RESUMO
To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.
Assuntos
Replicação do DNA , DNA Super-Helicoidal/biossíntese , DNA Super-Helicoidal/química , Escherichia coli/genética , Conformação de Ácido Nucleico , Plasmídeos/biossíntese , Plasmídeos/química , Southern Blotting , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/ultraestrutura , Eletroforese em Gel Bidimensional , Etídio , Microscopia Eletrônica , Modelos Genéticos , Plasmídeos/metabolismo , Plasmídeos/ultraestruturaRESUMO
In order to determine the time required for nucleosomes assembled on the daughter strands of replication forks to assume favoured positions with respect to DNA sequence, psoralen cross-linked replication intermediates purified from preparative two-dimensional agarose gels were analysed by exonuclease digestion or primer extension. Analysis of sites of psoralen intercalation revealed that nucleosomes in the yeast Saccharomyces cerevisiae rDNA intergenic spacer are positioned shortly after passage of the replication machinery. Therefore, both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands, suggesting that the positioning of nucleosomes is an initial step in the chromatin maturation process.
Assuntos
Replicação do DNA , Nucleossomos/metabolismo , Sequência de Bases , Cromatina/metabolismo , Primers do DNA , DNA Fúngico/genética , DNA Ribossômico/genética , Exonucleases/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
In the yeast S. cerevisiae, ARS (autonomously replicating sequence) elements located in the intergenic spacers of the rRNA gene locus are infrequently activated as origins of replication. We analyzed the rARS activation with a combination of neutral/neutral (N/N) two-dimensional (2D) gel electrophoresis and either the intercalating drug psoralen, which in vivo specifically marks the transcribing gene copies, or the selective accessibility of restriction sites in transcriptionally active genes. We found that initiation of replication starts at those rARSs placed immediately downstream of transcribing rRNA genes. This correlation between transcription and replication is consistent with the presence of nucleosome-free enhancers at each transcriptionally active gene copy and suggests that the transcription factor Abf1p is involved in replication initiation at the ARS in the rDNA gene locus.
Assuntos
Replicação do DNA , DNA Fúngico/genética , DNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , DNA Fúngico/efeitos dos fármacos , DNA Ribossômico/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Ficusina/farmacologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Origem de Replicação/genéticaRESUMO
Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand. A model explaining these findings is presented. Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules. This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm.
Assuntos
RNA Ribossômico/química , Leveduras/genética , Sequência de Bases , Replicação do DNA , Desoxirribonuclease HindIII/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/metabolismo , Dados de Sequência Molecular , RNA Ribossômico/isolamento & purificação , RNA Ribossômico/metabolismoRESUMO
In the pro-alpha1(I) collagen gene a number of cis-regulatory elements, which interact with a variety of trans-acting factors, are present in the promoter and first intron. We have undertaken a comprehensive study of Sp1, Ap1, and Ap2 binding in the region spanning -442 to +1697 nt. DNase I footprinting analysis revealed these factors bind with varying affinities to some of the potential sites: Sp1 binds to 16 of 34 potential sites, Ap2 binds to 22 of 40 potential binding sites, and Ap1 binds to its only potential site. The Sp1 sites were mostly clustered in the intron region, while the Ap2 sites were clustered in the promoter region. Transmission electron microscopic analysis of DNA-protein complexes not only confirmed these results, but also clearly showed that heterologous and/or homologous protein-protein interactions between Sp1 and/or Ap2 bring the promoter and intron in contact with each other, with the resulting looping out of the intervening DNA. This strongly suggests that the DNA-looping model is an explanation for the orientation preference of the enhancing element in the first intron as these interactions possibly create an optimum environment for the binding of the rest of the transcriptional machinery.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Pró-Colágeno/genética , Elementos de Resposta/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/química , DNA/genética , DNA/ultraestrutura , Pegada de DNA , Sondas de DNA/química , Sondas de DNA/genética , Sondas de DNA/metabolismo , Sondas de DNA/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Microscopia Eletrônica , Modelos Genéticos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Deleção de Sequência/genética , Fator de Transcrição Sp1/ultraestrutura , Termodinâmica , Fator de Transcrição AP-2 , Fatores de Transcrição/ultraestruturaRESUMO
Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.3-42.2 kbp) in Xenopus egg extracts by two-dimensional gel electrophoresis and electron microscopy. We confirm that replication initiates without sequence specificity on all plasmids. We demonstrate for the first time that multiple initiation events occur on large plasmids, but not on small (<10 kb) plasmids, at average intervals of approximately 10 kb. Origin interference may prevent multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on later than on earlier replication intermediates, showing that initiation frequency increases throughout S phase, speeding up replication of late intermediates. We suggest that potential origins are abundant and randomly distributed, but that the increase of initiation frequency during S phase, and possibly origin interference, regulate origin activation to ensure rapid completion of replication.
Assuntos
Replicação do DNA/genética , Embrião não Mamífero/metabolismo , Origem de Replicação/genética , Xenopus laevis , Animais , Extratos Celulares , DNA/biossíntese , DNA/química , DNA/genética , DNA/ultraestrutura , Eletroforese em Gel Bidimensional , Embrião não Mamífero/citologia , Cinética , Microscopia Eletrônica , Peso Molecular , Conformação de Ácido Nucleico , Plasmídeos/biossíntese , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/ultraestrutura , Fase S/genética , Zigoto/citologia , Zigoto/metabolismoRESUMO
In Escherichia coli, adenine methylation at the sequence GATC allows coupling of cellular processes to chromosome replication and the cell cycle. The transient presence of hemimethylated DNA after replication facilitates post-replicative mismatch repair, induces transcription of some genes and allows transposition of mobile elements. We were interested in estimating the half-life of hemimethylated DNA behind the replication fork in plasmid molecules and in determining whether Dam methyltransferase restores N6 adenine methylation simultaneously on both replicative arms. We show that remethylation takes place asynchronously on the leading and lagging daughter strands shortly after replication. On the leading arm the fully methylated adenine is restored approximately 2000 bp (corresponding to 2 s) behind the replication fork, while remethylation takes twice as long (at 3500-4000 bp or approximately 3.5-4 s) on the lagging replicative arm. This observation suggests that Dam remethylation of the lagging arm requires ligated Okazaki fragments.
Assuntos
Metilação de DNA , Replicação do DNA , Escherichia coli/genética , Plasmídeos/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Ciclo Celular , Cromossomos Bacterianos/genética , Escherichia coli/citologia , Proteínas de Escherichia coli , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , TransfecçãoRESUMO
Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction. Using the electron microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The electron microscopic data indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA. No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident.
Assuntos
DNA de Cadeia Simples/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio , Microscopia Eletrônica , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Animais , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Drosophila/química , Células HeLa , Humanos , Antígenos de Histocompatibilidade Menor , Ácidos Nucleicos Heteroduplexes/ultraestrutura , Plasmídeos/química , Ligação Proteica , Proteína de Replicação CRESUMO
We used the psoralen gel retardation assay and Northern blot analysis in an in vivo yeast system to analyze effects of rDNA enhancer deletions on the chromatin structure and the transcription of tagged rDNA units. We found that upon deletion of a single enhancer element, transcription of the upstream and downstream rRNA gene was reduced by about 50%. Although removing both flanking enhancers of an rRNA gene led to a further reduction in transcription levels, a significant amount of transcriptional activity remained, either resulting from the influence of more distantly located enhancer elements or reflecting the basal activity of the polymerase I promoter within the nucleolus. Despite the reduction of transcriptional activity upon enhancer deletion, the activation frequency (proportion of nonnucleosomal to nucleosomal gene copies in a given cell culture) of the tagged rRNA genes was not significantly altered, as determined by the psoralen gel retardation assay. This is a strong indication that, within the nucleolus, the yeast rDNA enhancer functions by increasing transcription rates of active rRNA genes and not by activating silent transcription units.
Assuntos
Cromatina , Cromossomos Fúngicos , Elementos Facilitadores Genéticos , RNA Fúngico , RNA Ribossômico , Saccharomyces cerevisiae/genética , Transcrição Gênica , DNA Fúngico , DNA Ribossômico , RNA Polimerase I/metabolismoRESUMO
Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.
Assuntos
Replicação do DNA/genética , DNA Bacteriano/química , Conformação de Ácido Nucleico , DNA Topoisomerases Tipo I/genética , DNA Bacteriano/ultraestrutura , DNA Super-Helicoidal/química , Escherichia coli/genética , Microscopia Eletrônica , Plasmídeos/química , Mapeamento por RestriçãoRESUMO
A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, we directly tested for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking as well as by in vitro transcription. While H1-containing control chromatin severely repressed transcription of our model hsp26 gene, highly acetylated chromatin was significantly less repressive. Acetylation of histones, and particularly of histone H4, affected transcription at the level of initiation. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, we found that heat shock factor, a crucial regulator of heat shock gene transcription, profited most from histone acetylation. These experiments demonstrate that histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin.
Assuntos
Cromatina/metabolismo , Genes de Insetos , Proteínas de Choque Térmico/genética , Histonas/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Acetilação , Animais , Cromatina/ultraestrutura , Drosophila/genética , Proteínas de Drosophila , Moldes GenéticosRESUMO
By the use of psoralen crosslinking and primer extension, a method was developed which allows the analysis of chromatin structure in vivo. Using a yeast minichromosome, >9 nucleosomes were mapped with a resolution of at least +/-30 bp.
Assuntos
Mapeamento Cromossômico/métodos , Nucleossomos/genética , Sequência de Bases , Cromossomos Fúngicos/genética , Reagentes de Ligações Cruzadas , Primers do DNA/genética , DNA Fúngico/genética , Fármacos Fotossensibilizantes , Saccharomyces cerevisiae/genética , Trioxsaleno , Raios UltravioletaRESUMO
The RNA phage Qbeta requires for the replication of its genome an RNA binding protein called Qbeta host factor or Hfq protein. Our previous results suggested that this protein mediates the access of replicase to the 3'-end of the Qbeta plus strand RNA. Here we report the results of an evolutionary experiment in which phage Qbeta was adapted to an Escherichia coli Q13 host strain with an inactivated host factor (hfq) gene. This strain initially produced phage at a titer approximately 10,000-fold lower than the wild-type strain and with minute plaque morphology, but after 12 growth cycles, phage titer and plaque size had evolved to levels near those of the wild-type host. RNAs isolated from adapted Qbeta mutants were efficient templates for replicase without host factor in vitro. Electron microscopy showed that mutant RNAs, in contrast to wild-type RNA, efficiently interacted with replicase at the 3'-end in the absence of host factor. The same set of four mutations in the 3'-terminal third of the genome was found in several independently evolved phage clones. One mutation disrupts the base pairing of the 3'-terminal CCCOH sequence, suggesting that the host factor stimulates activity of the wild-type RNA template by melting out its 3'-end.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/virologia , Conformação de Ácido Nucleico , Fagos RNA/genética , RNA Viral/química , Sequência de Bases , Proteínas de Transporte/fisiologia , Fator Proteico 1 do Hospedeiro , Fatores Hospedeiros de Integração , Microscopia Eletrônica , Dados de Sequência Molecular , Fenótipo , Fagos RNA/ultraestrutura , RNA Viral/biossíntese , RNA Viral/genética , Proteínas de Ligação a RNA/fisiologiaRESUMO
By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome- packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of Hpa II and Msp I restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single Hpa II site, located in the promoter region, showed particularly strong correlation with the transcriptional activity.
