RESUMO
Genomic propagation in both prokaryotes and eukaryotes is tightly regulated at the level of initiation, ensuring that the genome is accurately replicated and equally segregated to the daughter cells. Even though replication origins and the proteins that bind onto them (initiator proteins) have diverged throughout the course of evolution, the mechanism of initiation has been conserved, consisting of origin recognition, multi-protein complex assembly, helicase activation and loading of the replicative machinery. Recruitment of the multiprotein initiation complexes onto the replication origins is constrained by the dense packing of the DNA within the nucleus and unusual structures such as knots and supercoils. In this review, we focus on the DNA topological barriers that the multi-protein complexes have to overcome in order to access the replication origins and how the topological state of the origins changes during origin firing. Recent advances in the available methodologies to study DNA topology and their clinical significance are also discussed.
Assuntos
Replicação do DNA , DNA/biossíntese , DNA/química , Conformação de Ácido Nucleico , Origem de Replicação , Animais , DNA Topoisomerases/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismoRESUMO
Genome duplication relies on the timely activation of multiple replication origins throughout the genome during S phase. Each origin is marked by the assembly of a multiprotein pre-replication complex (pre-RC) and the recruitment of the replicative machinery, which can gain access to replication origins on the DNA through the barrier of specific chromatin structures. Inheritance of the genetic information is further accompanied by maintenance and inheritance of the epigenetic marks, which are accomplished by the activity of histone and DNA modifying enzymes traveling with the replisome. Here, we studied the changes in the chromatin structure at the loci of three replication origins, the early activated human lamin B2 (LB2) and monkey Ors8 (mOrs8) origins and the late-activated human homologue of the latter (hOrs8), during their activation, by measuring the abundance of post-translationally modified histone H3. The data show that dynamic changes in the levels of acetylated, methylated and phosphorylated histone H3 occur during the initiation of DNA replication at these three origin loci, which differ between early- and late-firing origins as well as between human- and monkey-derived cell lines. These results suggest that specific histone modifications are associated with origin firing, temporal activation and replication fork progression and underscore the importance of species specificity.
Assuntos
Cromatina/química , Período de Replicação do DNA , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Origem de Replicação , Acetilação , Animais , Western Blotting , Linhagem Celular , Chlorocebus aethiops , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Células HeLa , Humanos , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Metilação , Fosforilação , Fase S/efeitos dos fármacos , Especificidade da EspécieRESUMO
DNA replication in eukaryotes is initiated at multiple replication origins distributed over the entire genome, which are normally activated once per cell cycle. Due to the complexity of the metazoan genome, the study of metazoan replication origins and their activity profiles has been less advanced than in simpler genome systems. DNA replication in eukaryotes involves many protein-protein and protein-DNA interactions, occurring in multiple stages. As in prokaryotes, control over the timing and frequency of initiation is exerted at the initiation site. A prerequisite for understanding the regulatory mechanisms of eukaryotic DNA replication is the identification and characterization of the cis-acting sequences that serve as replication origins and the trans-acting factors (proteins) that interact with them. Furthermore, in order to understand how DNA replication may become deregulated in malignant cells, the distinguishing features between normal and malignant origins of DNA replication as well as the proteins that interact with them must be determined. Based on advances that were made using simple genome model systems, several proteins involved in DNA replication have been identified. This review summarizes the current findings about metazoan origins of DNA replication and their interacting proteins as well as the role of chromatin structure in their regulation. Furthermore, progress in origin identification and isolation procedures as well as potential mechanisms to inhibit their activation in cancer development and progression are discussed.
Assuntos
Cromatina/química , Replicação do DNA , Origem de Replicação , Animais , Neoplasias/etiologia , Neoplasias/genéticaRESUMO
14-3-3 proteins are conserved multifunctional molecules, involved in many biological processes. Several 14-3-3 isoforms were recently shown to be cruciform DNA-binding proteins, which is a new activity ascribed to the 14-3-3 family. As cruciform-binding proteins, 14-3-3 proteins are putatively involved in the regulation of DNA replication. Inverted repeat sequences that are able to extrude into cruciform structures are a common feature of replication origins in both prokaryotes and eukaryotes. The involvement of cruciform structures in the initiation of DNA replication has been demonstrated. A leading model of 14-3-3 function proposes that they facilitate critical protein-protein interactions, thus serving as a central component of a wide variety of cellular processes.
