RESUMEN
Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging-strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers.
Asunto(s)
ADN Intergénico/fisiología , Impresión Genómica , Schizosaccharomyces/genética , Secuencia de Bases , Southern Blotting , Ciclo Celular , Mapeo Cromosómico , Momento de Replicación del ADN/fisiología , ADN Intergénico/genética , Electroforesis en Gel Bidimensional , Sitios Genéticos , Datos de Secuencia Molecular , Schizosaccharomyces/crecimiento & desarrollo , Análisis de Secuencia de ADN , Transcripción Genética , Activación TranscripcionalRESUMEN
Genetic instability due to stalled replication forks is thought to underlie a number of human diseases, such as premature ageing and cancer susceptibility syndromes. In addition, site-specific stalling occurs at some genetic loci. A detailed understanding of the topology of the stalled replication fork gives a valuable insight into the causes and mechanisms of replication stalling. The method described here allows mapping of the position of the 3'-end of the nascent leading or lagging strand at the replication fork, stalled at a site-specific barrier. The replicating DNA is purified, digested with restriction enzymes, and enriched by BND-cellulose chromatography. The DNA is separated on a sequencing gel, transferred to a membrane, and hybridised to a strand-specific probe. The data obtained using this method allow determining the position of the 3'-end of the nascent strand at a stalled fork with a one-nucleotide resolution.
Asunto(s)
Replicación del ADN/genética , Mapeo Nucleótido/métodos , Sitios de Unión/genética , Cromatografía DEAE-Celulosa , DEAE-Celulosa/análogos & derivados , ADN de Hongos/biosíntesis , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Técnicas de Sonda Molecular , Hibridación de Ácido Nucleico , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismoRESUMEN
Bi-directionality is a common feature observed for genomic replication for all three phylogenetic kingdoms: Eubacteria, Archaea, and Eukaryotes. A consequence of bi-directional replication, where the two replication forks initiated at an origin move away from each other, is that the replication termination will occur at positions away from the origin sequence(s). The replication termination processes are therefore physically and mechanistically dissociated from the replication initiation. The replication machinery is a highly processive complex that in short time copies huge numbers of bases while competing for the DNA substrate with histones, transcription factors, and other DNA-binding proteins. Importantly, the replication machinery generally wins out; meanwhile, when converging forks meet termination occurs, thus preventing over-replication and genetic instability. Very different scenarios for the replication termination processes have been described for the three phylogenetic kingdoms. In eubacterial genomes replication termination is site specific, while in archaea and eukaryotes termination is thought to occur randomly within zones where converging replication forks meet. However, a few site-specific replication barrier elements that mediate replication termination have been described in eukaryotes. This review gives an overview about what is known about replication termination, with a focus on these natural site-specific replication termination sites.
Asunto(s)
Replicación del ADN/fisiología , Archaea/genética , Archaea/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sitios de Unión , Replicación del ADN/genética , ADN de Archaea/biosíntesis , ADN de Archaea/genética , ADN Bacteriano/biosíntesis , ADN Bacteriano/genética , ADN de Hongos/biosíntesis , ADN de Hongos/genética , ADN Ribosómico/biosíntesis , ADN Ribosómico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
Exclusive gene expression, where only one member of a gene or gene cassette family is selected for expression, plays an important role in the establishment of cell identity in several biological systems. Here, we compare four such systems: mating-type switching in fission and budding yeast, where cells choose between expressing one of the two different mating-type cassettes, and immunoglobulin and odorant receptor gene expression in mammals, where the number of gene choices is substantially higher. The underlying mechanisms that establish this selective expression pattern in each system differ in almost every detail. In all four systems, once a successful gene activation event has taken place, a feedback mechanism affects the fate of the cell. In the mammalian systems, feedback is mediated by the expressed cell surface receptor to ensure monoallelic gene expression, whereas in the yeasts, the expressed gene cassette at the mating-type locus affects donor choice during the subsequent switching event.
