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At least 109 records · Page 6Linked to original sources
PubMed1990

Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin.

Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.

Animals↗
PubMed1988

Deletion analysis of bacteriophage T4 tertiary origins. A promoter sequence is required for a rifampicin-resistant replication origin.

The DNA sequence requirements of two T4 tertiary replication origins have been characterized by a deletion analysis of origin-containing plasmids. Maximal replication of each origin-containing plasmid required both an intact gpmotA-dependent middle-mode promoter sequence and approximately 50 base pairs of the downstream region. In contrast, gpmotA-dependent transcription from the origin promoter was found to be independent of the downstream region. The requirement for a promoter element within the tertiary origins is striking, particularly since the replication of tertiary origin-containing plasmids is resistant to the RNA polymerase inhibitor rifampicin.

Base Sequence↗
PubMed1995

Modular structural elements in the replication origin region of Tetrahymena rDNA.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.

Animals↗
PubMed2001

A hybrid bacterial replication origin.

We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC. The AT-rich region could be unwound by E. coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble. The results show that species specificity, i.e. which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.

Bacillus subtilis↗
PubMed1994

A search for an essential function of the replication origin ARS1 in the life cycle of Saccharomyces cerevisiae.

We have investigated the significance of the chromosomal replication origin, ARS1, during the entire life cycle of yeast. This was done by substituting the chromosomal copy with a series of ars1 deletion mutants. It was shown that the ARS1 replication origin is not essential for mitotic or premeiotic DNA replication since no effect on growth, chromosomal loss rate and spore viability was observed in the ars1 mutant strains. We conclude that replication origins are abundantly, present in the yeast genome and that the removal of a single replication origin is compensated for by replication forks emanating from neighbouring origins.

Chromosomes, Fungal↗
PubMed1990

[Cloning and study on the replication origin of the integrated F plasmid in Escherichia coli].

The replication origin of the integrated F' plasmid was cloned by means of marker rescue. No difference in incompatibility and acridine orange sensitivity was found between the mini-F plasmid constructed from such an origin and the autonomous F' plasmid. Subcloning and comparative restriction enzyme analysis were carried out with the replication origin from the integrated F' plasmid and that from the autonomous F plasmid. No structural difference was found between them. These results suggest that the difference for the dependence of recA gene between the F and F' plasmids dose not come from any difference in structure of their replication origin, but probably from their site of integration on the chromosome.

Cloning, Molecular↗
PubMed1988

Influence of fd gene 2-protein and the viral replication origin on the compatibility of pfd-plasmids.

Plasmids with the replication origin of bacteriophage fd, the pfd-plasmids, were investigated for compatibility in E. coli cells expressing fd gene 2-protein. This was measured by transformation of Ca-treated cells with and without a residing pfd-plasmid. When the two plasmids contained the complete intergenic region of bacteriophage fd, they were fully compatible in contrast to the situation in which at least one plasmid had a shortened origin for viral strand replication. This incompatibility effect was partially compensated for by a pfd-plasmid with a short origin and with the fd gene 2. The fd replication origin on a colEl plasmid did not affect compatibility in polA+ cells indicating its idling in the presence of the colEl origin. It can be concluded that a short replication origin requires high amounts of gene 2-protein in contrast to the long origin. Accumulation of replication intermediates severely interferes with host cell metabolism.

Coliphages↗
PubMed1999

Conservation of ARS elements and chromosomal DNA replication origins on chromosomes III of Saccharomyces cerevisiae and S. carlsbergensis.

DNA replication origins, specified by ARS elements in Saccharomyces cerevisiae, play an essential role in the stable transmission of chromosomes. Little is known about the evolution of ARS elements. We have isolated and characterized ARS elements from a chromosome III recovered from an alloploid Carlsberg brewing yeast that has diverged from its S. cerevisiae homeologue. The positions of seven ARS elements identified in this S. carlsbergensis chromosome are conserved: they are located in intergenic regions flanked by open reading frames homologous to those that flank seven ARS elements of the S. cerevisiae chromosome. The S. carlsbergensis ARS elements were active both in S. cerevisiae and S. monacensis, which has been proposed to be the source of the diverged genome present in brewing yeast. Moreover, their function as chromosomal replication origins correlated strongly with the activity of S. cerevisiae ARS elements, demonstrating the conservation of ARS activity and replication origin function in these two species.

Conserved Sequence↗
PubMed2001

Conserved gene cluster at replication origins of the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii.

A 30-kb region surrounding the replication origin in Caulobacter crescentus was analyzed. Comparison to the genome sequence of another alpha-proteobacterium, Rickettsia prowazekii, revealed a conserved cluster of genes (RP001, hemE, hemH, and RP883) that overlaps the established origin of replication in C. crescentus and the putative origin of replication in R. prowazekii. The genes flanking this cluster differ between these two organisms. We therefore propose that this conserved gene cluster can be used to identify the origin of replication in other alpha-proteobacteria.

