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At least 919 records · Page 51Linked to original sources
PubMed1991

Analysis of polyomavirus enhancer-effect on DNA replication and early gene expression.

The polyomavirus enhancer is located adjacent to the origin of DNA replication and the transcriptional promoters. It has a cis-acting essential function in the initiation of both viral DNA synthesis and early transcription. The enhancer is activated by the binding of protein factors to specific sites in DNA. Mutants with deletions of the A- or the B-segment of the enhancer were constructed. In mouse 3T6 cells, the transcription of the viral early region was significantly decreased by deletion of the A-segment, but not by deletion of the B-segment. In contrast, the two deletions had a similar, moderately negative effect on viral DNA synthesis. However, the presence of DNA with a wild-type enhancer in doubly transfected cells resulted in very strong interference with the replication, but not with the transcription, of deletion mutant DNA. DNA of the deletion mutants were subjected to site-directed mutagenesis of the remaining enhancer segment. Three non-viable mutants were isolated. All three had base-pair changes in the A-segment affecting immediately adjacent binding sites of cellular protein factors. The mutants had lost the enhancer activity on the early promoter, but only one of them with multiple base substitutions had lost the capacity of DNA replication. Together, the results suggest that different aspects of enhancer function determine the activity in initiation of transcription and replication.

Amino Acid Sequence↗
PubMedDate not supplied

Role of DNA replication in the repression of silent mating type loci in yeast.

A putative origin of DNA replication is associated with the DNA sequences necessary for the repression of silent mating type loci in yeast. These sequences lie about a kilobase away from the affected promoters, so the repression must act at a distance. We show here that DNA replication is required for the onset of repression.

Animals↗
PubMed2005

Site-directed mutagenesis of large DNA palindromes: construction and in vitro characterization of herpes simplex virus type 1 mutants containing point mutations that eliminate the oriL or oriS initiation function.

Technical challenges associated with mutagenesis of the large oriL palindrome have hindered comparisons of the functional roles of the herpes simplex virus type 1 (HSV-1) origins of DNA replication, oriL and oriS, in viral replication and pathogenesis. To address this problem, we have developed a novel PCR-based strategy to introduce site-specific mutations into oriL and other large palindromes. Using this strategy, we generated three plasmids containing mutant forms of oriL, i.e., pDoriL-I(L), pDoriL-I(R), and pDoriL-I(LR), containing point mutations in the left, right, and both copies, respectively, of the origin binding protein (OBP) binding site (site I) which eliminate OBP binding. In in vitro DNA replication assays, plasmids with mutations in only one arm of the palindrome supported origin-dependent DNA replication, whereas plasmids with symmetrical mutations in both arms of the palindrome were replication incompetent. An analysis of the cloned mutant plasmids used in replication assays revealed that a fraction of each plasmid mutated in only one arm of the palindrome had lost the site I mutation. In contrast, plasmids containing symmetrical mutations in both copies of site I retained both mutations. These observations demonstrate that the single site I mutations in pDoriL-I(L) and pDoriL-I(R) are unstable upon propagation in bacteria and suggest that functional forms of both the left and right copies of site I are required to initiate DNA replication at oriL. To examine the role of oriL and oriS site I in virus replication, we introduced the two site I mutations in pDoriL-I(LR) into HSV-1 DNA to yield the mutant virus DoriL-I(LR) and the same point mutations into the single site I sequence present in both copies of oriS to yield the mutant virus DoriS-I. In Vero cells and primary rat embryonic cortical neurons (PRN) infected with either mutant virus, viral DNA synthesis and viral replication were efficient, confirming that the two origins can substitute functionally for one another in vitro. Measurement of the levels of oriL and oriS flanking gene transcripts revealed a modest alteration in the kinetics of ICP8 transcript accumulation in DoriL-I(LR)-infected PRN, but not in Vero cells, implicating a cell-type-specific role for oriL in regulating ICP8 transcription.

Animals↗
PubMed1993

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.

Antigens, Viral↗
PubMed1991

Mutations in a herpes simplex virus type 1 origin that inhibit interaction with origin-binding protein also inhibit DNA replication.