Assuntos
Replicação do DNA , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Animais , Ciclo Celular/fisiologia , Humanos , Origem de Replicação , Transdução de Sinais/fisiologiaRESUMO
Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.
Assuntos
Antígenos Nucleares , DNA Helicases , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Origem de Replicação , Ribonucleoproteínas , Animais , Sítios de Ligação , Ciclo Celular , Extratos Celulares , Linhagem Celular , Reagentes de Ligações Cruzadas , Proteínas de Ligação a DNA/genética , Formaldeído , Autoantígeno Ku , Mamíferos , Camundongos , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase/métodos , Testes de Precipitina , Fatores de Processamento de Serina-Arginina , Fator de Transcrição RelARESUMO
To study the possible relationships between origin activities and cellular processes leading to malignancy, we used an isogenic system of human embryo lung fibroblast cells WI38 and a SV40-transformed variant, WI38 VA13 2RA (WI38(SV40)). We found that the activities of all initiation sites at the c-myc locus were approximately two-fold as high in WI38(SV40) cells as in WI38 cells. Thus, higher initiation frequency of origins at certain loci is induced with cell immortalization, one of the steps in the multi-step process leading to malignancy. We measured the activities of the four c-myc promoters P0, P1, P2, and P3 with nuclear runon assay in the two cell lines in order to detect potential individual promoter changes that may be also associated with immortalization by SV40 virus. The results show that the activities of the promoters P0, P1, and P3 did not significantly change, but the activity of the major promoter P2 in WI38(SV40) cells was about 7.5- to 8.0-fold as high as that in WI38 cells. The increased activity of promoter P2, although approximately 600 bp downstream of one of the major DNA replication initiation sites, had no preferential influence on the major sites of origin activity. Since the distribution of nascent strand abundance was not significantly altered, binding of transcription factors does not seem to facilitate the assembly of pre-replication complex (pre-RC) or otherwise preferentially alter the activities of the DNA replication proteins at this major initiation site.
Assuntos
Ciclo Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes myc/genética , Origem de Replicação/genética , Antígenos Virais de Tumores/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/genética , DNA/análise , Fibroblastos/patologia , Humanos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genéticaRESUMO
DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122-417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , Sequência de Bases , Sítios de Ligação , Western Blotting , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferase 1 , Deleção de Genes , Humanos , Metilação , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Peptídeos/química , Testes de Precipitina , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
DNA replication initiation sites and initiation frequencies over 12. 5 kb of the human c-myc locus, including 4.6 kb of new 5' sequence, were determined based on short nascent DNA abundance measured by competitive polymerase chain reaction using 21 primer sets. In previous measurements, no comparative quantitation of nascent strand abundance was performed, and distinction of major from minor initiation sites was not feasible. Two major initiation sites were identified in this study. One predominant site has been located at approximately 0.5 kb upstream of exon 1 of the c-myc gene, and a second new major site is located in exon 2. The site in exon 2 has not been previously identified. In addition, there are other sites that may act as less frequently used initiation sites, some of which may correspond to sites in previous reports. Furthermore, a comparison of the abundance of DNA replication intermediates over this same region of the c-myc locus between HeLa and normal skin fibroblast (NSF) cells indicated that the relative distribution was very similar, but that nascent strand abundance in HeLa cells was approximately twice that in NSF relative to the abundance at the lamin B2 origin. This increased activity at initiation sites in the c-myc locus may mainly be influenced by regulators at higher levels in transformed cells like HeLa.
