RESUMEN
Pol2 is the leading-strand DNA polymerase in budding yeast. Here we describe an antagonism between its conserved POPS (Pol2 family-specific catalytic core peripheral subdomain) and exonuclease domain and the importance of this antagonism in genome replication. We show that multiple defects caused by POPS mutations, including impaired growth and DNA synthesis, genome instability, and reliance on other genome maintenance factors, were rescued by exonuclease inactivation. Single-molecule data revealed that the rescue stemmed from allowing sister replication forks to progress at equal rates. Our data suggest that balanced activity of Pol2's POPS and exonuclease domains is vital for genome replication and stability.
Asunto(s)
Replicación del ADN , Exonucleasas , Humanos , Exonucleasas/genética , Exonucleasas/metabolismo , Replicación del ADN/genética , Mutación , Inestabilidad Genómica/genética , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismoRESUMEN
Fundamental aspects of DNA replication, such as the anatomy of replication stall sites, how replisomes are influenced by gene transcription, and whether the progression of sister replisomes is coordinated, are poorly understood. Available techniques do not allow the precise mapping of the positions of individual replisomes on chromatin. We have developed a method called Replicon-seq that entails the excision of full-length replicons by controlled nuclease cleavage at replication forks. Replicons are sequenced using Nanopore, which provides a single-molecule readout of long DNA. Using Replicon-seq, we found that sister replisomes function autonomously and yet progress through chromatin with remarkable consistency. Replication forks that encounter obstacles pause for a short duration but rapidly resume synthesis. The helicase Rrm3 plays a critical role both in mitigating the effect of protein barriers and with facilitating efficient termination. Replicon-seq provides a high-resolution means of defining how individual replisomes move across the genome.
Asunto(s)
ADN Helicasas , Replicación del ADN , Cromatina/genética , Cromosomas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismoRESUMEN
Compaction of the genome into the nuclear space is achieved by wrapping DNA around octameric assemblies of histone proteins to form nucleosomes, the fundamental repeating unit of chromatin. Aside from providing a means by which to fit larger genomes into the cell, chromatinization of DNA is a crucial means by which the cell regulates access to the genome. While the complex role that chromatin plays in gene transcription has been appreciated for a long time, it is now also apparent that crucial aspects of DNA replication are linked to the biology of chromatin. This review will focus on recent advances in our understanding of how the chromatin environment influences key aspects of DNA replication.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Replicación del ADN , Animales , HumanosRESUMEN
Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1â¢H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1â¢histoneâ¢DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4)2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Replicación del ADN , ADN de Hongos/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Ribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Unión ProteicaRESUMEN
Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.
Asunto(s)
Cromatina/genética , Replicación del ADN , ADN de Hongos/genética , Nucleosomas/genética , Origen de Réplica , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Hongos/biosíntesis , Genotipo , Humanos , Nucleosomas/metabolismo , Fenotipo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
The primary task of developing embryos is genome replication, yet how DNA replication is integrated with the profound cellular changes that occur through development is largely unknown. Using an approach to map DNA replication at high resolution in C. elegans, we show that replication origins are marked with specific histone modifications that define gene enhancers. We demonstrate that the level of enhancer associated modifications scale with the efficiency at which the origin is utilized. By mapping replication origins at different developmental stages, we show that the positions and activity of origins is largely invariant through embryogenesis. Contrary to expectation, we find that replication origins are specified prior to the broad onset of zygotic transcription, yet when transcription initiates it does so in close proximity to the pre-defined replication origins. Transcription and DNA replication origins are correlated, but the association breaks down when embryonic cell division ceases. Collectively, our data indicate that replication origins are fundamental organizers and regulators of gene activity through embryonic development.
Asunto(s)
Caenorhabditis elegans/embriología , Replicación del ADN , Desarrollo Embrionario , Transcripción Genética , Animales , Origen de Réplica , Análisis Espacio-TemporalRESUMEN
During DNA replication, chromatin must be disassembled and faithfully reassembled on newly synthesized genomes. The mechanisms that govern the assembly of chromatin structures following DNA replication are poorly understood. Here, we exploited Okazaki fragment synthesis and other assays to study how nucleosomes are deposited and become organized in S. cerevisiae. We observe that global nucleosome positioning is quickly established on newly synthesized DNA in vivo. Importantly, we find that ATP-dependent chromatin-remodeling enzymes, Isw1 and Chd1, collaborate with histone chaperones to remodel nucleosomes as they are loaded behind a replication fork. Using a whole-genome sequencing approach, we determine that the positioning of newly deposited nucleosomes in vivo is specified by the combined actions of ATP-dependent chromatin-remodeling enzymes and select DNA-binding proteins. Altogether, our data provide in vivo evidence for coordinated "loading and remodeling" of nucleosomes behind the replication fork, allowing for rapid organization of chromatin during S phase.
