RESUMEN
Noncoding Y RNAs have recently been identified as essential factors for chromosomal DNA replication in human cell nuclei. Here, we investigate the expression of human Y RNAs in tumours and test their requirement for cell proliferation. Relative expression levels of all four human Y RNAs (hY1, hY3, hY4 and hY5 RNA) were determined by quantitative RT-PCR in extracts from human solid tumours, corresponding nonmalignant normal tissues and derived cultured cells. On average, all four hY RNAs are significantly overexpressed in solid tumours between 4- and 13-fold, compared to the corresponding normal tissues. In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22). A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi). Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation. We conclude that noncoding hY RNAs have potential both as new cancer biomarkers and as molecular targets for anti-proliferative intervention.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias/genética , ARN no Traducido/análisis , Línea Celular Tumoral , Proliferación Celular , Humanos , Antígeno Ki-67/genética , Neoplasias/patología , ARN Mensajero/análisis , ARN no Traducido/antagonistas & inhibidores , ARN no Traducido/fisiologíaRESUMEN
Trisomy 21 (Down syndrome, DS) is the most common genetic cause of mental retardation, resulting from triplication of the whole or distal part of human chromosome 21. Overexpression of genes located on chromosome 21, as a result of extra gene load, has been considered a central hypothesis for the explanation of the DS phenotype. This gene dosage hypothesis has been challenged, however. We have therefore decided to study proteins whose genes are encoded on chromosome 21 in brain of patients with DS and Alzheimer's disease (AD), as all patients with DS from the fourth decade show Alzheimer-related neuropathology. Using immunoblotting we determined Coxsackievirus and adenovirus receptor (CAR), Claudin-8, C21orf2, Chromatin assembly factor 1 p60 subunit (CAF-1 p60) in frontal cortex from DS, AD and control patients. Significant reduction of C21orf2 and CAF-1 p60, but comparable expression of CAR and claudin-8 was observed in DS but all proteins were comparable to controls in AD, even when related to NSE levels to rule out neuronal cell loss or actin to normalise versus a housekeeping protein. Reduced CAF-1 p60 may reflect impaired DNA repair most probably due to oxidative stress found as early as in fetal life continuing into adulthood. The decrease of C21orf2 may represent mitochondrial dysfunction that has been reported repeatedly and also data on CAR and claudin-8 are not supporting the gene-dosage hypothesis at the protein level. As aberrant expression of the four proteins was not found in brains of patients with AD, decreased CAF and C21orf2 can be considered specific for DS.
Asunto(s)
Corteza Cerebral/metabolismo , Proteínas Cromosómicas no Histona/biosíntesis , Cromosomas Humanos Par 21/metabolismo , Proteínas de Unión al ADN/biosíntesis , Síndrome de Down/metabolismo , Biosíntesis de Proteínas , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Encéfalo/patología , Corteza Cerebral/patología , Factor 1 de Ensamblaje de la Cromatina , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos Par 21/genética , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Síndrome de Down/genética , Síndrome de Down/patología , Regulación hacia Abajo/fisiología , Humanos , Masculino , Persona de Mediana Edad , Proteínas/genética , Estadísticas no ParamétricasRESUMEN
During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACE An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Replicación del ADN , Histonas/metabolismo , Fase S , Animales , Quinasa de la Caseína II , Proteínas de Ciclo Celular , División Celular , Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina , Quinasas Ciclina-Dependientes/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteína 1 de Ensamblaje de Nucleosomas , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismoRESUMEN
The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G(1)-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G(1)-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclinas/metabolismo , Replicación del ADN , Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Virales/metabolismo , Células 3T3 , Animales , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Fase G1 , Humanos , Ratones , Microscopía Fluorescente , Complejo de Reconocimiento del Origen , Fosforilación/efectos de los fármacos , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Purinas/farmacología , Roscovitina , Fase S , Transfección , Células Tumorales CultivadasRESUMEN
The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Cromosómicas no Histona , Ciclina A/fisiología , Quinasas Ciclina-Dependientes/fisiología , Proteínas de Unión al ADN/metabolismo , ADN/biosíntesis , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Ciclo Celular , Factor 1 de Ensamblaje de la Cromatina , Ciclina B/fisiología , Ciclina B1 , Ciclina D , Ciclina E/fisiología , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Ciclinas/fisiología , ADN/efectos de los fármacos , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Cinética , Mitosis , Modelos Biológicos , Nucleosomas/metabolismo , Ácido Ocadaico/farmacología , Fosforilación , Proteína Fosfatasa 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Purinas/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Roscovitina , Vanadatos/farmacologíaRESUMEN
Initiation of human DNA replication is investigated in vitro, using initiation-competent nuclei isolated from cells arrested in late G(1) phase by a 24-h treatment with 0.5 mm mimosine (Krude, T. (1999) Exp. Cell Res. 247, 148-159). Nuclei isolated from mimosine-arrested HeLa cells initiate semiconservative DNA replication upon incubation in cytosolic extracts from proliferating human cells. Initiation occurs in the absence and presence of a nuclear membrane. The cyclin-dependent kinase (Cdk) inhibitors roscovitine and olomoucine inhibit initiation of DNA replication, indicating a dependence of initiation on Cdk activity. Cell fractionation shows that cyclins A, E, and Cdk2 are bound to nuclei from mimosine-arrested cells. Exogenously added human cyclin A.Cdk2 and cyclin E.Cdk2 complexes, but not cyclin B1/Cdk1 or cyclin D2/Cdk6, can overcome inhibition of initiation by roscovitine in vitro. Depleting Cdk2 from cytosolic extract does not prevent initiation, demonstrating that cyclin.Cdk2 complexes are not required in the soluble extract, but are provided by the nuclei. Initiation depends further on an essential and soluble activity present in cytosolic extracts from proliferating cells, but not from mimosine-arrested cells, acting together with nuclear cyclin/Cdk2 activity.
Asunto(s)
Quinasas CDC2-CDC28 , Núcleo Celular/genética , Replicación del ADN , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Humanos , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
We discuss nuclear chaperones that bind correctly folded protein subunits and mediate molecular interactions, particularly between proteins and nucleic acids. The charge of these chaperones helps to prevent non-specific electrostatic interactions between the components. Thus, an ordered assembly of macromolecular complexes is mediated, most notably in the formation and maintenance of chromatin, though similar principles are likely to apply in ribonucleoprotein assembly. Here, we discuss roles for nuclear chaperones in mediating nucleosome assembly and remodelling during DNA replication and transcription, and upon fertilisation.
Asunto(s)
Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiología , Proteínas Nucleares/fisiología , Animales , Cromatina/metabolismo , Cromatina/ultraestructura , Humanos , Sustancias Macromoleculares , Modelos Biológicos , Proteínas Nucleares/metabolismo , Nucleoplasminas , Nucleoproteínas/biosíntesis , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologíaRESUMEN
Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Replicación del ADN/fisiología , Proteínas de Ciclo Celular , Factor 1 de Ensamblaje de la Cromatina , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Nucleares , Proteína 1 de Ensamblaje de Nucleosomas , Nucleosomas/metabolismo , Proteínas/metabolismo , Fase SRESUMEN
Nucleosomes are preferentially assembled on replicating DNA by chromatin assembly factor 1; recent studies have shown that replicated DNA is marked for assembly into chromatin by the replication-fork-associated protein PCNA.
Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Factor 1 de Ensamblaje de la Cromatina , HumanosRESUMEN
The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Mimosina/farmacología , División Celular/efectos de los fármacos , Núcleo Celular/genética , Replicación del ADN/genética , Relación Dosis-Respuesta a Droga , Fase G1/efectos de los fármacos , Células HeLa , Humanos , Fase S/efectos de los fármacos , Factores de Tiempo , Células Tumorales Cultivadas , Neoplasias de la Vejiga UrinariaRESUMEN
We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Fase S/fisiología , Proteínas de Saccharomyces cerevisiae , Células 3T3 , Animales , Sistema Libre de Células , Cromatina/metabolismo , Citosol/metabolismo , Replicación del ADN , Fase G1 , Células HeLa , Humanos , Ratones , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Permeabilidad , Unión Proteica , Fase de Descanso del Ciclo CelularRESUMEN
We characterized changes of nucleosome assembly activity, intracellular localization, and reversible phosphorylation of the human chromatin assembly factor CAF-1 during the somatic cell division cycle. HeLa cells were synchronized in the G1, S, G2, and M phases of the cell cycle. All three subunits of human CAF-1 (p150, p60, and p48) are present during the entire cell cycle. In interphase, p150 and p60 are bound to the nucleus, but they predominantly dissociate from chromatin during mitosis. During S phase, p150 and p60 are concentrated at sites of intranuclear DNA replication. Only a fraction of total p48 is associated with p150 and p60, and the majority is present in other high molecular weight complexes. The other nucleosome assembly protein, NAP-1, is predominantly cytosolic throughout the cell cycle. Human CAF-1 efficiently mediates nucleosome assembly during complementary DNA strand synthesis in G1, S, and G2 phase cytosolic extracts. Active CAF-1 can be isolated as a 6.5 S complex from G1, S, and G2 phase nuclei. In contrast, CAF-1 isolated from mitotic cytosol does not support nucleosome assembly during DNA synthesis. In mitosis, the p60 subunit of inactive CAF-1 is hyperphosphorylated, whereas active CAF-1 in interphase contains hypophosphorylated and/or phosphorylated forms of p60.
Asunto(s)
Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/química , Nucleosomas/metabolismo , Proteínas de Ciclo Celular , Cromatina/metabolismo , Factor 1 de Ensamblaje de la Cromatina , Replicación del ADN/fisiología , Citometría de Flujo , Células HeLa , Humanos , Nucleasa Microcócica/metabolismo , Mitosis/fisiología , Proteínas Nucleares/análisis , Proteína 1 de Ensamblaje de Nucleosomas , Fosforilación , Proteínas/metabolismo , Factores de TranscripciónRESUMEN
We describe a cell-free system from HeLa cells that initiates DNA replication under cell cycle control. G1 but not G2 phase nuclei initiate replication when coincubated with S phase nuclei in cytosolic extracts from S phase but not from G1 or G2 phase HeLa cells. S phase nuclei or an S phase nuclear extract are required for the initiation of semiconservative DNA replication in G1 nuclei but not for elongation in S phase nuclei. S phase nuclear extract could be replaced by recombinant human cyclins A and E complexed to Cdk2 but not by Cdk2 alone or by human cyclin B1 complexed to Cdc2. In S phase cytosol, cyclins A/Cdk2 and E/Cdk2 triggered initiation synergistically.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/fisiología , Replicación del ADN/fisiología , Fase G1/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Fase S/fisiología , Extractos Celulares , Núcleo Celular/metabolismo , Sistema Libre de Células , Quinasa 2 Dependiente de la Ciclina , Fase G2/fisiología , Células HeLa , HumanosRESUMEN
Nucleosomes assembled on regulatory DNA sites in chromatin repress gene expression; protein factors have now been identified that can help overcome such repression by excluding or remodelling nucleosomes so regulatory sites are accessible to transcription factors.