Assuntos
Mapeamento Cromossômico , Replicação do DNA/genética , Genes myc/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Células Cultivadas , DNA/biossíntese , Primers do DNA/química , Eletroforese em Gel de Ágar , Éxons/genética , Fibroblastos/metabolismo , Células HeLa/metabolismo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-myc/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Pele/metabolismoRESUMO
Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Replicação do DNA , Animais , Divisão Celular , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Inibidores Enzimáticos/farmacologia , Humanos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Reação em Cadeia da PolimeraseRESUMO
We have recently cloned the human nucleosome assembly protein 2 (NAP-2). Here, we demonstrate that casein kinase 2 (CKII) from HeLa cell nuclear extracts interacts with immobilized NAP-II, and phosphorylates both NAP-2 and nucleosome assembly protein 1 (NAP-1) in vitro. Furthermore, NAP-1 and NAP-2 phosphorylation in crude HeLa cell extracts is abolished by heparin, a specific inhibitor of CKII. Addition of core histones can stimulate phosphorylation of NAP-1 and NAP-2 by CKII. NAP-2 is also a phosphoprotein in vivo. The protein is phosphorylated at the G0/G1 boundary but it is not phosphorylated in S-phase. Here, we show that NAP-2 is a histone chaperone throughout the cell cycle and that its cell-cycle distribution might be governed by its phosphorylation status. Phosphorylated NAP-2 remains in the cytoplasm in a complex with histones during the G0/G1 transition, whereas its dephosphorylation triggers its transport into the nucleus, at the G1/S-boundary, with the histone cargo, suggesting that binding to histones does not depend on phosphorylation status. Finally, indirect immunofluorescence shows that NAP-2 is present during metaphase of HeLa and COS cells, and its localization is distinct from metaphase chromosomes.
Assuntos
Ciclo Celular , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Animais , Transporte Biológico , Caseína Quinase II , Proteínas de Ciclo Celular , Extratos Celulares , Linhagem Celular , Núcleo Celular/metabolismo , Cromatografia de Afinidade , Cromossomos/metabolismo , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fase G1 , Células HeLa , Heparina/farmacologia , Histonas/farmacologia , Humanos , Metáfase , Proteína 1 de Modelagem do Nucleossomo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Fase SRESUMO
pYACneo, a 15.8-kb plasmid, contains a bacterial origin, G418-resistance gene, and yeast ARS, CEN, and TEL elements. Three mammalian origins have been cloned into this circular vector: 343, a 448-bp chromosomal origin from a transcribed region of human chromosome 6q; X24, a 4.3-kb element containing the hamster DHFR origin of bidirectional replication (oribeta), and S3, a 1.1-kb human anti-cruciform purified autonomously replicating sequence. The resulting constructs have been transfected into HeLa cells, and G418-resistant subcultures were isolated. The frequency of G418-resistant transformation was 1.7-8.7 times higher with origin-containing YACneo than with vector alone. After >45 generations under G418 selection, the presence of episomal versus integrated constructs was assessed by fluctuation assay and by PCR of supercoiled, circular, and linear genomic cellular DNAs separated on ethidium bromide-cesium chloride gradients. In stable G418-resistant subcultures transfected with vector alone or with linearized constructs, as well as in some subcultures transfected with circular origin-containing constructs, resistance was conferred by integration into the host genome. However, several examples were found of G418-resistant transfectants maintaining the Y.343 and the YAC.S3 circular constructs in a strictly episomal state after long-term culture in selective medium, with 80-90% stability per cell division. The episomes were found to replicate semiconservatively in a bromodeoxyuridine pulse-labeling assay for =130 cell generations after transfection. Furthermore, after =172 cell generations rescued episomal DNA could be isolated intact and unrearranged, and could be used to retransform bacteria. These versatile constructs, containing mammalian origins, have the capacity for further modification with human telomere or large putative centromere elements, in an effort to move towards construction of a human artificial chromosome.
