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At least 937 records · Page 52Linked to original sources
PubMed2004

Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli.

In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC-DNA and DnaC-DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6*DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.

Adenosine Diphosphate↗
PubMed2004

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication.

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1979

Membrane enrichment of genetic markers close to the origin and terminus during the deoxyribonucleic acid replication cycle in Bacillus subtilis.

A temperature-sensitive Bacillus subtilis initiation mutant was used to achieve one cycle of synchronized deoxyribonucleic acid (DNA) replication. Markers near the origin of replication and the terminus were assayed for association with the cell membrane at intervals during the DNA replication cycle. DNA near the origin and terminus was found to be enriched in the membrane fraction throughout the DNA replication cycle. The magnitude of membrane enrichment or origin and terminus markers varied coincidentally, possibly as a consequence of incubating the cells at 45 degrees C.

Bacillus subtilis↗
PubMed2006

Mouse pre-replicative complex proteins colocalise and interact with the centrosome.

Initiation of eukaryotic DNA replication is achieved by the sequential binding of different proteins to origins of DNA replication. Using EGFP-tagged initiator proteins and immunofluorescence techniques we found that most of the ORC and the MCM subunits are localised at centrosomes and are colocalised with the polo-like protein kinase, Plk1. Yeast two-hybrid studies revealed interactions of Plk1 with the Mcm2 as well as the Orc2 protein. Co-immunoprecipitations showed an interaction of Plk1 with Mcm2 as well as interactions of gamma-tubulin with Mcm3 and Orc2, respectively. An in vitro phosphorylation assay showed that the Orc2 protein is a substrate of Plk1. Depletion of Orc2 and Mcm3 by siRNA leads to an inhibition of cell proliferation, an altered cell cycle distribution as well as to multinucleated cells with insufficiently organised microtubules. These results indicate an important role of the MCM and ORC proteins in mitosis besides their described role in the establishment of the pre-replicative complex.

Animals↗
PubMed1984

Replication of adenovirus mini-chromosomes.

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear mini-chromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.

Adenoviridae↗
PubMed2006

DNA replication timing: random thoughts about origin firing.

Regions of metazoan genomes replicate at defined times within S phase. This observation suggests that replication origins fire with a defined timing pattern that remains the same from cycle to cycle. However, an alterative model based on the stochastic firing of origins may also explain replication timing. This model assumes varying origin efficiency instead of a strict origin-timing programme. Here, we discuss the evidence for both models.

Animals↗
PubMedDate not supplied

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro.

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.

Amino Acid Sequence↗
PubMed1983

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.

Adenosine Triphosphatases↗
PubMed1992

DNA binding activity is required for EBNA 1-dependent transcriptional activation and DNA replication.

Epstein-Barr virus nuclear antigen 1 (EBNA 1) has been shown to be a sequence-specific DNA binding protein that is required for the replication of episomal elements carrying the viral origin of DNA replication, oriP, as well as for the activation of a specific transcriptional enhancer. We have constructed and analyzed a series of deletion and nonsense mutants in a cloned copy of the EBNA 1 gene and have tested mutant peptides for the ability (a) to bind to a synthetic oligonucleotide containing a consensus EBNA 1 binding site, (b) to activate the EBNA 1-specific enhancer, and (c) to drive replication of an oriP-bearing plasmid in a transient replication assay. The presence of a DNA binding domain in the carboxy-terminal third of the protein was confirmed. Interestingly, neither the acidic tail nor the Gly-Ala copolymer of EBNA 1 contributes significantly to binding. In addition to sequences in the carboxy-terminal portion of the protein, our data indicate that sequences in the amino-terminal portion of the polypeptide affect the binding of EBNA 1 to its target sequence. Further, we show that EBNA 1 binds to its recognition sequence as a dimer. Results of transient expression assays indicate that the ability of EBNA 1 species to activate the transcriptional enhancer and to drive the replication of oriP plasmids is directly dependent on the ability of the polypeptides to bind to the EBNA 1 consensus binding sequence.

Antigens, Viral↗
PubMed2001

Ori-somes, nucleoprotein complexes descending from origin regions of animal chromosomal DNA replication. A micromorphological study.