The herpes simplex virus type 1 genome contains three origins of replication: OriL and a diploid OriS. The origin-binding protein, the product of the UL9 gene, interacts with two sites within OriS, box I and box II. A third site, box III, which is homologous to boxes I and II, may also be a binding site for the origin-binding protein. Mutations in these three sites significantly reduce OriS-directed plasmid replication measured in transient replication assays. The reduction in replication efficiency of the mutants correlates well with the decrease in the ability to bind to the origin-binding protein, as determined by Elias et al. (P. Elias, C. M. Gustafsson, and O. Hammarsten, J. Biol. Chem. 265: 17167-17173, 1990). The effect of multiple mutations in boxes I, II, and III on plasmid replication suggests that there are multiple binding sites in OriS for the origin-binding protein. These studies indicate that proper interaction of the origin-binding protein with the OriS sequence is essential for OriS-directed DNA replication.

Animals↗
PubMed1986

Autonomous replicating sequences from intron of human ras gene in a simian virus 40 T antigen dependent system.

Of the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.

Antigens, Polyomavirus Transforming↗
PubMedDate not supplied

[Attachment of DNA loops to an artificial matrix does not affect the origin of replication initiation in early development of African clawed frog].

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.

Animals↗
PubMed1993

The transactivator proteins VP16 and GAL4 bind replication factor A.

Many transcription factors can activate the initiation of DNA replication. We have used affinity chromatography to show that the acidic activation domains of the transcription factors VP16, GAL4, and p53 each bind selectively to human and yeast replication factor A (RPA). The binding is direct and to the largest subunit of the trimeric RPA complex, RPA-1. Mutations in VP16 that reduce the ability of GAL4-VP16 to activate polyomavirus DNA replication also compromise the binding of VP16 to RPA. We suggest that transcription factors may interact with RPA either to stabilize single-stranded DNA at a replication origin or to recruit DNA polymerase alpha to the replication initiation complex.

DNA-Binding Proteins↗
PubMed2004

DNA replication licensing.

The DNA replication licensing system ensures that chromosomal DNA is replicated precisely once before cell division occurs. A DNA helicase must be loaded on origin DNA for replication to initiate. Considerable evidence suggests that the MCM complex acts as a replicative helicase in eukaryotes. When the MCM complex is loaded on the chromatin, the replication origin is formally defined as being licensed for replication. Licensing takes place several hours before origins are activated to undergo replication in S-phase. Genetic and biochemical studies show that the licensing process is well conserved in eukaryotes. Cyclin Dependent Kinases (CDKs), the master regulators of the cell cycle, coordinate the initiation of the two key cell cycle events, replication of DNA and its segregation at mitosis. Eukaryotes have developed complex regulatory mechanisms to ensure that origin licensing is coordinated with these events so that genome integrity is preserved during successive cell divisions.

Animals↗
PubMed1990

Unique hairpin structures occurring at the replication origin of phage G4 DNA.

Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure. The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on. Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined. It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions. The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein.

Base Sequence↗
PubMed1982

Fragment spanning the SV40 replication origin is the only DNA sequence required in cis for viral excision.

A 311-base pair fragment containing the SV40 origin of replication was linked to the chicken thymidine kinase gene on a recombinant plasmid. This molecule was transfected into human 143 thymidine kinase-deficient (TK-) cells, and colonies positive for thymidine kinase were selected. When cell lines derived from these colonies were fused to permissive simian cells that produce SV40 T antigen, the recombinant plasmid excised itself from the human cellular genome and replicated with a high copy number per cell. These results show that this segment of the viral genome is the only sequence required in cis to mediate SV40 excision and replication upon fusion to permissive cells. In addition, we have shown that excised plasmids apparently identical to the input DNA can be efficiently rescued in Escherichia coli. SV40 excision and replication may therefore be useful for the recovery of cloned genes from eukaryotic cells.

Animals↗
PubMed1996

The plasmid RK2 initiation protein binds to the origin of replication as a monomer.