Alphaproteobacteria↗
PubMed2000

Cell cycle regulator phosphorylation stimulates two distinct modes of binding at a chromosome replication origin.

In Caulobacter crescentus, the global response regulator CtrA controls chromosome replication and determines the fate of two different cell progenies. Previous studies proposed that CtrA represses replication by binding to five sites, designated [a-e], in the replication origin. We show that phosphorylated CtrA binds sites [a-e] with 35- to 100-fold lower K(d) values than unphosphorylated CtrA. CtrA phosphorylation stimulates two distinct modes of binding to the replication origin. Phosphorylation stimulates weak intrinsic protein-protein cooperation between half-sites and does not stimulate CtrA-P binding unless protein-DNA contacts are made at both half-sites. CtrA phosphorylation also stimulates cooperative binding between complete sites [a] and [b]. However, binding to each of the other CtrA-binding sites [c], [d] and [e] is completely independent and suggests a modular organization of replication control by CtrA. We therefore propose a model where a phosphorelay targets separate biochemical activities inside the replication origin through both cooperative and independent CtrA-binding sites.

Bacterial Proteins↗
PubMed1986

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

Animals↗
PubMed1999

DNA methylation at mammalian replication origins.

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene. Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.

Animals↗
PubMed1985

Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K.

Plasmid pMM3 is a pBR322 derivative carrying the gamma origin of replication of the naturally occurring plasmid R6K. We have produced a gamma-origin mutant bank of this plasmid using the single-strand-specific mutagen sodium bisulfite. Members of this bank contain single or multiple mutations in the seven direct repeats and the flanking sequences in the gamma origin. Three mutants with defective gamma origins have been isolated from this mutant bank. Two of these direct repeat mutants, gamma 117 and gamma 120, are unable to replicate and also have lost the ability to bind the R6K initiation protein pi in vitro at one of the seven 22-base-pair direct repeats within their respective origins. Precise deletion of the damaged repeat of either of these mutants restores origin function, suggesting that the primary defect of these mutants involves a disruption of the normal spacing of pi binding and flanking sequences within the gamma origin. The third mutant, gamma 111, binds pi normally but replicates at a greatly reduced copy number due to a mutation near the seventh repeat. This mutation falls within a short sequence that appears to be conserved among a number of other plasmids that contain direct repeats within their origins of replication.

Base Sequence↗
PubMed1998

Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae.

BACKGROUND: The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis-element, ACS (ARS Consensus Sequence), essential for DNA replication. The protein-DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression. Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S. cerevisiae. RESULTS: Topological changes in the replication origins of the S. cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding. The footprinting method detected the inhibition and enhancement of UV-induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits. Furthermore, footprint patterns were reproduced in vitro with purified ORC. The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase. Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1-arrested cells. CONCLUSION: The application of in vivo UV photofootprinting in studies of topological changes of S. cerevisiae replication origins revealed the presence of two modes of topological ORC-ACS interaction. The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC-ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication.

Base Sequence↗
PubMed1986

Illegitimate recombination occurs between the replication origin of the plasmid pC194 and a progressing replication fork.

Hybrids between plasmids pC194, pBR322 and the bacteriophage f1 undergo deletions in Escherichia coli. The deletions end most often between nucleotides 1445 and 1446 of pC194. That site probably corresponds to a nick in the replication origin of this plasmid. The localization of the other deletion end appears to be determined by the position of the f1 replication fork. Two models accounting for these data are discussed.

Bacillus subtilis↗
PubMed1996

Multiple determinants controlling activation of yeast replication origins late in S phase.

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

Base Sequence↗
PubMed1997

Identification of a predominant replication origin in fission yeast.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.

Chromosomes, Fungal↗
PubMed1997

The iteron bases and spacers of the P1 replication origin contain information that specifies the formation of a complex structure involved in initiation.

The origin of replication of the P1 plasmid contains five direct, imperfect repeats (iterons) of a 19 bp sequence that binds the P1-encoded RepA initiator protein. RepA binding to these iterons triggers origin initiation and represses transcription from the repA promoter that is nested within the iterons. The origin iterons were replaced with ligated oligonucleotides that insert five perfect 19 bp repeats with identical spacer sequences. This eliminates the natural variation in the iteron and spacer sequences and removes the repA promoter. The reconstructed origin is functional, showing that the repA promoter is not essential for origin function. The method used to make the reconstructed origin allows substitution of identical iterons with altered sequence or spacer length. Single changes of conserved iteron bases gave reduced or non-existent origin activity, as did an increase in spacer length. Like the wild type, most of these mutant arrays retain avid primary binding activity for the RepA protein. However, although the wild-type arrays readily form a mature complex in which all iterons are saturated, the most replication-defective mutants were completely unable to do this, even at very high RepA concentrations. It appears that iteron spacing and contacts involving at least three of the conserved iteron bases play an important role in the assembly of the mature structure in which all sites are occupied. A model is presented in which an allosteric interaction between the DNA site and protein is needed for the saturated, mature complex required for initiation.

Bacteriophage P1↗