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At least 1,207 records · Page 67Linked to original sources
PubMed1999

Subcellular localization of plasmids containing the oriC region of the Escherichia coli chromosome, with or without the sopABC partitioning system.

Fluorescence in situ hybridization (FISH) analysis has revealed the subcellular localization of specific chromosomal segments and plasmid molecules during the cell division cycle in Escherichia coli: the replication origin (oriC) segments on the chromosome are localized at nucleoid borders, and actively partitioning mini-F plasmid molecules are localized at the 1/4 and 3/4 positions of the cell. In contrast, mini-F plasmid molecules lacking the sopABC segment are randomly localized in cytoplasmic areas at cell poles. In this study, we analysed the subcellular localization of an oriC plasmid that contains the minimum E. coli chromosomal replication origin and its flanking regions. These oriC plasmid molecules were mainly localized in cytosolic areas at cell poles. On the other hand, oriC plasmid DNA molecules carrying the sopABC segment of F plasmid were localized at cell quarter sites, as were actively partitioning mini-F plasmid DNA molecules. Therefore, we conclude that oriC itself and its flanking regions are not sufficient for positioning the replication origin domain of the E. coli chromosome within the cell.

Bacterial Proteins↗
PubMed1977

The frequency of P1 transduction of the genes of Escherichia coli as a function of chromosomal position: preferential transduction of the origin of replication.

The frequencies with which the generalized transducing phage P1 transduced 26 selected markers on the E. coli. chromosome were measured. The frequencies were found to vary relative to argH+ = 1 from a maximum of 6.8 near the origin of replication to a minimum of 0.23 for a marker not far from the terminus. The low frequencies obtained for some markers were shown not to result from poor expression under the selective conditions employed. When plotted as a function of marker position on the chromosome the frequencies were found to exhibit a series of peaks and troughs which correspond to those in gene density noted by Bachmann et al. (1976). The possible relationship of these results to the structure of the E. coli chromosome and to the mechanism of generalized transduction are discussed.

Chromosome Mapping↗
PubMed1984

A 140 base-pair DNA segment from the kanamycin resistance region of plasmid R1 acts as an origin of replication and promotes site-specific recombination.

A 140 base-pair DNA segment situated just upstream of the kanamycin resistance gene of transposon Tn2350, a transposon carried by the plasmid R1, was found to act as an origin of replication and allow autonomous replication of a plasmid composed only of the segment and of the tetracycline resistance gene of pBR322. This segment also promotes site-specific recombination: when cloned in pBR322 it promotes multimer formation in a recA- strain. If two copies are cloned on the same plasmid they promote either deletion or inversion of the intervening region, depending on their orientation relative to each other. DNA gyrase seems to be involved in this process since the inversion rate, in a plasmid carrying sequences in opposite orientations, varies in different nalidixic acid-resistant strains (gyr A mutants) independently isolated.

Base Composition↗
PubMed1985

Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution.

Large tumor (T) antigen and its bound multimeric states are positioned by scanning transmission electron microscopy (STEM) within a few base pairs at control sequences of the simian virus 40 DNA origin of replication region. Proximal and distal edge positions for each multimer group match the end positions of previously mapped fragments protected from DNase cleavage. Since chance correspondence is shown to be extremely unlikely, STEM mass measurements, obtained concurrently with STEM map positions, indicate that the DNase fragments arise from bound monomers, dimers, trimers, and tetramers in binding region II and monomers, dimers, and trimers in binding region I. Simultaneous binding of seven monomer-equivalent masses is observed, three in region I and four in region II, with an ordered and interpretable mass distribution in the plane of the foil. Although this observation does not prove that the six G-A-G-G-C and one T-A-G-G-C sequences, similarly distributed, function as recognition sequences for T-antigen monomer, it provides strong support for such a model. The stable existence in solution of low-and intermediate-mass structures, observed at lower T-antigen concentrations, suggests a role as assembly intermediates.

Antigens, Polyomavirus Transforming↗
PubMed2002

Identification of Tah11/Sid2 as the ortholog of the replication licensing factor Cdt1 in Saccharomyces cerevisiae.

Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Amino Acid Sequence↗
PubMed1992

cis-inhibitory elements in the pT181 replication system.

We report here the existence of a pair of sequence elements in plasmid cointegrates that together block the function of pT181 plasmid replication origins in cis. The study is an outgrowth of the use of plasmid pE194 as a vector for the analysis of the pT181 replication system. We have observed that whereas the isolated pT181 replication origin is fully functional when cloned to pE194, it is inactive when the entire pT181 plasmid genome is cloned. This cis-inhibition is relieved by deletion of all or part of the pE194 palA element or of the pT181 countertranscript promoter. The inhibitory effect of pE194 palA is independent of distance and orientation, whereas the inhibitory effect of the countertranscript promoter is lost when the promoter is moved to a distance of 1.5 kb from the replication origin or inverted in situ. We found that the cis-inhibited pT181 origin expresses origin-specific (Inc3B) incompatibility, which involves competition for the initiator protein. This finding suggests that the cis-inhibited origin binds the initiator protein and therefore that the inhibition affects a step in the initiation process subsequent to initiator binding.