Asunto(s)
Cromatina/fisiología , Nucleosomas , Animales , HumanosRESUMEN
Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Replicación del ADN , Proteínas de Unión al ADN , Proteínas Nucleares/metabolismo , Factores de Transcripción , Proteínas de Xenopus , Animales , Núcleo Celular/metabolismo , Fraccionamiento Químico , Células HeLa , Humanos , Interfase , Componente 3 del Complejo de Mantenimiento de Minicromosoma , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Conejos , Fase SAsunto(s)
Proteínas Cromosómicas no Histona , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Chaperonas Moleculares/metabolismo , Nucleosomas/fisiología , Animales , Cromatina , Factor 1 de Ensamblaje de la Cromatina , Proteínas de Unión al ADN/química , Histonas/metabolismo , Humanos , Chaperonas Moleculares/químicaRESUMEN
In the S-phase of the eukaryotic cell cycle, newly replicated DNA is assembled into chromatin. I used indirect immunofluorescence microscopy to localize the sites of chromatin assembly in respect to DNA replication. Replication foci in the nuclei of permeabilized HeLa cells were labeled by incorporation of biotin-16-dUTP and detected by fluorescent streptavidin. Prelabeling of replication foci in vivo with bromodeoxyuridine showed that replication in permeabilized cells proceeds at preexisting replication forks. The localization of chromatin assembly factor 1 (CAF-1) was determined with subunit-specific monoclonal antibodies. CAF-1 is not detectable in mitotic cells and is detectable only at background levels in about 60% of all interphase nuclei. The other interphase nuclei show an intense punctate immunostaining of CAF-1. These sites of CAF-1 colocalize with replication foci during all stages of the S-phase. No other discrete sites of CAF-1 are observed. Human replication protein A (RPA) colocalizes with these replication/chromatin assembly sites. In addition, extra nuclear sites of RPA are observed that probably represent prereplication foci, poised for initiation of DNA replication.
Asunto(s)
Ciclo Celular , Núcleo Celular/fisiología , Cromatina/fisiología , Proteínas Cromosómicas no Histona , Replicación del ADN , Proteínas de Unión al ADN/análisis , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Permeabilidad de la Membrana Celular , Núcleo Celular/ultraestructura , Factor 1 de Ensamblaje de la Cromatina , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Interfase , Sustancias Macromoleculares , Microscopía Fluorescente , Proteína de Replicación A , Fase SRESUMEN
The template activities of protein-free SV40 DNA and SV40 minichromosomes for DNA re-replication are compared in in vitro replication assays. Density substitution experiments and two-dimensional gel electrophoresis show that protein-free DNA can replicate for at least two cycles whereas salt-treated minichromosomes replicate only once. Re-replication of minichromosomes is blocked at the stage of replicative chain elongation suggesting that replicatively assembled chromatin has structural features that prevent a second round of replication.
Asunto(s)
Replicación del ADN/fisiología , ADN Viral/biosíntesis , Virus 40 de los Simios/fisiología , Replicación Viral/fisiología , Cromosomas , Citosol/química , ADN Superhelicoidal/análisis , ADN Viral/química , Electroforesis en Gel de Agar , Electroforesis en Gel Bidimensional , Genoma Viral , Células HeLa , Humanos , Cinética , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Moldes GenéticosRESUMEN
Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.
Asunto(s)
Cromosomas Humanos , Replicación del ADN , Nucleosomas , Centrifugación , Cromatina/fisiología , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/genética , ADN Viral , Células HeLa , Humanos , Virus 40 de los Simios/genéticaRESUMEN
Circular single-stranded phage M13 DNA is used as a template for complementary strand synthesis in cytosolic extracts from proliferating HeLa cells. DNA synthesis is initiated by one or maximally two priming events and typically leads to covalently closed double-stranded reaction products. When carried out in the presence of the nuclear chromatin assembly factor CAF-1, complementary strand synthesis is accompanied by nucleosome assembly. This novel system is very useful for the study of basic biochemical aspects concerning the assembly of nucleosomes. The activity of CAF-1 completely depends on complementary strand synthesis and acts stoichiometrically to promote the assembly of nucleosomes in a noncooperative manner. Apparently, CAF-1 activity is coupled to DNA synthesis via a structural feature of replicating DNA, most likely its partial single strandedness.