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PubMed1984

Gene A protein interacting with recombinant plasmid DNAs containing 25-30 b.p. of the phi X174 replication origin.

Synthetic oligodeoxyribonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the 30 b.p. origin region of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the kanamycin resistance gene of pACYC177 (AmpR, KmR). Transformants were picked up by antibiotic selection and filter-hybridization using one of the oligodeoxyribonucleotides as a probe. Approximate lengths of the inserts were determined by restriction enzyme analysis. Exact length and orientation of each insert was determined by DNA sequencing. Plasmid DNA with an insert homologous to the first 25 b.p. of the phi X174 origin is not nicked by the gene A protein. However, plasmid DNA containing the 27 b.p. fragment in either orientation is nicked by the gene A protein, as well as plasmid DNAs containing the first 28 b.p. or the complete 30 b.p. conserved origin region of the isometric phages.

Bacteriophage phi X 174↗

PubMed1988

Minimal region necessary for autonomous replication of pTAR.

The native 44-kilobase-pair plasmid pTAR, discovered in a grapevine strain of Agrobacterium tumefaciens, contains a single origin of DNA replication confined to a 1.0-kilobase-pair region of the macromolecule. This region (ori) confers functions sufficient for replication in Agrobacterium and Rhizobium species but not in Pseudomonas solanacearum, Pseudomonas glumae, Pseudomonas syringae pv. savastanoi, Xanthomonas campestris pv. campestris, and Escherichia coli. ori contains a repA gene that encodes a 28,000-dalton protein required for replication. Nucleotide sequencing of repA and its promoter region revealed four 8-base-pair palindromic repeats upstream of the repA coding region. Deletion of these repeats alters repA expression and plasmid copy number. Downstream of repA are three additional repeats in a region essential for replication. A locus responsible for plasmid partitioning (parA) and a putative second locus regulating plasmid copy number are part of the origin region and are required for stable plasmid maintenance.

Amino Acid Sequence↗

PubMed2002

Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import.

During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly, our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.

Active Transport, Cell Nucleus↗

PubMed1990

Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene.

The c-myc genes of HeLa cells are preferentially replicated in the transcriptional direction, from chromosomal origin sequences which display cell type-specific activity. Using a run-off replication assay involving in vitro extension of replication forks initiated in intact HeLa cells, bidirectional replication was observed to begin within a 3.5 kb domain 5' to the c-myc gene. To characterize the replication origin further a 2.4 HindIII-Xhol subfragment of the c-myc 5' flanking DNA was cloned in a selectable vector and transfected into HeLa cells. The resulting pNeo.Myc-2.4 construct persisted as a circular extrachromosomal element for more than 300 cell generations under selection, with recovery of approximately 500-1000 times the mass of plasmid initially introduced into the cells. Extrachromosomal circular pNeo.Myc-2.4 monomer was reisolated in supercoiled form, along with oligomeric and miniplasmid variants which had been generated in vivo; however, chromosomally integrated copies of the plasmid were not detectable in cultures containing extrachromosomal pNeo.Myc-2.4. The recovered pNeo.Myc-2.4 plasmid was resistant to Dpnl digestion and sensitive to Mbol digestion. After transfection with pNeo.Myc-2.4 BrUdR pulse labeling of long-term or short-term cultures demonstrated that the plasmid replicated semiconservatively, under controls similar to those imposed on chromosome replication. Bisection of the pNeo.Myc-2.4 insert suggested that c-myc 5' flanking DNA within 1.2 kb 5' to promoter P1 was sufficient to confer autonomously replicating sequence activity on the plasmid vector in transient replication assays.

Chromosomes, Human↗

PubMed1989

Rate, origin, and bidirectionality of Caulobacter chromosome replication as determined by pulsed-field gel electrophoresis.

Cell division in Caulobacter crescentus yields progeny cells that differ with respect to cell structure and developmental program. Chromosome replication initiates in the daughter stalked cell but is repressed in the daughter swarmer cell until later in the cell cycle. To study cell-type-specific DNA initiation, chromosome replication was directly analyzed by pulsed-field gel electrophoresis. Analysis of Dra I restriction fragments of DNA taken at various times from synchronized cell cultures labeled with 2'-deoxy[3H]guanosine has allowed us to determine the origin of DNA replication, the rate and direction of fork movement, and the order of gene replication. The first labeled Dra I fragment to appear contains the site of replication initiation. Based on the correlation of the physical and genetic maps derived by Ely and Gerardot [Ely, B. & Gerardot, C. J. (1988) Gene 68, 323-333], the origin was localized to a 305-kilobase fragment containing the rrnA gene. Furthermore, the sequential replication through unmapped Dra I fragments has enabled us to localize their positions on the genome. The order of appearance of labeled restriction fragments revealed that the chromosome replicates bidirectionally at a fork movement rate of 21 kilobases per minute.