Base Sequence↗
PubMed2002

Initiation sites are distributed at frequent intervals in the Chinese hamster dihydrofolate reductase origin of replication but are used with very different efficiencies.

Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less.

Animals↗
PubMed1999

Characterization of the oriI and oriII origins of replication in phage-plasmid P4.

In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region. The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the alpha protein did not bind ori2. Thus, the alpha protein appears to act differently at the two origins of replication.

Bacterial Proteins↗
PubMed2001

Mammalian recombination hot spot in a DNA loop anchorage region: a model for the study of common fragile sites.

We analyzed the replication pattern and the topological organization of a 200 kb long Chinese hamster polygenic locus, which spans the boundary of two isochores. One of them is G + C rich while the second one is highly A + T rich. Previous analysis of mutants amplified for this locus had identified, within the A + T rich isochore, a mitotic recombination hotspot and a replication origin separated by some 7 kb. The recombination hotspot exhibits structural features repeatedly observed at common fragile sites, including a typical enrichment in peaks of enhanced DNA helix flexibility. By studying the replication pattern of the same locus in the non-amplified CHO cells, we confirm here the localization of the replication origin and show that the mitotic recombination hotspot does not correspond to a replicon junction. This finding makes questionable current hypotheses correlating replication termination regions with recombination prone sequences. Using topoisomerase II-mediated DNA cleavage at matrix attachment sites, we identified a 40 kb-long DNA anchorage region extending all along a transcription unit nested within the A + T rich isochore. Both the recombination hotspot and the replication origin lie within this topoisomerase II sensitive region, which suggests that features essential for initiation of recombination and initiation of DNA replication cluster within DNA anchorage regions. Features common to this region and to common fragile sites are discussed. J. Cell. Biochem. Suppl. 36: 170-178, 2001.

Animals↗
PubMed1975

T antigen binds to simian virus 40 DNA at the origin of replication.

A technique employing ferritin-conjugated antibody has been developed to visualize specific protein-DNA complexes in the electron microscope and has been used to demonstrate the preferential binding of simian virus 40 (SV40) T antigen at or near the origin of replication of SV40 DNA, 0.67 fractional length clockwise from the EcoRI restriction endonuclease cleavage site. urified covalently closed supercoiled circles of SV40DNA were treated with partially purified T antigen and the complex was stabilized by crosslinking with glutaraldehyde. Hamster antiT antigen gamma-globulin, ferritin-labeled goat anti-hamster gamma-globulin, and glutaraldehyde were then added sequentially. The location of the bound ferritin cores was measured with respect to the EcoRI cleavage site and the orientation of the cores relative to the ends of the DNA was determined with respect to the locations of Escherichia coli DNA unwinding protein, which binds to covalently closed supercoiled SV40 DNA at either of two preferred sites, 0.46 or 0.90 fractional length clockwide from the EcoRI cleavage site.

Antigens, Viral↗
PubMed1998

An oligonucleotide inhibits oligomerization of a rolling circle initiator protein at the pT181 origin of replication.

A large number of plasmids have been shown to replicate by a rolling circle (RC) mechanism. The initiators encoded by these plasmids have origin-specific, nicking-closing activity that is required for the initiation and termination of RC replication. Since the initiators of many RC plasmids are rate-limiting for replication, these proteins are usually inactivated after supporting one round of replication. In the case of the pT181 plasmid, inactivation of the initiator RepC protein occurs by the attachment of an oligonucleotide to its active tyrosine residue. We have generated the inactivated form of RepC, termed RepC*, in vitro and investigated the effects of attachment of the oligonucleotide on its various biochemical activities. Our results demonstrate that while RepC* is inactive in nicking-closing and replication activities due to the blockage of its active tyrosine residue, it is competent in origin DNA binding and DNA religation activities. We have investigated the oligomeric state of RepC and RepC* and found that RepC exists as a dimer in solution and can oligomerize on the DNA. We have generated heterodimers in vitro between the wild-type and epitope-tagged RepC proteins. In electrophoretic mobility shift experiments, the initiator heterodimers generated a novel DNA-protein complex, demonstrating that it binds to DNA as a dimer. We have shown that a DNA binding mutant of RepC can be targeted to the origin in the presence of the wild-type protein primarily through a protein-protein interaction. Interestingly, RepC* is defective in its ability to oligomerize on the DNA. RepC* inhibited the DNA binding and replication activity of wild-type RepC to only a very limited extent, suggesting that it may play only a minor regulatory role in replication in vivo. Based on these and earlier results, we propose a model for the role of RepC during the initiation and termination of pT181 RC replication.