Assuntos
Cromatina/química , Formaldeído/química , Furocumarinas/química , RNA Ribossômico/genética , Animais , Linhagem Celular , Núcleo Celular/química , Reagentes de Ligações Cruzadas/química , Desoxirribonuclease EcoRI/química , Desoxirribonuclease HpaII/química , Elementos Facilitadores Genéticos , Fígado/química , Metilação , Regiões Promotoras Genéticas , RatosRESUMO
RNA-protein interactions between bacteriophage Qbeta plus strand RNA and the components of the Qbeta replicase system were studied by deletion analysis. Internal, 5'-terminal and 3'-terminal deletions were assayed for template activity with replicase in vitro. Of the two internal binding sites previously described for replicase, we found that the S-site (map position 1247 to 1346) could be deleted without any significant effect on template activity, whereas deletion of the M-site (map position 2545 to 2867) resulted in a strong inactivation and a high salt sensitivity of the residual activity. Binding complexes of the deletion mutant RNAs with the different proteins involved in Qbeta RNA replication were analysed by electron microscopy. The formation of looped complex structures, previously reported and explained as simultaneous interactions with replicase at the S and the M-site, was abolished by deleting the S-site but, surprisingly, not by deleting the M-site. The same types of complexes observed with replicase were also formed with purified protein S1 (the alpha subunit of replicase), suggesting that these internal interactions with Qbeta RNA are mediated by the S1 protein. The Qbeta host factor, a protein required for the template activity of the Qbeta plus strand, was reported earlier to form similar complexes by binding to the S and M-sites (or adjacent sites) and in addition to the 3'-end, resulting in double-looped structures. The patterns of looped complexes observed with the deletion mutant RNAs suggest that the binding of host factor might not involve the S and M-sites themselves but adjacent downstream sites. An additional internal host factor interaction near map position 2300 was detected with several mutant RNAs. Qbeta RNA molecules with 3'-truncations formed 3'-terminal loops with similar efficiency as wild-type RNA, indicating that recognition of the 3'-end by host factor is not dependent on a specific 3'-terminal base sequence.
Assuntos
Allolevivirus/enzimologia , Proteínas de Transporte/metabolismo , Q beta Replicase/metabolismo , RNA Viral/metabolismo , Proteínas Ribossômicas/metabolismo , Allolevivirus/genética , Sítios de Ligação , Proteínas de Transporte/ultraestrutura , Fatores Hospedeiros de Integração , Mutagênese , Conformação de Ácido Nucleico , Q beta Replicase/ultraestrutura , RNA Viral/ultraestrutura , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Deleção de Sequência , Especificidade por Substrato , Transcrição GênicaRESUMO
Purified simian virus (SV40) minichromosomes were photoreacted with psoralen under various conditions that moderately destabilize nucleosomes. This assay allows indirect distinction between stable nucleosomes, partially unravelled nucleosomes and nucleosomes containing (or lacking) histone H1. In replicating molecules the passage of the replication machinery destabilizes the nucleosomal organization of the chromatin fiber over a distance of 650 to 1100 bp. In front of the fork, an average of two nucleosomes are destabilized presumably by the dissociation of histone H1 and the advancing replication machinery. On daughter strands, the first nucleosome is detected at a distance of about 260 nucleotides from the elongation point. This nucleosome is interpreted to contain no histone H1, while no stepwise association of (H3-H4)2 tetramers with H2A/H2B dimers on nascent DNA can be detected in vivo. The second nucleosome after the replication fork appears to contain histone H1. The prolonged nuclease sensitivity of newly replicated chromatin described in the literature therefore may not be due to a slow reassociation of histone H1.
