RESUMEN
In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation-whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated naphthoylated DEAE (BND)-cellulose in high salt concentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA, and are thus suitable for analysis on 2-D gels.
Asunto(s)
Replicación del ADN , Enzimas de Restricción del ADN/metabolismo , ADN/aislamiento & purificación , Electroforesis en Gel Bidimensional/métodos , Genoma , Células 3T3 , Animales , Southern Blotting , Células CHO , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Células HeLa , Humanos , Ratones , SuspensionesRESUMEN
The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently activated origins of replication from asynchronous cell cultures. In one of these, nascent strands are melted from the long template DNA, and the small, origin-centered strands are isolated on sucrose gradients. The second method involves the selective entrapment of bubble-containing fragments in gelling agarose and their subsequent recovery and isolation by molecular cloning. Libraries prepared by this method from Chinese hamster and human cells have been shown to be extremely pure, and provide a renewable resource of origins that can be used as probes on microarrays or sequenced by high-throughput techniques to localize them within the genomic source. The bubble-trapping method is described here for asynchronous mammalian cells that grow with reasonable doubling times and from which nuclear matrices can be reliably prepared. The method for nuclear matrix preparation and enrichment of replication intermediates is described in an accompanying chapter entitled "Purification of restriction fragments containing replication intermediates from mammalian cells for 2-D gel analysis" (Chapter 16 ).
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , ADN/aislamiento & purificación , Genoma , Origen de Réplica , Animales , Células CHO , Cricetinae , Cricetulus , ADN Recombinante/metabolismo , Humanos , SefarosaRESUMEN
Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.
Asunto(s)
Cryptococcus neoformans/genética , Genoma Fúngico/genética , ARN de Hongos/genética , Transcriptoma/genética , Virulencia/genética , Cromosomas Fúngicos/genética , ADN de Hongos/genética , Intrones/genéticaRESUMEN
We have devised a method for isolating virtually pure and comprehensive libraries of restriction fragments that contained replication initiation sites (bubbles) in vivo. We have now sequenced and mapped the bubble-containing fragments from GM06990, a near-normal EBV-transformed lymphoblastoid cell line, and have compared origin distributions with a comprehensive replication timing study recently published for this cell line. We find that early-firing origins, which represent â¼32% of all origins, overwhelmingly represent zones, associate only marginally with active transcription units, are localized within large domains of open chromatin, and are significantly associated with DNase I hypersensitivity. Origin "density" falls from early- to mid-S-phase, but rises again in late S-phase to levels only 17% lower than in early S-phase. Unexpectedly, late origin density calculated on the 1-Mb scale increases as a function of increasing chromatin compaction. Furthermore, the median efficiency of origins in late-replicating, heterochromatic domains is only 25% lower than in early-replicating euchromatic loci. Thus, the activation of early- and late-firing origins must be regulated by quintessentially different mechanisms. The aggregate data can be unified into a model in which initiation site selection is driven almost entirely by epigenetic factors that fashion both the long-range and local chromatin environments, with underlying DNA sequence and local transcriptional activity playing only minor roles. Importantly, the comprehensive origin map we have prepared for GM06990 overlaps moderately well with origin maps recently reported for the genomes of four different human cell lines based on the distributions of small nascent strands.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Genoma Humano , Origen de Réplica/genética , Activación Transcripcional , Línea Celular Tumoral , Momento de Replicación del ADN , Desoxirribonucleasa I/metabolismo , Epigenómica , Sitios Genéticos , Células HeLa , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
BACKGROUND: Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. FINDINGS: Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. CONCLUSIONS: We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.
RESUMEN
Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4-5 MCM hexamers, and 2 Cdc6. Relative to these subunits, â¼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain â¼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Replicón , Animales , Anticuerpos/inmunología , Línea Celular , Cromatina/metabolismo , Humanos , Estabilidad Proteica , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Origen de Réplica , Reproducibilidad de los Resultados , Fase SRESUMEN
We have used a novel bubble-trapping procedure to construct nearly pure and comprehensive human origin libraries from early S- and log-phase HeLa cells, and from log-phase GM06990, a karyotypically normal lymphoblastoid cell line. When hybridized to ENCODE tiling arrays, these libraries illuminated 15.3%, 16.4%, and 21.8% of the genome in the ENCODE regions, respectively. Approximately half of the origin fragments cluster into zones, and their signals are generally higher than those of isolated fragments. Interestingly, initiation events are distributed about equally between genic and intergenic template sequences. While only 13.2% and 14.0% of genes within the ENCODE regions are actually transcribed in HeLa and GM06990 cells, 54.5% and 25.6% of zonal origin fragments overlap transcribed genes, most with activating chromatin marks in their promoters. Our data suggest that cell synchronization activates a significant number of inchoate origins. In addition, HeLa and GM06990 cells activate remarkably different origin populations. Finally, there is only moderate concordance between the log-phase HeLa bubble map and published maps of small nascent strands for this cell line.