Bacterial Proteins↗
PubMed1997

[Molecular mechanism of lytic phase of Epstein-Barr virus DNA replication].

Epstein-Barr virus(EBV) is a human herpesvirus with a linear double stranded DNA, 172 kbp in length. The EBV has both a latent state and a lytic replicative cycle. Latency replication is dependent on cellular DNA replication machinery except for a single EBV-encoded protein, EBNA1. The EBV genome is maintained as a circular plasmid molecule and ori P, the latent phase replication origin of EBV, mediates this type of replication. The immediate-early BZLF1 protein disrupts viral latency through transactivation of early EBV genes. After induction of the lytic phase of viral replication, the EBV genome is amplified 100- to 1000-fold. The replication product is a head to tail concatemer which is presumed to be synthesized via a rolling circle mechanism initiated from the lytic phase replication origin, ori Lyt. EBV encodes seven viral genes that are essential for ori Lyt-dependent DNA replication. A number of features of EBV DNA replication make it a unique model system for the study of eukaryotic DNA replication. Here, the molecular mechanism of the lytic phase of the EBV DNA replication is discussed.

DNA Replication↗
PubMed2001

Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae.

Cdc45, which binds to the minichromosomal maintenance (Mcm) proteins, has a pivotal role in the initiation and elongation steps of chromosomal DNA replication in eukaryotes. Here we show that throughout the cell cycle in Saccharomyces cerevisiae, Cdc45 forms a complex with a novel factor, Sld3. Consistently, Sld3 and Cdc45 associate simultaneously with replication origins in the chromatin immunoprecipitation assay: both proteins associate with early-firing origins in G(1) phase and with late-firing origins in late S phase. Moreover, the origin associations of Sld3 and Cdc45 are mutually dependent. The temperature-sensitive sld3 mutation confers a defect in DNA replication at the restrictive temperature and reduces an interaction not only between Sld3 and Cdc45, but also between Cdc45 and Mcm2. These results suggest that the Sld3-Cdc45 complex associates with replication origins through Mcm proteins. At the restrictive temperature in sld3-5 cells, replication factor A, a single-strand DNA binding protein, does not associate with origins. Therefore, the origin association of Sld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.

Carrier Proteins↗
PubMed1990

Identification of the origin of replication of the eukaryote Dictyostelium discoideum nuclear plasmid Ddp2.

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryote Dictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication in D. discoideum, provided that the Rep gene, which acts in trans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp "TGTCATGACA" palindromes separated by a poly(T.A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.

Base Sequence↗
PubMed1998

Initiation of DNA replication in eukaryotic chromosomes.

Our understanding of the process by which eukaryotes regulate initiation of DNA replication has made remarkable advances in the past few years, thanks in large part to the explosion of genetic and biochemical information on the budding yeast, Saccharomyces cerevisiae. At least three major concepts have emerged: 1) The sequence of molecular events that determines when replication begins and how frequently each replication site is used are conserved among most, if not all, eukaryotes; 2) specific replication origins are used in most, if not all, eukaryotes that consist of a flexible modular anatomy; and 3) epigenetic factors such as chromatin structure and nuclear organization determine which of many potential replication origins are used at different stages in animal development. Thus, the current state of our knowledge suggests a simple unifying concept--all eukaryotes utilize the same basic proteins and DNA sequences to initiate replication, but the metazoa can change both the number and locations of replication origins in response to the demands of animal development.

Animals↗
PubMed1999

A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.

Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

Where does DNA replication start in archaea?

Genome-wide measures of DNA strand composition have been used to find archaeal DNA replication origins. Archaea seem to replicate using a single origin (as do eubacteria) even though archaeal replication factors are more like those of eukaryotes.

Archaea↗
PubMed1991

Site-dependent inhibition by single O6-methylguanine bases of SV40 T-antigen interactions with the viral origin of replication.

The effects of O6-methylguanine on the reactions involved in initiation of DNA replication were investigated by measuring the interactions of SV40 T antigen with oligonucleotides substituted with the methylated base. O6-Methylguanine residues were positioned in either binding site I or binding site II of the SV40 origin of replication. Binding of purified T antigen, measured by both nitrocellulose filter binding and delayed oligonucleotide migration, was unaffected by the presence of seven methylated bases in binding site II. Single substitutions within binding site I were sufficient to inhibit T-antigen binding, and the extent of inhibition was dependent on the position of O6-methylguanine in the DNA sequence. Unwinding by T antigen was analyzed by measuring displacement of a single-stranded oligonucleotide from similarly substituted, partially duplex substrates. The presence of three O6-methylguanine residues in binding site I facilitated the helicase activity of T antigen. In contrast, single O6-methylguanine bases inhibited unwinding. A correlation was observed between the position of the methylated base and the inhibition of both binding and unwinding by T antigen.

Animals↗