Assuntos
Replicação do DNA , DNA Viral/biossíntese , Nucleossomos/fisiologia , Vírus 40 dos Símios/genética , Replicação Viral , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , DNA Viral/efeitos dos fármacos , DNA Viral/ultraestrutura , Ficusina/farmacologia , Haplorrinos , Histonas , Concentração de Íons de Hidrogênio , Nucleossomos/ultraestrutura , Vírus 40 dos Símios/fisiologia , Vírus 40 dos Símios/ultraestruturaRESUMO
In growing yeast cells, about half of the 150 tandemly repeated rRNA genes are transcriptionally active and devoid of nucleosomes. By using the intercalating drug psoralen as a tool to mark accessible sites along chromatin DNA in vivo, we found that the active rRNA gene copies are rather randomly distributed along the ribosomal rRNA gene locus. Moreover, results from the analysis of a single, tagged transcription unit in the tandem array are not consistent with the presence of a specific subset of active genes that is stably maintained throughout cell divisions. In the rRNA intergenic spacers of yeast cells, an enhancer is located at the 3' end of each transcription unit, 2 kb upstream of the next promoter. Analysis of the chromatin structure along the tandem array revealed a structural link between transcription units and adjacent, 3' flanking enhancer sequences: each transcriptionally active gene is flanked by a nonnucleosomal enhancer, whereas inactive, nucleosome-packed gene copies are followed by enhancers regularly packaged in nucleosomes. From the fact that nucleosome-free enhancers were also detected in an RNA polymerase I mutant strain, we interpret these open chromatin structures as being the result of specific protein-DNA interactions that can occur before the onset of transcription. In contrast, in this mutant strain, all of the rRNA coding sequences are packaged in nucleosomal arrays. This finding indicates that the establishment of the open chromatin conformation on the activated gene copies requires elongating RNA polymerase I molecules advancing through the template.
Assuntos
Cromatina , DNA Ribossômico/genética , Elementos Facilitadores Genéticos/genética , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica/fisiologia , Divisão Celular , Reagentes de Ligações Cruzadas , Replicação do DNA/genética , DNA Fúngico/genética , DNA Fúngico/ultraestrutura , DNA Ribossômico/ultraestrutura , Genes Fúngicos/genética , Família Multigênica/genética , RNA Fúngico/genética , Mapeamento por Restrição , Saccharomyces cerevisiae/citologia , TrioxsalenoRESUMO
To understand the mechanism of action of the two eukaryotic replication auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C), we constructed a plasmid for producing PCNA which could be 32P labelled in vitro. This allowed us to analyze the assembly of the auxiliary proteins directly on DNA and to examine this process in the absence of DNA synthesis. By using closed circular double-stranded DNA or gapped circular DNA for protein-DNA complex formation, the following results were obtained, (i) RF-C can load PCNA in an ATP-dependent manner directly on double-stranded DNA, and no 3'-OH ends are required for this reaction; (ii) the RF-C-PCNA complex assembled on closed circular DNA differs from those assembled on gapped or nicked circular DNA; (iii) the stable RF-C-PCNA complex can be assembled on circular but not on linear DNA; and (iv) only gapped DNA can partially retain the assembled RF-C-PCNA complex upon the linearization of the template. We propose that RF-C first binds unspecifically to double-stranded DNA in the presence of ATP and then loads PCNA onto DNA to yield a protein complex able to track along DNA. The RF-C-PCNA complex could slide along the template until it encounters a 3'-OH primer-template junction, where it is likely transformed into a competent clamp. The latter complex, finally, might still be able to slide along double-stranded DNA.
Assuntos
DNA Circular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Replicação do DNA , DNA Circular/ultraestrutura , DNA Polimerase Dirigida por DNA/metabolismo , Mamíferos , Microscopia Eletrônica , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/ultraestrutura , Ligação Proteica , Proteínas Recombinantes/metabolismo , Proteína de Replicação CRESUMO
In eukaryotic cells, active genes and their regulatory sequences are organized into open chromatin conformations in which nucleosomes can be modified, disrupted or totally absent. It has been proposed that these characteristic chromatin structures and their associated factors might be directly inherited by the newly synthesized daughter strands during chromosome duplication. Here we show that in the yeast Saccharomyces cerevisiae, replication machinery entering upstream of a transcriptionally active ribosomal RNA gene generates two newly replicated coding regions regularly packaged into nucleosomes, indicating that the active chromatin structure cannot be directly inherited at the replication fork. Whereas the establishment of an exposed chromatin conformation at some newly replicated rRNA gene promoters can occur shortly after the passage of the replication fork, regeneration of the active chromatin structure along the coding region is always a post-replicative process involving disruption of preformed nucleosomes.