Asunto(s)
Regulación de la Expresión Génica/genética , Familia de Multigenes/genética , Animales , Humanos , Activación TranscripcionalRESUMEN
Eukaryotic replication termination generally occurs randomly in the region between two active origins. However, termination, or pausing of the replication forks has been observed at specific loci. Recently, a site-specific terminator of replication named RTS1 was shown to play an important role in mating-type switching in Schizosaccharomyces pombe. Mating-type switching in S. pombe relies on an imprinting event that chemically modifies one strand of the DNA at the mating-type locus mat1. This imprint, that is formed only when mat1 is replicated in a specific direction, marks the DNA for a rearrangement leading to mating-type switching. The RTS1 element ensures that mat1 is replicated in the correct direction for imprinting and initiation of the subsequent mating-type switching event. This is the first replication terminator shown to play a role in cellular differentiation.
Asunto(s)
Replicación del ADN/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Animales , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/metabolismo , Impresión Genómica , Humanos , Datos de Secuencia MolecularRESUMEN
Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase.
Asunto(s)
Daño del ADN/efectos de los fármacos , ADN Polimerasa I/metabolismo , Reparación del ADN/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Alquilantes/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/efectos de la radiación , ADN Polimerasa I/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Inestabilidad Genómica , Hidroxiurea/farmacología , Metilmetanosulfonato/farmacología , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Rayos UltravioletaRESUMEN
The fission yeast clade--comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus--occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.
Asunto(s)
Genoma Fúngico , Schizosaccharomyces/genética , Centrómero/genética , Centrómero/fisiología , Centrómero/ultraestructura , Elementos Transponibles de ADN , Evolución Molecular , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes del Tipo Sexual de los Hongos , Genómica , Glucosa/metabolismo , Meiosis , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Filogenia , ARN sin Sentido/genética , ARN de Hongos/genética , ARN Interferente Pequeño/genética , ARN no Traducido/genética , Elementos Reguladores de la Transcripción , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Análisis de Secuencia de ADN , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The imprint at the mat1 locus of Schizosaccharomyces pombe acts to initiate the replication-coupled recombination event that underlies mating-type switching. However, the nature of the imprint has been an area of dispute. Two alternative models have been proposed: one stated that the imprint is a nick in the DNA, whereas our data suggested that it consists of one or two ribonucleotides incorporated into the otherwise intact DNA duplex. Here, we verify key predictions of the RNA model by characterization of wild-type genomic DNA purified under conditions known to hydrolyse DNA-RNA-DNA hybrid strands. First, we observe one-nucleotide gap at the hydrolysed DNA, as expected from the presence of two ribonucleotides. Second, using a novel assay based on ligation-mediated PCR, a 3'-terminal ribonucleotide is detected at the hydrolysed imprint. Our observations allow the unification of available data sets characterizing the wild-type imprint.
Asunto(s)
ADN de Hongos , Genes del Tipo Sexual de los Hongos , Ribonucleótidos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Secuencia de Bases , Estructura Molecular , Recombinación Genética , Ribonucleótidos/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Mating-type switching in fission yeast depends on an imprint at the mat1 locus. Previous data showed that the imprint is made in the DNA strand replicated as lagging. We now identify this imprint as an RNase-sensitive modification and suggest that it consists of one or two RNA residues incorporated into the mat1 DNA. Formation of the imprint requires swi1- and swi3-dependent pausing of the replication fork. Interestingly, swi1 and swi3 mutations that abolish pausing do not affect the use of lagging-strand priming site during replication. We show that the pausing of replication and subsequent formation of the imprint occur after the leading-strand replication complex has passed the site of the imprint and after lagging-strand synthesis has initiated at this proximal priming site. We propose a model in which a swi1- and swi3-dependent signal during lagging-strand synthesis leads to pausing of leading-strand replication and the introduction of the imprint.