Bacteria↗

PubMed2002

Functional domains involved in the interaction between Orc1 and transcriptional repressor AlF-C that bind to an origin/promoter of the rat aldolase B gene.

The promoter of the rat aldolase B (AldB) gene functions in vivo as an origin of DNA replication in the cells in which transcription of the gene is repressed. Previously, we identified two closely related DNA-binding proteins, AlF-C1 and AlF-C2, which repressed the AldB gene promoter. We also reported that the binding site of these proteins, site C, is one of the required DNA elements of the AldB gene origin/promoter for autonomously replicating activity in transfected cells. In the present study, we show that AlF-C1 and AlF-C2 bind directly to Orc1, a subunit of the origin recognition complex (ORC). Deletion analyses revealed a functional domain in AlF-C2 for binding to Orc1, which is located separately from the DNA-binding domain. In addition, we found a novel protein-interacting domain in Orc1 required for the binding of AlF-C2, which was conserved in human, mouse and Chinese hamster, but not in Drosophila, frog and yeast. Thus, it is assumed that in mammalian cells, sequence- specific DNA-binding proteins are involved in recruiting ORC to regulate replication initiation and/or transcription repression.

Amino Acid Sequence↗

PubMed2002

The expansion of a hypervariable, non-hr ori-like region in the genome of Cryptophlebia leucotreta granulovirus provides in vivo evidence for the utilization of baculovirus non-hr oris during replication.

In this report a naturally occurring hypervariable region within the genome of different Cryptophlebia leucotreta granulovirus genotypes is characterized. The region consists of a stretch of direct repeats, short palindromes and an unusual AT-rich region. Although the organization of these repeat sequences is unique to baculoviruses, it has the structural features of a 'non-hr' origin of DNA replication (ori). Restriction analysis and Southern hybridization revealed that this region is expanded during virus replication. Sequence comparison of different isolated genotypes indicated that the expansion is caused by concatenation of short repeats within the region or by concatenation of the complete region. These findings indicate that the expansion of non-hr origin-like regions is not restricted to defective-interfering particles, as was found previously for baculoviruses propagated in cell culture. Moreover, it appears that non-hr complexity contributes to the natural heterogeneity and genetic plasticity of baculovirus genomes. Also, circumstantial evidence is discussed that hr oris might have developed from internal rearrangement and multiplication of a non-hr ori during baculovirus evolution.

Amino Acid Sequence↗

PubMed2000

Think global, act local--how to regulate S phase from individual replication origins.

All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved. Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins. Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases. The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins. Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states.

Animals↗

PubMed1989

Transcriptional activation with concurrent or nonconcurrent template replication has differential effects on transient expression from herpes simplex virus promoters.

We have used two methods to induce template replication in order to assess the effect on expression of marker genes controlled by herpes simplex virus type 1 (HSV-1) promoters. One method used the HSV-1 origin of DNA replication from the short repeat region of the viral genome (HSV-1 oris), and allowed simultaneous replication and transcriptional activation of the plasmid-borne template. The other, using the simian virus 40 origin of replication (SV40 ori) allowed plasmid template replication prior to activation of transcription by HSV-1 infection. The two regimes had markedly different effects upon the levels of reporter gene activity induced by HSV-1 superinfection. Replication of reporter plasmids using the SV40 ori yielded levels of reporter gene activity proportional to plasmid copy number when cells were superinfected with HSV-1. In contrast, our results indicated that sequences containing, or in close proximity to, the HSV-1 oris in the reporter plasmid had a significant inhibitory effect on expression from all viral promoters whether or not the plasmid was allowed to replicate. Still, the early (beta) promoter-controlled reporter enzyme activity declined at late times while that controlled by the strict late (gamma) promoter was significantly higher following HSV-1 oris-mediated template replication.

Animals↗

PubMedDate not supplied

Intraspecific nucleotide sequence variability surrounding the origin of replication in human mitochondrial DNA.