Asunto(s)
Cromatina/metabolismo , Análisis por Micromatrices/métodos , Origen de Réplica , Transcripción Genética , Ciclo Celular/genética , Cromatina/genética , Análisis por Conglomerados , Replicación del ADN , ADN Intergénico/metabolismo , Femenino , Biblioteca de Genes , Genoma Humano , Células HeLa , Humanos , Sistemas de Lectura Abierta/genética , Regiones Promotoras GenéticasRESUMEN
Studies in our laboratory over the last three decades have shown that the Chinese hamster dihydrofolate reductase (DHFR) origin of replication corresponds to a broad zone of inefficient initiation sites distributed throughout the spacer between the convergently transcribed DHFR and 2BE2121 genes. It is clear from mutational analysis that none of these sites is genetically required for controlling origin activity. However, the integrity of the promoter of the DHFR gene is needed to activate the downstream origin, while the 3' processing signals prevent invasion and inactivation of the downstream origin by transcription forks. Several other origins in metazoans have been shown to correspond to zones of inefficient sites, while a different subset appears to be similar to the fixed replicators that characterize origins in S. cerevisiae and lower organisms. These observations have led us to suggest a model in which the mammalian genome is dotted with a hierarchy of degenerate, redundant, and inefficient replicators at intervals of a kilobase or less, some of which may have evolved to be highly circumscribed and efficient. The activities of initiation sites are proposed to be largely regulated by local transcription and chromatin architecture. Recently, we and others have devised strategies for identifying active origins on a genome-wide scale in order to define their distributions between fixed and dispersive origin types and to detect relationships among origins, genes, and epigenetic markers. The global pictures emerging are suggestive but far from complete and appear to be plagued by some of the same uncertainties that have led to conflicting views of individual origins in the past (particularly DHFR). In this paper, we will trace the history of origin discovery in mammalian genomes, primarily using the well-studied DHFR origin as a model, because it has been analyzed by nearly every available origin mapping technique in several different laboratories, while many origins have been identified by only one. We will address the strengths and shortcomings of the various methods utilized to identify and characterize origins in complex genomes and will point out how we and others were sometimes led astray by false assumptions and biases, as well as insufficient information. The goal is to help guide future experiments that will provide a truly comprehensive and accurate portrait of origins and their regulation. After all, in the words of George Santayana, "Those who do not learn from history are doomed to repeat it."
Asunto(s)
Origen de Réplica , Tetrahidrofolato Deshidrogenasa/genética , Animales , Cricetinae , Cricetulus , Electroforesis en Gel BidimensionalRESUMEN
In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation - whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated napthoylated DEAE (BND)-cellulose in high salt conCentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA and are thus suitable for analysis on 2-D gels.
Asunto(s)
Replicación del ADN , ADN/aislamiento & purificación , Electroforesis en Gel Bidimensional/métodos , Animales , Southern Blotting , Células CHO , Ciclo Celular , Fraccionamiento Celular , Cromatografía DEAE-Celulosa/métodos , Cricetinae , Cricetulus , DEAE-Celulosa/análogos & derivados , ADN/biosíntesis , ADN/genética , Células HeLa , Humanos , ReplicónRESUMEN
The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently activated origins of replication from asynchronous cell cultures. In one of these, nascent strands are melted from the long template DNA, and the small, origin-centered strands are isolated on sucrose gradients. The second method involves the selective entrapment of bubble-containing fragments in gelling agarose and their subsequent recovery and isolation by molecular cloning. Libraries prepared by this method from Chinese hamster and human cells have been shown to be extremely pure, and provide a renewable resource of origins that can be used as probes on microarrays or sequenced by high-throughput techniques to localize them within the genomic source. The bubble-trapping method is described here for asynchronous mammalian cells that grow with reasonable doubling times and from which nuclear matrices can be reliably prepared. The method for nuclear matrix preparation and enrichment of replication intermediates is described in an accompanying chapter entitled, "Purification of Restriction Fragments Containing Replication Intermediates from Mammalian Cells for 2-D Gel Analysis").
