencing) and the formation of centromeric heterochromatin. In metazoans, the expression of non-coding RNAs is often associated with the formation of silent chromatin domains but it remains to be determined if RNAi is involved.


Assuntos
Cromatina/fisiologia , Inativação Gênica/fisiologia , RNA Interferente Pequeno/fisiologia , Animais , Elementos de DNA Transponíveis/fisiologia , Interferons/fisiologia , Nucleossomos/fisiologia , Plantas/genética , Interferência de RNA/fisiologia , Tetrahymena thermophila/genética , Tetrahymena thermophila/fisiologia
4.
Curr Biol ; 12(9): 762-6, 2002 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-12007422

RESUMO

Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, we provide here evidence for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, we provide in vivo evidence that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues PEV modification, telomeric silencing of SAS2-deficient yeast cells, nor lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels.


Assuntos
Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/enzimologia , Inativação Gênica , Proteínas de Saccharomyces cerevisiae/química , Animais , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/química , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/fisiologia , Histona Acetiltransferases , Histonas/metabolismo , Masculino , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Asas de Animais/fisiologia
6.
J Mol Biol ; 326(4): 1081-94, 2003 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-12589755

RESUMO

We have characterized spSet1p, the Schizosaccharomyces pombe ortholog of the budding yeast histone H3 methyltransferase Set1p. SpSet1p catalyzes methylation of H3 at K4, in vivo and in vitro. Deleting spset1 partially affects telomeric and centromeric silencing. Strikingly, lack of spSet1p causes elongation of telomeres in wild-type cells and in most DNA damage checkpoint rad mutant cells, but not in cells lacking the ATM kinase Rad3 or its associated protein Rad26. Interestingly, spset1 deletion specifically causes a reduction in sensitivity to ultraviolet radiation of the PCNA-like checkpoint mutants hus1 and rad1, but not of cells devoid of Rad3. This partial suppression was not due to restoration of checkpoint function or to transcriptional induction of DNA repair genes. Moreover, spset1 allows recovery specifically of the crb2 checkpoint mutant upon treatment with the replication inhibitor hydroxyurea but not upon UV irradiation. Nevertheless, the pathway induced in spset1 cells cannot substitute for the Mus81/Rqh1 DNA damage tolerance pathway. Our results suggest that SpSet1p and the ATM kinase Rad3 function in a common genetic pathway linking chromatin to telomere length regulation and DNA repair.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metiltransferases/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Telômero/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , Centrômero/metabolismo , Quinase do Ponto de Checagem 2 , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Inativação Gênica/fisiologia , Genes cdc , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Hidroxiureia/metabolismo , Metilação , Metiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Raios Ultravioleta
7.
J Cell Biol ; 204(1): 45-59, 2014 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-24379413

RESUMO

DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding yeast Saccharomyces cerevisiae and the avian DT40 cell line as model systems for studying DNA anaphase bridges and show that TopBP1/Dpb11 plays an evolutionarily conserved role in their metabolism. Together with the single-stranded DNA binding protein RPA, TopBP1/Dpb11 binds to UFBs, and depletion of TopBP1/Dpb11 led to an accumulation of chromatin bridges. Importantly, the NoCut checkpoint that delays progression from anaphase to abscission in yeast was activated by both UFBs and chromatin bridges independently of Dpb11, and disruption of the NoCut checkpoint in Dpb11-depleted cells led to genome instability. In conclusion, we propose that TopBP1/Dpb11 prevents accumulation of anaphase bridges via stimulation of the Mec1/ATR kinase and suppression of homologous recombination.


Assuntos
Anáfase/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Galinhas , Cromatina/genética , Cromatina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
8.
Science ; 301(5636): 1069-74, 2003 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-12869699

RESUMO

The expression of short hairpin RNAs in several organisms silences gene expression by targeted mRNA degradation. This RNA interference (RNAi) pathway can also affect the genome, as DNA methylation arises at loci homologous to the target RNA in plants. We demonstrate in fission yeast that expression of a synthetic hairpin RNA is sufficient to silence the homologous locus in trans and causes the assembly of a patch of silent Swi6 chromatin with cohesin. This requires components of the RNAi machinery and Clr4 histone methyltransferase for small interfering RNA generation. A similar process represses several meiotic genes through nearby retrotransposon long terminal repeats (LTRs). These analyses directly implicate interspersed LTRs in regulating gene expression during cellular differentiation.


Assuntos
Cromatina/metabolismo , Inativação Gênica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Retroelementos , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Sequências Repetidas Terminais , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase , Meiose , Metiltransferases/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Genéticos , Conformação de Ácido Nucleico , RNA Interferente Pequeno/genética , Transcrição Gênica
9.
EMBO J ; 22(7): 1697-706, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12660175

RESUMO

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1-independent mechanism regulates the number of yeast telomeric repeats (TG(1-3)) and of vertebrate repeats (T(2)AG(3)) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T(2)AG(3)-only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1-independent manner, both in TEL1 and in tel1Delta humanized yeast cells. Altogether, these findings shed light on multiple repeat-counting mechanisms, which may share critical features between lower and higher eukaryotes.


Assuntos
Saccharomyces cerevisiae/ultraestrutura , Telômero , Proteínas rap1 de Ligação ao GTP/fisiologia , Sequência de Bases , Primers do DNA , Humanos , Plasmídeos , Saccharomyces cerevisiae/genética
10.
Chromosome Res ; 11(2): 137-46, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12733640

RESUMO

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast (Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat (dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi.


Assuntos
Centrômero/genética , Centrômero/fisiologia , Interferência de RNA , Schizosaccharomyces/genética , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Metilação de DNA , Corantes Fluorescentes , Perfilação da Expressão Gênica , Inativação Gênica , Histonas/química , Indóis , Cinetocoros , Testes de Precipitina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Schizosaccharomyces pombe/química , Sequências Repetidas Terminais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA