Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Replication Origin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 829 records · Page 46Linked to original sources
PubMed1992

Initiation of DNA replication in ColE1 plasmids containing multiple potential origins of replication.

We have investigated the frequency of replication origin usage in bacterial plasmids containing more than one potential origin. Escherichia coli recA- cells were selectively transformed with pBR322 monomers, dimers, or trimers. Plasmid DNA was isolated and digested with a restriction enzyme that cut the monomer only once, and the replicative intermediates (RIs) were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis. Evidence for initiation outside the linearized plasmid was found only for oligomers. Moreover, in dimers, the intensity of the signal indicative for external initiation was equivalent to that reflecting internal initiation, whereas it was approximately twice as strong in trimers. To determine whether initiation could occur simultaneously at two origins in a single plasmid, we studied the replication of a neodimer in which both units could be unambiguously distinguished. The results showed that although both origins were equally competent to initiate replication, only one was active per plasmid. These observations strongly suggest that in ColE1 plasmids, replication initiates at a single site even when there are several identical potential origins per plasmid. In addition to the conventional two-dimensional gel patterns, novel specific patterns were observed with intensities that varied from one DNA sample to another. These unique patterns were the result of breakage of the RIs at a replication fork. This type of breakage changes both the mass and shape of RIs. When the entire population of RIs is affected, a new population of molecules is formed that may generate a novel pattern in two-dimensional gels.

DNA Replication↗
PubMed1988

Origin and direction of replication in mitochondrial plasmid DNAs of broad bean, Vicia faba.

Broad bean (Vicia faba) mitochondrial DNA (mtDNA) includes three circular plasmids: mt-plasmid 1 (1,704 ntp), mt-plasmid 2 (1,695 ntp) and mt-plasmid 3 (1,476 ntp). Partially replicated circular forms of these mt-plasmids have been observed in electron microscope preparations. Restriction enzymes that cleave either mt-plasmid 2 (but not mt-plasmids 1 and 3) or mt-plasmid 3 (but not mt-plasmids 1 and 2) were used to generate linear forms of partially replicated mt-plasmid 2 and mt-plasmid 3 molecules. Analyses of these linearized replicative intermediates, observed by electron microscopy, indicated that in both mt-plasmid 2 and mt-plasmid 3 replication originates at a specific location and proceeds in the same, single direction around the molecules. The replication origins of mt-plasmid 2 and mt-plasmid 3 map close to sequences that can fold into hairpin structures.

DNA Replication↗
PubMed1992

Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region.

A global analysis of the 230-kilobase-pair (kbp) human cytomegalovirus genome revealed three regions that were very rich in repeated sequences. The region with the highest content of inverted and direct repeats lies between 92,100 and 93,500 bp, upstream of the gene encoding the single-stranded DNA binding protein. Cloned restriction fragments containing this region were able to replicate when trans-acting factors were provided by virus infection in a transient replication assay. With this assay, the region between 92,210 and 93,715 bp on the viral genome was defined as the minimal replication origin, oriLyt. The sequence composition and repeats within oriLyt were used to divide the region into two domains that may be important in origin function. Sequences flanking either the left or right side of the minimal oriLyt contributed to efficient replication; however, these sequences were not essential for origin function. Thus, the region of the viral genome with the most striking concentration of direct and inverted repeats corresponds to the oriLyt of human cytomegalovirus.

Base Sequence↗
PubMed2004

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function.

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component of oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.

DNA Helicases↗
PubMed2000

Mitochondria--tool for taxonomic identification of yeasts from Saccharomyces sensu stricto complex.

Mitochondrial genomes of Saccharomyces and close relatives previously used for transplacement of mitochondria to S. cerevisiae were examined. The origins of replication in mitochondrial DNA, the presence of nuclear and mitochondrial polymorphic loci and the ability to produce mitochondrial respiration-deficient mutants were used to reclassify some collection yeasts and to assign others into four separate subgroups. The first included isolates identical to Saccharomyces cerevisiae (S. italicus, S. oviformis, S. chevalieri and S. capensis) which possess 5 or more replication origins. The second group consists of S paradoxus (var douglasii) mitochondrial genome with the equal number of ori sequences but incompatible mitochondria. The third group represents Saccharomyces sensu stricto petite-positive species (S. carlsbergensis, S. heterogenicus, S. uvarum, S. willianus) with 1-2 origins of replication significantly different from S. cerevisiae. In addition, the locus between tRNA(fMet) and tRNA(Pro) is about one-half of the 1400 bp members of S. cerevisiae complex. The last group includes isolates that do not belong to Saccharomyces sensu stricto group as they are petite-negative and devoid of any S. cerevisiae-like replication origins.

DNA, Fungal↗
PubMed1983

Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA.

Nuclear factor I, a 47-kilodalton protein, purified from nuclear extracts of uninfected HeLa cells, is involved in the initiation and possibly the elongation of replicating adenovirus (Ad) DNA in vitro. The binding of nuclear factor I to DNA has been monitored by a filter binding assay of nuclear factor I to DNA has been monitored by a filter binding assay using plasmid pLA1 DNA, which contains a 3,290 base-pair fragment derived from the left-hand terminus (coordinates, 0-9.4 map units) of Ad serotype 5 DNA. Nuclear factor I binds selectively to a double-stranded fragment spanning nucleotides 0-451 to the Ad genome. The retention of the 451-base-pair DNA fragment-nuclear factor I complex on nitrocellulose filters does not require Mg2+ or ATP and is resistant to high ionic strength. DNase I protection experiments revealed that nuclear factor I binds to a nucleotide sequence located at position 17-48, close to the terminus of Ad DNA. This 32-nucleotide sequence contains four "consensus" sequences present in various serotypes of Ad DNA and is capable of forming higher ordered structures. The role of nuclear factor I and this DNA sequence in the generation of Ad preterminal protein-dCMP initiation complex is discussed.

Adenoviridae↗
PubMed1992

Inhibition of structural changes in the simian virus 40 core origin of replication by mutation of essential origin sequences.

Mutation of the simian virus 40 (SV40) origin of replication (ori) has revealed the presence of three critical domains needed for DNA replication. The outer two domains, the AT tract and early palindrome element (EP), colocalize with DNA regions that become structurally altered in the presence of the SV40 large tumor antigen (T antigen) and ATP. Mutations within each domain were examined for their effect on the distortion of ori DNA by T antigen, as assayed by the sensitivity of DNA to KMnO4 oxidation. We have found that mutations in the AT tract that inhibit SV40 DNA replication also inhibit the distortion of the AT tract. Similarly, mutations in the EP inhibited the generation of structural changes in this element by T antigen. Although AT-tract mutations or mutations on the late side of ori affected structural changes only in the AT tract, certain EP mutations or mutations on the early side of ori also inhibited AT-tract distortion. Mutation of the flanking regions did not significantly affect either the affinity of T antigen for ori or the rate of binding to ori. We conclude from these results that the primary function of the flanking ori domains is to undergo structural changes required during the initiation of SV40 DNA replication. Moreover, our results suggest that the efficiency of replication initiation is significantly affected by the degree to which the flanking elements undergo a structural transition.

Antigens, Polyomavirus Transforming↗
PubMed1990

The origin binding protein of herpes simplex virus 1 binds cooperatively to the viral origin of replication oris.

The origin binding protein (OBP) or herpes simplex virus 1 has been expressed in Escherichia coli and used to study the role of multiple OBP binding sites in the herpes simplex virus #1 origin of replication, oris. Our results showed that the sequence CGTTCGCACTT was required for the binding of OBP to duplex DNA with high affinity. The minimal oris contains three repeats of this sequence or close derivatives thereof. Filter binding experiments have demonstrated that specific binding occurs to two of these repeats, box I and box II. An investigation using the DNase I footprinting technique revealed that the binding of OBP to box I and box II was cooperative and led to the formation of a highly organized complex in which the entire oris sequence was induced. We observed furthermore that the AT-rich sequence of the oris dyad was readily accessible to macromolecules even in the OBP.oris complex. The DNase I cleavage pattern of this sequence was, however, altered radically, indicating that a significant conformational change had occurred. A tentative structural model for the OBP-oris interaction is discussed on the basis of these observations.

Base Sequence↗
PubMed1994

An asymmetric nucleotide in the parvoviral 3' hairpin directs segregation of a single active origin of DNA replication.

The 3' telomere of the linear single-stranded DNA genome of minute virus of mice (MVM), a murine parvovirus, can assume a complex hairpin structure. This contains a stem in which there is a mismatched 'bubble' sequence where a GA doublet opposes a GAA triplet. During replication, this hairpin is copied to form an imperfect palindrome which bridges adjacent genomes in a dimer duplex intermediate, leaving the two 'bubble' sequences embedded in potential replication origins on either side of the axis of symmetry. Such junctions are resolved asymmetrically in vitro in a DNA synthetic reaction which requires the viral initiator protein NS1. We show that the sequence surrounding the doublet is a potent origin, but the analogous region containing the triplet is completely inactive. The active origin is approximately 50 bp long, extending from an Activated Transcription Factor binding site at one end to a position some 7 bp beyond the major initiation site, to which NS1 ultimately becomes covalently attached. The actual sequence of the GA doublet is unimportant, but insertion of any third nucleotide here inactivates the origin, indicating that it represents a critical spacer element. Segregation of this asymmetry, therefore, allows the virus to confine replication initiation to one particular telomeric configuration.

Base Sequence↗
PubMed1990

Identification of the lytic origin of DNA replication in human cytomegalovirus by a novel approach utilizing ganciclovir-induced chain termination.

Infection with human cytomegalovirus in the presence of the antiviral nucleotide analog ganciclovir results in continuing low-level viral DNA synthesis and the accumulation of relatively small fragments of double-stranded progency DNA. These fragments consistently proved to represent amplification of sequences from only one small section of the viral genome (EcoRI-V) lying near the center of the unique L segment. Further mapping revealed that the viral sequences represented in these fragments occurred in gradients of abundance that decreased in both directions from a point near 0.35 to 0.4 map unit. The proportion of amplified sequences increased with both time after infection and dosage of ganciclovir used. We conclude that the primary lytic cycle replication origin of human cytomegalovirus lies within a 3- to 4-kb region immediately upstream and to the right of the promoter for the single-stranded DNA-binding protein (DB140). The amplified origin-containing DNA molecules appeared to arise by continuing rounds of bidirectional initiation on truncated fragments of the genome that were generated as a result of chain termination effects induced by the incorporation of ganciclovir into the viral DNA. Inspection of the DNA sequence in the vicinity of ori-Lyt revealed a large complex upstream region that may be a noncoding intergenic domain and that bears no homology to any previously described herpesvirus origin. This 2.5-kb region includes many duplicated and inverted sequences, together with consensus CRE/ATF and other transcription factor-binding sites, and an interesting set of 23 copies of an interspersed decamer consensus element AAAACACCGT that is also conserved at the equivalent locus in simian cytomegalovirus. This work represents the first identification of an origin domain in a cytomegalovirus genome and is the first demonstration of a bidirectional mechanism for any herpesvirus lytic cycle origin.

Base Sequence↗
PubMed2006

Identification of new human origins of DNA replication by an origin-trapping assay.

Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr virus latent origin oriP. oriP contains two elements, an origin (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G(1) phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.

Base Sequence↗
PubMed1988

Identification of the origin and direction of replication of the broad-host-range plasmid pLS1.

The replication origin of the fully sequenced broad-host-range streptococcal plasmid pLS1 has been determined by the use of an in vitro replication system prepared from Escherichia coli, a host in which the plasmid can be established. Replicative intermediates were isolated from reaction mixtures that contained dideoxythymidine triphosphate, thus limiting the average extent of in vitro synthesis. Analysis of HinfI-cleaved intermediates demonstrated that the origin of replication is included within a 443-bp fragment. Replication proceeds unidirectionally in the same direction as transcription of plasmid mRNAs. Isolation of deletion derivatives allowed us to define the replication origin of pLS1 within a region of 284 bp. Replication of pLS1 occurs through single-stranded intermediates by a rolling circle mechanism. Cleavage of supercoiled plasmid DNAs with endonuclease S1 followed by restriction mapping, allowed the positioning of three major specific S1 sites in regions of high potential to form secondary structures. One of these inverted repeats is located in the region where the origin of replication of pLS1 has been defined.

Base Sequence↗
PubMed1988

In vitro transcription of the origin region of replication of the Escherichia coli chromosome.

Transcription was carried out in vitro by RNA polymerase on supercoiled minichromosome DNA containing the replication origin of the Escherichia coli chromosome (oriC) and the products were analyzed. Leftward transcription starting from the 16-kDa gene promoter located adjacent to the right of oriC was inhibited by the DnaA protein, a protein essential for initiation of replication. This inhibition is due to binding of DnaA protein to the 9-base pair sequence (dnaA box) located just upstream of the 16-kDa gene promoter. The inhibition was observed at levels of DnaA protein and RNA polymerase comparable to those required for replication of oriC plasmids in vitro and was abolished at high levels of RNA polymerase. These results suggest that a balance of DnaA protein and RNA polymerase is important in regulating transcription and that this regulation operates under conditions that allow initiation of replication. The transcription traversed oriC with very few transcripts terminating before or within it. DnaA protein did not affect the chain length of these transcripts. Furthermore, we identified leftward transcripts which start close to position 177 in the oriC sequence.