Micromorphology of nucleoprotein (NP) complexes designated according to their descent and shape as Ori-somes is presented. These NP complexes of three different types harbor molecules of cytoplasmic "small" polydisperse DNA, which descend from origin regions of chromosomal DNA replication and are equipped, as shown previously, with early DNA-synthesizing activities. By negative staining the Ori-somes are visualized as particles of irregular shape, sometimes of a subunit-like structure. Micromorphological differences in size and structural compactness noted among individual Ori-somes are dependent on their type similarly as earlier shown physico-chemically and biochemically. Such differences were also confirmed by two different spreading techniques. The most unravelled structures with electron diffuse centers belong to Ori-somes of component B associated with most active DNA synthesis. In contrast, the Ori-somes of components A and C, associated with pronounced RNA synthesis, revealed large electron-dense centers. The incidence of replicative structures present in Ori-somes corresponds with the level of their DNA-synthesizing activities.

Animals↗
PubMed1975

Unidirectional replication of a minority of polyoma virus and SV40 DNAs.

Poyoma DNA replication is initiated predominatly at a site which is 29% from the EcoRI cleavage site. Molecules replicating from this site, after digestion with EcoRI appear as linear structures with a double stranded loop centered at the origin of replication. These forms constitute 90% of all replicating intermediates. Approx. 10% of the replicating intermediates of polyoma and SVP40 DNAs occur as Y-forms after treatment with EcoRI. These structures have probably resulted from unidirectional replication initiated at an additional origin of DNA replication which is located near the EcoRI cleavage site on the genomes of these viruses.

Animals↗
PubMed1995

Domains of the BPV-1 E1 replication protein required for origin-specific DNA binding and interaction with the E2 transactivator.

The viral E1 and E2 proteins are required for replication of bovine papillomavirus type 1 DNA. Both proteins bind as a complex to the replication origin, which consists of an E1 binding site flanked on either side by E2 binding sites. The E1 protein has properties common to replication initiator proteins such as sequence-specific origin binding and DNA helicase activities. The E2 protein is a transcriptional transactivator that forms a complex with the E1 protein and enhances binding of E1 to the replication origin. We have mapped the regions of the E1 protein required for sequence-specific DNA binding, for cooperative binding with the E2 protein to the origin region, and for interaction with the E2 protein. These studies demonstrate that a region between amino acids 162 and 378 of the E1 protein is important for origin-specific DNA binding. The C-terminal half of the E1 protein is required in addition to the DNA binding domain (residues 162 to 605) for cooperative binding to the origin with the E2 protein. Binding studies confirmed that this region is also required for efficient complex formation with the E2 protein.

Amino Acid Sequence↗
PubMed2004

Essentiality of the early transcript in the replication origin of the lactococcal prolate phage c2.

The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter P(E)1 abolished origin function. Introduction of mutations into P(E)1 which did not eliminate promoter activity or replacement of P(E)1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of P(E)1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the P(E)1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3' end of the DNA sequence coding for the transcript. The P(E)1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of P(E)1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

Bacteriophages↗
PubMed2005

Susceptibility to superhelically driven DNA duplex destabilization: a highly conserved property of yeast replication origins.

Strand separation is obligatory for several DNA functions, including replication. However, local DNA properties such as A+T content or thermodynamic stability alone do not determine the susceptibility to this transition in vivo. Rather, superhelical stresses provide long-range coupling among the transition behaviors of all base pairs within a topologically constrained domain. We have developed methods to analyze superhelically induced duplex destabilization (SIDD) in genomic DNA that take into account both this long-range stress-induced coupling and sequence-dependent local thermodynamic stability. Here we apply this approach to examine the SIDD properties of 39 experimentally well-characterized autonomously replicating DNA sequences (ARS elements), which function as replication origins in the yeast Saccharomyces cerevisiae. We find that these ARS elements have a strikingly increased susceptibility to SIDD relative to their surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do their immediately surrounding sequences, making them more than 2,000 times easier to open. Statistical analysis shows that the probability of this strong an association between SIDD sites and ARS elements arising by chance is approximately 4 x 10(-10). This local enhancement of the propensity to separate to single strands under superhelical stress has obvious implications for origin function. SIDD properties also could be used, in conjunction with other known origin attributes, to identify putative replication origins in yeast, and possibly in other metazoan genomes.

Journal Article↗
PubMed1978

Nucleotide sequence of the origin of replication in bacteriophage phiX174 RF DNA.

The gene A protein of bacteriophage phiX174 has been used in vitro to convert phiX RFI DNA into the relaxed RFII form by nicking the viral strand. The nucleotide sequence at the 3' end of the nick has been determined as -- T G C T C C C C C A A C T T Goh. This sequence gives the exact position of the origin of phiX RF DNA replication.

Base Sequence↗