The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (iterons) present at the plasmid replication origin to initiate DNA replication. Purified TrfA protein is largely in the form of a dimer, and using a dimerization test system that involves the fusion of the amino-terminal domain of the lambda cI repressor protein to TrfA, we show that the TrfA protein forms dimers in vivo. Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and different sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation. The results of gel mobility shift assays using in vitro or in vivo formed heterodimers indicated that the TrfA protein binds to the iteron DNA as a monomer. Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the chromatographic position of the monomeric form demonstrated significant DNA binding activity. These results indicate that only the monomer form of the TrfA protein is active for binding to the iterons at the RK2 replication origin.

Bacterial Proteins↗
PubMed2004

Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication.

The chromosomal origin and terminus of replication are precisely localized in bacterial cells. We examined the cellular position of 112 individual loci that are dispersed over the circular Caulobacter crescentus chromosome and found that in living cells each locus has a specific subcellular address and that these loci are arrayed in linear order along the long axis of the cell. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. Thus, the remarkable organization of the chromosome is being established while DNA replication is still in progress. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites.

Caulobacter crescentus↗
PubMed2005

Overexpression of ORC subunits and increased ORC-chromatin association in transformed mammalian cells.

The origin recognition complex (ORC) is a conserved heterohexamer required for the formation of pre-replication (pre-RC) complexes at origins of DNA replication. Many studies of ORC subunits have been carried out in transformed human cell lines but the properties of ORC in primary cells have not been addressed. Here, we compare the expression levels and chromatin-association of ORC subunits in HeLa cells to the primary human cell line, WI38, and a virally transformed derivative of WI38, VA13. ORC subunits 2 and 4 were highly overexpressed in both HeLa and VA13, whereas ORC1 levels were elevated in VA13 but considerably higher in HeLa cells. Cellular extraction revealed that the proportion of ORC2 and ORC4 subunits bound to chromatin was similar in all three cell lines throughout the cell-cycle. In contrast, very little ORC1 was associated with chromatin after extraction of primary WI38 cells, whereas the majority of overexpressed ORC1 in both HeLa and VA13 co-fractionated with chromatin throughout the cell-cycle. Although none of the cell lines displayed significant changes in the levels or chromatin-association of ORC during the cell-cycle, the chromatin-associated fraction of ORC1 displayed an increase in apparent molecular weight during S-phase. Similar experiments comparing immortalized CHO cells to an isogenic virally transformed derivative revealed no changes in levels of ORC subunits but an increase in the proportion of all three ORC subunits associated with chromatin. These results demonstrate a complex influence of cellular immortalization and transformation properties on the expression and regulation of ORC subunits. These results extend the potential link between cancer and deregulation of pre-RC proteins, and underscore the importance of considering the transformation status of cell lines when working with these proteins.

Animals↗
PubMed2001

The nuclear location and chromatin organization of active chorion amplification origins.

It remains unclear how certain regions on metazoan chromosomes are selected to initiate DNA replication. In recent years a number of origins of DNA replication have been mapped, but there is still no DNA consensus for predicting where replication will initiate. Evidence suggests that the higher order structure of the nucleus and chromosome influences origin activity. Chromosomal DNA replication is proposed to occur in special compartments in the nucleus called replication foci. Foci in different regions of the nucleus initiate replication at different times of S-phase, suggesting nuclear position may contribute to where and when replication begins. Here we test the contribution of nuclear compartments for well-defined origins, those involved in amplification of the chorion (eggshell) genes during Drosophila oogenesis. The results of three-dimensional confocal microscopy indicate that chorion DNA replication origins are highly active in diverse positions within the nucleus. We also find that chorion replication origins inserted at ectopic chromosomal sites can amplify highly in diverse nuclear locations distinct from the endogenous loci, including when they are buffered against genomic position effects. We used fluorescence in situ hybridization to analyze chromosome structure during amplification. Contrary to the replication factory model, we find no evidence for spooling of DNA toward a replication center. We discuss the implications of these results for understanding the role of higher order structure in amplification and chromosome duplication.

Animals↗
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↗