Asunto(s)
Replicación del ADN/genética , Genómica/métodos , Origen de Réplica/genética , Animales , ADN/biosíntesis , ADN/genética , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Humanos , Mapeo Nucleótido/métodos , Replicón/genéticaRESUMEN
It is generally believed that DNA replication in most eukaryotes proceeds according to a precise program in which there is a defined temporal order by which each chromosomal region is duplicated. However, the regularity of this program at the level of individual chromosomes, in terms of both the relative timing and the size of the DNA domain, has not been addressed. Here, the replication of chromosome VI from synchronized budding yeast was studied at a resolution of approximately 1 kb with DNA combing and fluorescence microscopy. Contrary to what would be expected from cells following a rigorous temporal program, no two molecules exhibited the same replication pattern. Moreover, a direct evaluation of the extent to which the replication of distant chromosomal segments was coordinated indicates that the overwhelming majority of these segments were replicated independently. Importantly, averaging the patterns of all the fibers examined recapitulates the ensemble-averaged patterns obtained from population studies of the replication of chromosome VI. Thus, rather than an absolutely defined temporal order of replication, replication timing appears to be essentially probabilistic within individual cells, exhibiting only temporal tendencies within extended domains.
Asunto(s)
Cromosomas Fúngicos , Replicación del ADN/fisiología , ADN de Hongos/análisis , ADN de Hongos/genética , Saccharomyces cerevisiae/fisiología , Bromodesoxiuridina/metabolismo , ADN de Hongos/química , Fase G1/efectos de los fármacos , Dosificación de Gen , Genes Fúngicos , Factor de Apareamiento , Péptidos/metabolismo , Péptidos/farmacología , Regiones Promotoras Genéticas , Fase S , Saccharomyces cerevisiae/genética , Timidina Quinasa/genética , Factores de TiempoRESUMEN
Because of the complexity of higher eukaryotic genomes and the lack of a reliable autonomously replicating sequence (ARS) assay for isolating potential replicators, the identification of origins has proven to be extremely challenging and time consuming. We have developed a new origin-trapping method based on the partially circular nature of restriction fragments containing replication bubbles and have prepared a library of approximately 1,000 clones from early S phase CHO cells. When 15 randomly selected clones were analyzed by a stringent two-dimensional (2D) gel replicon mapping method, all were shown to correspond to active, early firing origins. Furthermore, most of these appear to derive from broad zones of potential sites, and the five that were analyzed in a time-course study are all inefficient. This bubble-trapping scheme will allow the construction of comprehensive origin libraries from any complex genome so that their natures and distributions vis-a-vis other chromosomal markers can be established.
Asunto(s)
Biblioteca de Genes , Genómica/métodos , Origen de Réplica/genética , Animales , Células CHO , Cricetinae , Cricetulus , Electroforesis en Gel Bidimensional , Rodopsina/genética , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
DNA replication is tightly regulated at the initiation step by both the cell cycle machinery and checkpoint pathways. Here, we discuss recent advances in understanding how replication is initiated in metazoans at the correct chromosome positions, at the appropriate time, and only once per cell cycle.
Asunto(s)
Replicación del ADN/fisiología , Mitosis/fisiología , Origen de Réplica/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Epigénesis Genética/fisiología , Retroalimentación Fisiológica/fisiología , Genes cdc/fisiología , HumanosRESUMEN
The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a 55-kb zone of potential initiation sites lying between the convergently transcribed DHFR and 2BE2121 genes. Two subregions within this zone (ori-beta/ori-beta' and ori-gamma) are preferred. In the DHFR-deficient variant, DR8, which has deleted a 14-kb sequence straddling the 3' end of the DHFR gene, early-firing origin activity in the downstream ori-beta/ori-beta' and ori-gamma regions is completely suppressed. We show that the critical deleted sequences reside within a 168-bp segment encompassing the intron 5/exon 6 boundary, exon 6, 54 bp of the 3' untranslated region (UTR), but not the three natural polyA sites. In wild-type cells, this sequence efficiently arrests transcription in a region a few kilobases downstream, which coincides with the 5' boundary of the replication initiation zone. In DR8, DHFR-specific transcripts efficiently use an alternative sixth exon (6c) and polyA signals near the middle of the former intergenic region to process primary transcripts. However, transcription proceeds to a position almost 35 kb downstream from these signals, and replication initiation can only be detected beyond this point. When the wild-type 168-bp 3' element is inserted into DR8 at the same position as alternative exon 6c, transcription is arrested efficiently and initiations occur almost immediately downstream. Thus, the normal 3' end of the DHFR gene constitutes a boundary element not only for the gene but also for the local origin of replication.