RESUMEN
Nucleosome repositioning at gene promoters is a fundamental aspect of the regulation of gene expression. However, the extent to which nucleosome repositioning is used within eukaryotic genomes is poorly understood. Here we report a comprehensive analysis of nucleosome positions as budding yeast transit through an ultradian cycle in which expression of >50% of all genes is highly synchronized. We present evidence of extensive nucleosome repositioning at thousands of gene promoters as genes are activated and repressed. During activation, nucleosomes are relocated to allow sites of general transcription factor binding and transcription initiation to become accessible. The extent of nucleosome shifting is closely related to the dynamic range of gene transcription and generally related to DNA sequence properties and use of the coactivators TFIID or SAGA. However, dynamic gene expression is not limited to SAGA-regulated promoters and is an inherent feature of most genes. While nucleosome repositioning occurs pervasively, we found that a class of genes required for growth experience acute nucleosome shifting as cells enter the cell cycle. Significantly, our data identify that the ATP-dependent chromatin-remodeling enzyme Snf2 plays a fundamental role in nucleosome repositioning and the expression of growth genes. We also reveal that nucleosome organization changes extensively in concert with phases of the cell cycle, with large, regularly spaced nucleosome arrays being established in mitosis. Collectively, our data and analysis provide a framework for understanding nucleosome dynamics in relation to fundamental DNA-dependent transactions.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases/genética , Cromatina/química , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading. Using purified proteins we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. Similarly, in yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a redistribution of Mcm2-7 complexes along the chromosomes, resulting in a corresponding shift in DNA replication initiation sites. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict.
Asunto(s)
Proteínas de Mantenimiento de Minicromosoma/metabolismo , Origen de Réplica , Saccharomyces cerevisiae/genética , ADN Helicasas/metabolismo , Replicación del ADN , ADN de Hongos/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
We have previously demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA. Using a strain of S. cerevisiae in which DNA ligase I can be conditionally depleted, we can enrich and purify Okazaki fragments. We delineate a method to extract, end label, and visualize Okazaki fragments using denaturing agarose gel electrophoresis. Furthermore, we describe an ion-exchange chromatographic method for purification of fragments and preparation of strand-specific sequencing libraries. Deep sequencing of Okazaki fragments generates a comprehensive, genomic map of DNA synthesis, starting from a single asynchronous culture. Altogether this approach represents a tractable system to investigate key aspects of DNA replication and chromatin assembly.
Asunto(s)
ADN/genética , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN/métodos , ADN de Hongos/metabolismo , Electroforesis en Gel de Agar , Genoma Fúngico , Coloración y EtiquetadoRESUMEN
Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.
Asunto(s)
Replicación del ADN , ADN de Hongos/biosíntesis , ADN/biosíntesis , Genoma Fúngico , Origen de Réplica , Saccharomyces cerevisiae/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Cinética , Biología de SistemasRESUMEN
Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin's DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.
Asunto(s)
Acetiltransferasas/genética , Replicación del ADN/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Intercambio de Cromátides Hermanas/genética , Acetilación , Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
Before each division, eukaryotic cells face the daunting task of completely and accurately replicating a heterogeneous, chromatinized genome and repackaging both resulting daughters. Because replication requires strand separation, interactions between the DNA and its many associated proteins--including histones--must be transiently broken to allow the passage of the replication fork. Here, we will discuss the disruption and re-establishment of chromatin structure during replication, and the consequences of these processes for epigenetic inheritance.
Asunto(s)
Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Replicación del ADN/genética , Epigénesis Genética , Animales , Cromatina/ultraestructura , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Eucromatina/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Nucleosomas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis--to our knowledge--of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Replicación del ADN , ADN/biosíntesis , Nucleosomas/metabolismo , Saccharomyces cerevisiae/genética , ADN/genética , ADN/metabolismo , ADN Ligasa (ATP) , ADN Ligasas/deficiencia , ADN Ligasas/metabolismo , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Nucleosomas/genética , Unión Proteica , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Mutations in the chromatin-remodeling protein ATRX cause alpha thalassaemia and mental retardation, but the severity of the disorder is independent of the specific mutation. In this issue of Cell, Law et al. (2010) demonstrate that ATRX alters gene expression by binding to G-rich tandem repeats, and the degree of transcriptional silencing caused by ATRX mutations correlates with the number of repeats.
RESUMEN
Nucleosome-free regions (NFRs) at the 5' and 3' ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5' and 3' ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Nucleosomas/genética , Nucleosomas/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Eliminación de Gen , Genes Fúngicos , Sistemas de Lectura Abierta/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Recent mapping of nucleosome positioning has added a new dimension to the study of transcriptional regulation. Hartley and Madhani (2009) now demonstrate the power of this approach and show that a chromatin regulator alters nucleosome positioning in the promoters of a large number of genes in the budding yeast Saccharomyces cerevisiae.
Asunto(s)
Genoma Fúngico , Saccharomyces cerevisiae/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Nucleosomas/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, we have investigated the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes. We find that Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. Here we show that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. Our analyses reveal how chromatin is organized on a global scale and advance our understanding of how transcription is regulated.
Asunto(s)
Elementos sin Sentido (Genética)/genética , Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Transcripción Genética/genética , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Fúngica de la Expresión Génica , Nucleosomas/genética , Nucleosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
ATP-dependent chromatin remodeling complexes are implicated in many areas of chromosome biology. However, the physiological role of many of these enzymes is still unclear. In budding yeast, the Isw2 complex slides nucleosomes along DNA. By analyzing the native chromatin structure of Isw2 targets, we have found that nucleosomes adopt default, DNA-directed positions when ISW2 is deleted. We provide evidence that Isw2 targets contain DNA sequences that are inhibitory to nucleosome formation and that these sequences facilitate the formation of nuclease-accessible open chromatin in the absence of Isw2. Our data show that the biological function of Isw2 is to position nucleosomes onto unfavorable DNA. These results reveal that antagonistic forces of Isw2 and the DNA sequence can control nucleosome positioning and genomic access in vivo.
Asunto(s)
Nucleosomas/fisiología , Nucleosomas/ultraestructura , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Cromatina/ultraestructura , Cromosomas Fúngicos/genética , ADN de Hongos/química , ADN de Hongos/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Transcripción GenéticaRESUMEN
The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5- or 10-bp gaps in the 3'-5' strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3'-5'-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.