Assuntos
Cromossomos Artificiais de Levedura/genética , DNA Circular/genética , Vetores Genéticos/genética , Plasmídeos/genética , Origem de Replicação/genética , Bromodesoxiuridina/metabolismo , Divisão Celular/genética , Cromossomos Humanos Par 6 , Clonagem Molecular , Replicação do DNA , Genes Bacterianos , Genes Fúngicos , Gentamicinas/farmacologia , Células HeLa , Humanos , Transfecção , Transformação GenéticaRESUMO
Ors12, a mammalian autonomously replicating sequence (812 bp), was previously isolated by extrusion of African green monkey (CV-1 cells) nascent DNA from active replication bubbles. It contains a region of alpha-satellite extending 168-bp from the 5'-end, and a nonrepetitive portion extending from nucleotide position 169 to nucleotide 812 that is present in less than nine copies per haploid genome. Ors12 is capable of transient autonomous DNA replication in vivo and in vitro, associates with the nuclear matrix in a cell cycle-dependent manner, and hybridizes at the centromeric region of six CV-1 cell chromosomes as well as a marker chromosome. To demonstrate that DNA replication initiates at ors12 at a native chromosomal locus, a 14.2 kb African green monkey genomic clone was isolated and sequence information was obtained that allowed us to generate eight sets of PCR primers spanning a region of 8 kb containing ors12. One set of primers occurred inside ors12. These primers were used to amplify nascent DNA strands from asynchronously growing CV-1 and African green monkey kidney (AGMK) cells, using noncompetitive and competitive PCR-based mapping methodologies. Both assays showed that DNA replication in vivo initiates preferentially in a 2.3 kb region containing ors12, as well as at a second site located 1.7 kb upstream of ors12. This study provides the first demonstration of genomic function for a centromeric mammalian origin of DNA replication, originally isolated by nascent strand extrusion.
Assuntos
Centrômero/genética , Replicação do DNA/genética , Origem de Replicação , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Mapeamento Cromossômico , Primers do DNA/genética , Genoma , Reação em Cadeia da PolimeraseRESUMO
Mammalian polynucleotide kinases (PNKs) carry out 5'-phosphorylation of nucleic acids. Although the cellular function(s) of these enzymes remain to be delineated, important suggestions have included a role in DNA repair and, more recently, in DNA replication. Like T4 PNK, some preparations of mammalian PNKs have been reported to have an associated 3'-phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3'-phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI-PNK, with a slightly acidic pH optimum that copurifies with a 3'-phosphatase activity. The enzyme appears to be a monomer of 60 kDa. Mammalian DNA replication reactions were supplemented with T4 PNK or SNQI-PNK, and no significant effect on DNA replication in vitro was observed. Database searches support the earlier mapping of the 3'-phosphatase activity of T4 PNK to the C-terminus and suggest that the 3'-phosphatase domain of T4 PNK is related to the protein superfamily of L-2-haloacid dehalogenases. Exopeptidase digestion experiments were carried out to compare the SNQI-PNK enzyme with T4 PNK and led to the inference that the domain organization of the bovine polypeptide may differ from that of the T4 enzyme.
Assuntos
Bacteriófago T4/enzimologia , Replicação do DNA , Monoéster Fosfórico Hidrolases/química , Polinucleotídeo 5'-Hidroxiquinase/isolamento & purificação , Timo/enzimologia , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Dados de Sequência Molecular , Polinucleotídeo 5'-Hidroxiquinase/química , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4-OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of approximately 92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication.
Assuntos
Antígenos Nucleares , DNA Helicases , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Anticorpos/farmacologia , Sequência de Bases , Ligação Competitiva , Bioquímica/métodos , Sequência Conservada , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa/metabolismo , Humanos , Autoantígeno Ku , Mamíferos/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Oligonucleotídeos/genética , Oligonucleotídeos/farmacologia , Plasmídeos/genética , Plasmídeos/metabolismo , Origem de Replicação/genética , Especificidade por SubstratoRESUMO
Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Replicação do DNA , Mapeamento Cromossômico , Ilhas de CpG , Metilação de DNA , Humanos , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Origem de ReplicaçãoRESUMO
One of the fundamental characteristics of life is the ability of an entity to reproduce itself, which stems from the ability of the DNA molecule to replicate itself. The initiation step of DNA replication, where control over the timing and frequency of replication is exerted, is poorly understood in eukaryotes in general, and in mammalian cells in particular. The cis-acting DNA element defining the position and providing control over initiation is the replication origin. The activation of replication origins seems to be dependent on the presence of both a particular sequence and of structural determinants. In the past few years, the development of new methods for identification and mapping of origins of DNA replication has allowed some understanding of the fundamental elements that control the replication process. This review summarizes some of the major findings of this century, regarding the mechanism of DNA replication, emphasizing what is known about the replication of mammalian DNA. J. Cell. Biochem. Suppls. 32/33:1-14, 1999.