Bacterial Proteins↗
PubMed1994

Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication.

Adenovirus preterminal protein (pTP) exists as a heterodimer with the viral DNA polymerase (AdPol) and becomes covalently linked to a dCMP residue during initiation of DNA replication. The in vivo phosphorylation of pTP could be demonstrated when pTP is overproduced using recombinant vaccinia viruses, or by a large scale metabolic labeling of adenovirus 2 (Ad2)-infected HeLa cells. Phosphoserine was the only phosphoamino acid obtained by acid hydrolysis of 32P-labeled pTP immunoprecipitated from metabolically labeled HeLa cells infected with either Ad2 or recombinant vaccinia virus. Tryptic peptide maps of pTP expressed using recombinant vaccinia virus system in HeLa cells revealed that phosphorylation of pTP occurred on multiple sites. Dephosphorylation of pTP with calf intestinal alkaline phosphatase resulted in a significant decrease in its activity in the in vitro DNA replication initiation assays. Further characterization of the phosphatase-treated pTP indicated that although dephosphorylation did not affect its interaction with AdPol, the specific recognition of the DNA replication origin by pTP was significantly reduced as determined by gel electrophoresis-based DNA mobility shift assays.

Adenoviruses, Human↗
PubMed1990

Replication of bovine papillomavirus type 1 DNA initiates within an E2-responsive enhancer element.

When bovine papillomavirus transforms cells in vitro, it maintains its genome as a multicopy nuclear plasmid. Plasmid DNA extracted from such transformed cells was analyzed by the two-dimensional gel electrophoresis technique of Brewer and Fangman (B. Brewer and W. Fangman, Cell 51:463-471, 1987). The replication intermediates detected in these assays were found to be the sums of the oligomeric and monomeric forms of the replicating plasmids. The multimeric DNAs were shown by field inversion gel electrophoresis and partial restriction digestion to be head-to-tail concatemers of the monomeric forms. Furthermore, the multimers progressed in size by steps of one monomer, indicating that they did not arise by replication segregation mistakes of the unit length, which would predict a ladder spaced by integrals of two monomers. To map the plasmid DNA replication origin, the replication intermediates of the monomers were isolated by successive sucrose gradient centrifugation and then examined by the two-dimensional gel electrophoresis method. The patterns detected show that bovine papillomavirus type 1 replicates in these cells bidirectionally and that one replication origin site in the viral genome is utilized. By employing several restriction enzymes and specific viral DNA probes to dissect the replication intermediates, we were able to map the origin of initiation site with some precision. The initiation site, which maps to bovine papillomavirus type 1 DNA position 7730 +/- 100 bp, places the origin within that region of the viral upstream regulatory region which contains the major cluster of transcription factor E2-binding sites, E2RE1. Thus, the actual viral plasmid origin of replication maps near, but outside, genetic elements previously shown to be important for plasmid maintenance.

Animals↗
PubMed2003

Two separate replication modes of the bovine papillomavirus BPV1 origin of replication that have different sensitivity to p53.

We have shown previously that transient amplificational replication of reporter plasmids that carry the papillomavirus origin of replication is efficiently blocked by p53 protein in several cell lines. We demonstrate now that the replication of stably maintained episomal bovine papillomavirus BPV1 URR (upstream regulatory region) reporter plasmid is not sensitive to p53. In addition, these two replication modes--initial transient amplificational replication and stable maintenance replication of essentially the same BPV1 URR reporter plasmid--can take place in the same cells, where amplificational replication does not interfere with the stable maintenance replication. These data suggest that BPV1 replicons could follow two clearly separable replication mechanisms during initial amplification and during stable extrachromosomal maintenance.

Animals↗
PubMed1983

Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication.

Soluble protein fractions from Escherichia coli dnaA+ cells but not dnaA temperature-sensitive cells replicate plasmids containing the E. coli chromosomal origin of replication (oriC). Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC. From a strain (constructed in vitro) that overproduces the dnaA protein more than 200-fold, the 52,000-dalton polypeptide was purified to near homogeneity. Although the protein tends to aggregate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication. It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Millipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence. In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis.

Bacterial Proteins↗
PubMed2005

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells.

Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy.

Adenosine Triphosphatases↗