We have cloned the major noncoding region of human mitochondrial DNA (mtDNA) from 11 human placentas. Partial nucleotide sequences of five of these clones have been determined and they share a maximum of 900 bp around the origin of H-strand replication. Alignment of these sequences with others previously determined has revealed a striking pattern of nucleotide substitutions and insertion/deletion events. The level of sequence divergence significantly exceeds the reported estimates of divergence in coding regions. Two particularly hypervariable regions have also been defined. More than 96% of the base changes are transitions, and length alterations have occurred exclusively by addition or deletion of mono-or dinucleotide segments within serially repeating stretches. This region of the mitochondrial genome, which contains the initiation sites for replication and transcription, is the least conserved among species with respect to both sequence and length (Anderson et al., 1981; Walberg and Clayton, 1981). Despite this overall lack of primary sequence conservation, several consistencies appear among the available mammalian mtDNA sequences within this region. Between species, a conserved linear array of characteristic stretches exists which nonetheless differ in primary sequence. Among humans, several conserved blocks of nucleotides appear within domains deleted from the mtDNA of other species. These observations are consistent with both a species-specificity of nucleotide sequence, and a preservation of the necessary genetic functions among species. This provides a model for the evolution of protein-nucleic acid interactions in mammalian mitochondria.

Amino Acid Sequence↗

PubMed2004

The replicon revisited: an old model learns new tricks in metazoan chromosomes.

The origins of DNA replication were proposed in the replicon model to be specified genetically by replicator elements that coordinate the initiation of DNA synthesis with gene expression and cell growth. Recent studies have identified DNA sequences in mammalian cells that fulfil the genetic criteria for replicators and are beginning to uncover the sequence requirements for the initiation of DNA replication. Mammalian replicators are com- posed of non-redundant modules that cooperate to direct initiation to specific chromosomal sites. Conversely, replicators do not show strong sequence similarity, and their ability to initiate replication depends on the chromosomal context and epigenetic factors, as well as their primary sequence. Here, we review the properties of metazoan replicators, and discuss the genetic and epigenetic factors that determine where and when DNA replication is initiated.

Animals↗

PubMed1977

Properties of the relaxation complex of supercoiled deoxyribonucleic acid and protein of R plasmid NR1 in Escherichia coli.

Some properties of the supercoiled deoxyribonucleic acid (DNA)-protein relaxation complex of the R plasmid NR1, which contains more than one origin for DNA replication, were examined. The percentage of complexed NR1 molecules that can be converted to the relaxed (nicked) form appeared to be unaffected by the conditions under which the host cells were cultured. However, the percentage of supercoiled NR1 DNA that can be relaxed was highly dependent on the method used to prepare the DNA and the agents used to induce relaxation. Our data suggest that 100% of NR1 molecules may exist in situ as DNA-protein relaxation complexes. An RTF-Tc segregant of NR1, which has deleted the r-determinants component of the NR1 and therefore does not contain the two origins of replication located in the r-determinants, has indistinguishable relaxation properties in comparison with NR1 itself.

Bacterial Proteins↗

PubMed1992

Organization of specific DNA sequence elements in the region of the replication origin and matrix attachment site in the chicken alpha-globin gene domain.

The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site (MAR) of the alpha-globin gene domain was investigated. The fragment was shown to contain a CR1-type repetitive element and two stably bent DNA sequences. One of them colocalizes with the previously described MAR element and with the recognition site for a proliferating-cell-specific, DNA-binding protein. The melting pattern of a set of subfragments of the region proved to be non random. No correlation between the distribution of readily melting sequences and bent DNA was found. The possible importance of curved, low-melting and repetitive DNA sequences for the organization of the upstream boundary of the alpha-globin gene domain and the function of the replication origin is discussed.

Animals↗

PubMed1997

Fine mapping of replication origins (ori A and ori B) in Nicotiana tabacum chloroplast DNA.