Assuntos
Replicação do DNA/genética , Animais , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA/biossíntese , DNA/química , DNA/genética , Metilação de DNA , Humanos , Modelos Genéticos , Complexos Multienzimáticos/metabolismo , Conformação de Ácido Nucleico , Origem de Replicação/genética , Fatores de TempoRESUMO
Cruciform DNA has been implicated in the initiation of DNA replication. Recently, we identified and purified from human (HeLa) cells a protein, CBP, with binding specificity for cruciform DNA. We have reported previously that the CBP activity sediments at approximately 66 kDa in a glycerol gradient. Here, photochemical cross-linking studies and Southwestern analyses confirm that a 70 kDa polypeptide interacts specifically with cruciform DNA. Microsequence analysis of tryptic peptides of the 70 kDa CBP reveals that it is 100% homologous to the 14-3-3 family of proteins and shows that CBP contains the epsilon, beta, gamma, and zeta isoforms of the 14-3-3 family. In addition to polypeptides with the characteristic molecular mass of 14-3-3 proteins (30 and 33 kDa), CBP also contains a polypeptide of 35 kDa which is recognized by an antibody specific for the epsilon isoform of 14-3-3. Cruciform-specific binding activity is also detected in 14-3-3 proteins purified from sheep brain. Immunofluorescene studies confirm the presence of the epsilon, beta, and zeta isoforms of 14-3-3 proteins in the nuclei of HeLa cells. The 14-3-3 family of proteins has been implicated in cell cycle control, and members of this family have been shown to interact with various signaling proteins. Cruciform binding is a new activity associated with the 14-3-3 family.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Conformação de Ácido Nucleico , Proteínas/química , Tirosina 3-Mono-Oxigenase , Proteínas 14-3-3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Química Encefálica , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Núcleo Celular/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isomerismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos da radiação , Fotoquímica , Proteínas/isolamento & purificação , Ovinos , Raios UltravioletaRESUMO
There is now convincing evidence associating estrogens with an increased risk of some cancers. However, the absence of a complete correlation between estrogen receptor binding and the biological activity of these estrogens has suggested the possibility of other mechanisms of action. The effect on DNA replication of several hormones that are putatively involved in breast cancer was tested at a physiological concentration. The studies were conducted in a HeLa cell-free system by using a plasmid containing a specific mammalian origin of replication (DHFR ori beta) as template DNA. A series of related steroids produced an entire range of activity from enhancement to inhibition of in vitro DNA replication. These studies indicate a new possible target, which may help to better understand the effect of these hormones in breast cancer. Furthermore, the results show that this in vitro DNA replication system provides an evaluative assay for the effects of compounds on hormone-responsive cancers independent of some hormone receptors.
Assuntos
Replicação do DNA/efeitos dos fármacos , Estrogênios/farmacologia , Receptores de Estrogênio/análise , Sistema Livre de Células , DNA/biossíntese , Desoxirribonucleases de Sítio Específico do Tipo II , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Estriol/farmacologia , Estrona/farmacologia , Células HeLa , Humanos , Nucleotídeos/metabolismo , Progesterona/farmacologia , Testosterona/farmacologiaRESUMO
One of the fundamental characteristics that help define life is the ability to propagate. At the basest level in the act of propagation is replication of the genetic information as the databank and architectural plans for each particular life form. Thus propagation of life requires the replication of the genome--for the purposes of our review, eukaryotic DNA replication. In this critical review, we have chosen to present the issues and supporting experimental evidence in question-and-answer format. Over the past 3 to 4 years, the research domain of eukaryotic DNA replication has developed a new dynamism. This new force in discovery of the fundamental elements and mechanisms for DNA replication in higher eukaryotes has been propelled by accepted methodologies for mapping (identification) of origins of DNA replication, applicable to mammalian DNA replication, and by the discovery of the origin recognition complex (ORC) in yeast, which has served as a model in the search for the mammalian equivalent.
Assuntos
Replicação do DNA/genética , Replicação do DNA/fisiologia , Animais , HumanosRESUMO
Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.