Asunto(s)
Región de Flanqueo 3'/genética , Cricetulus/genética , Origen de Réplica/genética , Tetrahidrofolato Deshidrogenasa/genética , Transcripción Genética/genética , Animales , Northern Blotting , Southern Blotting , Línea Celular , Cricetinae , Cartilla de ADN , Electroforesis en Gel Bidimensional , Componentes del Gen , Hibridación de Ácido Nucleico , Recombinación Genética/genética , Transcripción Genética/fisiologíaRESUMEN
Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression.
Asunto(s)
Quinasas CDC2-CDC28/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Fase S , Células 3T3 , Animales , Células CHO , Cricetinae , Cricetulus , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina , Replicación del ADN , Fibroblastos/metabolismo , Células HeLa , Histonas/genética , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Fosforilación , Pruebas de PrecipitinaRESUMEN
The dihydrofolate reductase (DHFR) and 2BE2121 genes in the Chinese hamster are convergently transcribed in late G1 and ea ly S phase, and bracket an early-firing origin of replication that consists of a 55-kb zone of potential initiation sites. To test whether transcription through the DHFR gene is required to activate this origin in early S phase, we examined the two-dimension (2D) gel patterns of replication intermediates from several variants in which parts or all of the DHFR promote had been deleted. In those variants in which transcription was undetectable, initiation in the intergenic space was markedly suppressed (but not eliminated) in early S phase. Further more, replication of the locus required virtually the entire S period, as opposed to the usual 3-4 h. However, restoration of transcription with either the wild-type Chinese hamster promote or a Drosophila-based construct restored origin activity to the wild-type pattern. Surprisingly, 2D gel analysis of promote less variants revealed that initiation occurs at a low level in ea ly S phase not only in the intergenic region, but also in the body of the DHFR gene. The latter phenomenon has never been observed in the wild-type locus. These studies suggest that transcription through the gene normally increases the efficiency of origin firing in early S phase, but also suppresses initiation in the body of the gene, thus helping to define the boundaries of the downstream origin.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/genética , Ovario/enzimología , Regiones Promotoras Genéticas/genética , Tetrahidrofolato Deshidrogenasa/genética , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Femenino , Variación Genética , Mutagénesis , Plásmidos/genética , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
Ectopically expressed Cdc6 is translocated from the nucleus during S phase in a cyclin A-Cdk2-dependent process, suggesting that reinitiation of DNA replication is prevented by removal of phosphorylated Cdc6 from chromatin after origin firing. However, whether endogenous Cdc6 translocates during S phase remains controversial. To resolve the questions regarding regulation of endogenous Cdc6, we cloned the cDNA encoding the Chinese hamster Cdc6 homolog and specifically focused on analyzing the localizations and chromatin affinities of endogenous and exogenous proteins during S phase and following overexpression of cyclin A. In agreement with other reports, ectopically expressed Cdc6 translocates from the nucleus during S phase and in response to overexpressed cyclin A. In contrast, using a combination of biochemical and immunohistochemical assays, we show convincingly that endogenous Cdc6 remains nuclear and chromatin bound throughout the entire S period, while Mcm5 loses chromatin affinity during S phase. Overexpression of cyclin A is unable to alter the nuclear localization of Cdc6. Furthermore, using a phosphospecific antibody we show that phosphoserine-54 Cdc6 maintains a high affinity for chromatin during the S period. Considering recent in vitro studies, these data are consistent with a proposed model in which Cdc6 is serine-54 phosphorylated during S phase and functions as a chromatin-bound signal that prevents reformation of prereplication complexes.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Ciclina A/metabolismo , Fosfoserina/metabolismo , Fase S , Secuencia de Aminoácidos , Animales , Células CHO , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Clonación Molecular , Cricetinae , Ciclina A/genética , Citoplasma/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Transporte de ProteínasRESUMEN
Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less.
Asunto(s)
Replicación del ADN , Origen de Réplica/genética , Tetrahidrofolato Deshidrogenasa/genética , Animales , Células CHO , Cricetinae , ADN/análisis , ADN/biosíntesis , ADN/genética , Replicación del ADN/efectos de los fármacos , Electroforesis en Gel Bidimensional , Mimosina/farmacología , Mapeo Físico de Cromosoma , Fase S/genética , Factores de TiempoRESUMEN
Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.