Using a partially purified replication complex from tobacco chloroplasts, replication origins have been localized to minimal sequences of 82 (pKN8, positions 137 683-137 764) and 243 bp (pKN3, positions 130 513-130 755) for ori A and ori B respectively. Analysis of in vitro replication products by two-dimensional agarose gel electrophoresis showed simple Y patterns for single ori sequence-containing clones, indicative of rolling circle replication. Double Y patterns were observed when a chloroplast DNA template containing both ori s (pKN9) was tested. Dpn I analysis and control assays with Escherichia coli DNA polymerase provide a clear method to distinguish between true replication and DNA repair synthesis. These controls also support the reliability of this in vitro chloroplast DNA replication system. EM analysis of in vitro replicated products showed rolling circle replication intermediates for single ori clones (ori A or ori B), whereas D loops were observed for a clone (pKN9) containing both ori s. The minimal ori regions contain sequences which are capable of forming stem-loop structures with relatively high free energy and other sequences which interact with specific protein(s) from the chloroplast replication fraction. Apparently the minimal ori sequences reported here contain all the necessary elements for support of chloroplast DNA replication in vitro.

Base Sequence↗

PubMed1990

Replication of an adenovirus type 34 mutant DNA containing tandem reiterations of the inverted terminal repeat.

A mutant of human adenovirus type 34 (Ad34) has been isolated which contains DNA molecules with tandem reiterations of from two to eight copies of a 131-bp sequence within the right-sided inverted terminal repetition. Terminal heterogeneity was not eliminated by repeated plaque purifications indicating that the population of DNA molecules with various numbers of reiterations could rapidly evolve from the DNA of a single virus particle. These enlarged DNA molecules were capable of replication both in vivo and in vitro. The nucleotide sequence of the mutant Ad34 inverted terminal repetitions contained most of the essential features of the Ad origin of DNA replication. These features include the ATAATATACC sequence which is present between the highly conserved bases 9-18 in all human adenoviruses, as well as the consensus sequences for the binding of nuclear factor I and nuclear factor III. However, the reiterated sequences lacked a dG appropriately placed on the template strand to serve as a potential site for internal initiation. It appears that the rapid amplification of two to eight copies of the reiterated terminal sequences does not arise from internal initiation during replication but probably from homologous recombination.

Adenoviruses, Human↗

PubMed1986

Permanent attachment of replication origins to the nuclear matrix in BHK-cells.

The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, showed that after brief pulses grains were exclusively found over the central core of the residual nuclei, which corresponds to positions in the nuclear matrix. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.

Animals↗

PubMed1995

The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators.

Replicators are genetically defined elements within chromosomes that determine the location of origins of DNA replication. In the yeast Saccharomyces cerevisiae, the ARS1 replicator contains multiple functional DNA elements: an essential A element and three important B elements--B1, B2, and B3. Functionally similar A, B1, and B2 elements are also present in the ARS307 replicator. The B3 element binds a replication and transcription enhancer protein Abf1p, whereas the A element is required for binding the origin recognition complex (ORC). The function of the B1 and B2 elements remains to be defined. We have used a gel-based DNA binding assay to study the interaction between replicators and the putative initiator protein ORC. In addition to the established requirements for ATP and the A element for ORC-DNA interaction, the new data demonstrate that sequences in the B1 element are also important for ORC-DNA association. This conclusion is supported by DNase I footprint analyses and demonstrates that ORC binds to a bipartitite recognition element within the DNA. Furthermore, mutation of nucleotides in the B1 element suggests that this element has other functions in the initiation of DNA replication besides participating in the ORC-DNA interaction.

Base Sequence↗

PubMed1994

Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori- plasmids and their in vivo chromosomal integration and retrieval.

We have constructed a plasmid system designed for the insertion of cloned DNA (e.g., genes, gene fusions, regulatory elements, etc.) into the Escherichia coli genome. Its principal feature is the presence of two tandem lox sites on the plasmids, which upon Cre-mediated in vitro recombination resolve the plasmids into ori- and ori+ DNA circles. The non-replicating ori- circles contain the lambda attP site, several unique restriction sites for cloning, a NotI site and KmR, a kanamycin-resistance-encoding gene. The ori+ circles carry the origin of DNA replication (ori) together with several cleavage sites not present in the ori- circles, including the rare site for the very efficient I-SceI enzyme, that are used to inactivate the ori+ circles and any unresolved plasmid DNA. We have used this system to insert cloned DNA into the host genome at (i) the attB site, by Int-mediated integration and (ii) at any predetermined sequence, as mediated by the Rec system(s) of the host. The genomes of the resulting transformants were analyzed by NotI digestion of the chromosomal DNA, embedded in agarose microbeads, followed by pulsed-field gel electrophoresis. A system for the retrieval of DNA fragments inserted at the attB site was also developed.

Base Sequence↗