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PubMed1993

Relationship between scaffold-attached regions, sequences replicating autonomously in yeast, and a chromosomal replication origin in the Drosophila rDNA.

The potential relationship between anchorage of Drosophila rDNA to a nuclear substructure and replication mechanisms was studied. First, two scaffold-attached regions (SARs) were identified, in the internally transcribed spacer and in the region spanning both the intergenic spacer (IGS) and the externally transcribed spacer (ETS), respectively. These SARs define two possible loops containing the sequence coding for the 18S rRNA and part of that coding for the 28S rRNA, respectively. They also bind yeast scaffolds. Then, the presence of sequences able to promote extrachromosomal replication in yeast (ARSs) was tested. The identified ARSs comap with SARs. The tight relationship between SARs and ARSs was further investigated in the IGS-ETS region which contains a chromosomal replication origin. The topological correlation observed between SARs, ARSs, and a chromosomal replication origin suggests the physical association of the replication origin to the nuclear substructure.

Animals↗
PubMed1999

Replication origins in metazoan chromosomes: fact or fiction?

The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.

Animals↗
PubMed2003

Developmentally regulated usage of Physarum DNA replication origins.

To determine the extent to which eukaryotic replication origins are developmentally regulated in transcriptionally competent cells, we compared origin use in untreated growing amoebae and plasmodia of Physarum polycephalum. At loci that contain genes transcribed in both developmental stages, such as the ribosomal RNA genes and two unlinked actin genes, we show that there is a similar replicational organization, with the same origins used with comparable efficiencies, as shown by two-dimensional agarose-gel electrophoresis. By contrast, we found cell-type-specific replication patterns for the homologous, unlinked profilin A (proA) and profilin P (proP) genes. proA is replicated from a promoter-proximal origin in amoebae, in which it is highly expressed, and is replicated passively in the plasmodium, in which it is not expressed. Conversely, proP is replicated passively and is not expressed in amoebae, but coincides with an efficient origin when highly expressed in the plasmodium. Our results show a reprogramming of S phase that is linked to the reprogramming of transcription during Physarum cell differentiation. This is achieved by the use of two classes of promoter-associated replication origins: those that are constitutively active and those that are developmentally regulated. This suggests that replication origins, like genes, are under epigenetic control associated with cellular differentiation.

Actins↗
PubMed1989

Replication properties of mini-Rts1 derivatives deleted for DnaA boxes in the replication origin.

Mini-Rts1 was found to be unable to replicate in a dnaA-null mutant. However, a mini-Rts1 derivative lacking entire tandem DnaA boxes in the replication origin retained the replication ability in a dnaA+ host although its copy number was about half that of the mini-Rts1 having complete DnaA boxes. Mini-Rts1cop1 that contains a high copy number mutation in repA was found to replicate more efficiently than mini-Rts1 of wild repA when DnaA boxes were deleted. In addition, the copy number of mini-Rts1cop1 without DnaA boxes increased 1.5-fold upon removal of incI iterons, whereas that of mini-Rts1 without DnaA boxes did not increase after the iterons were deleted. These indicate that the RepAcop1 protein can initiate the replication of mini-Rts1 efficiently even when DnaA boxes are absent from the origin of replication.

Bacterial Proteins↗
PubMed2005

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

Animals↗
PubMed2006

The Chinese hamster dihydrofolate reductase replication origin decision point follows activation of transcription and suppresses initiation of replication within transcription units.

Chinese hamster ovary (CHO) cells select specific replication origin sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G1 phase, the origin decision point (ODP). Origin selection is sensitive to transcription but not protein synthesis inhibitors, implicating a pretranslational role for transcription in origin specification. We have constructed a DNA array covering 121 kb surrounding the DHFR locus, to comprehensively investigate replication initiation and transcription in this region. When nuclei isolated within the first 3 h of G1 phase were stimulated to initiate replication in Xenopus egg extracts, replication initiated without any detectable preference for specific sites. At the ODP, initiation became suppressed from within the Msh3, DHFR, and 2BE2121 transcription units. Active transcription was mostly confined to these transcription units, and inhibition of transcription by alpha-amanitin resulted in the initiation of replication within transcription units, indicating that transcription is necessary to limit initiation events to the intergenic region. However, the resumption of DHFR transcription after mitosis took place prior to the ODP and so is not on its own sufficient to suppress initiation of replication. Together, these results demonstrate a remarkable flexibility in sequence selection for initiating replication and implicate transcription as one important component of origin specification at the ODP.

Animals↗
PubMed2002

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast.

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.

Animals↗
PubMed1988

Methylation is co-ordinated on the putative replication origins of Physarum ribosomal DNA.

In Physarum polycephalum, the ribosomal DNA is found as 60,000 base-pair palindromes. Each rDNA has four symmetrically arranged replication origins flanked by ribosomal RNA genes. A particular sequence, the putative replication origin, is repeated at the approximate position of each origin and nowhere else in the molecule. On a typical rDNA molecule, only one origin is active per replication cycle. We show that both the level and co-ordination of methylation result in asymmetrically methylated rDNA molecules that are particularly hypomethylated at one of their four putative replication origins. This pattern of methylation on a typical rDNA molecule is consistent with a model where hypomethylation is a determinant of origin activity.

Cell Cycle↗
PubMed2002

Single replication origin of the archaeon Methanosarcina mazei revealed by the Z curve method.

The genomic sequence of the archaeon Methanosarcina mazei has been analyzed by the Z curve method. The Z curve is a three-dimensional curve that uniquely represents the given DNA sequence. The three-dimensional Z curve and its x and y components for the genome of M. mazei show a sharp peak and relatively broad peak, respectively. The cdc6 gene is located exactly at the position of the sharp peak. Based on the known behavior of the Z curves for the archaea whose replication origins have been identified, we hypothesize that the replication origin and termination sites correspond to the positions of the sharp peak and broad peak, respectively. We have located an intergenic region that is between the cdc6 gene (MM1314) and the gene for an adjacent protein (MM1315), which shows strong characteristics of the known replication origins. This region is highly rich in AT and contains multiple copies of consecutive repeats. Our results strongly suggest that the single replication origin of M. mazei is situated at the intergenic region between the cdc6 gene and the gene for the adjacent protein, from 1,564,657 to 1,566,241 bp of the genome.

Base Sequence↗
PubMed1983

Replication origin region of Bacillus subtilis chromosome contains two rRNA operons.

The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon. A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region. Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction. The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon. A novel operon at the B7-B3 junction was termed rrnO. Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment. From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B. subtilis chromosome. The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter.

Bacillus subtilis↗
PubMed1995

Stimulation of DNA synthesis by mouse DNA helicase B in a DNA replication system containing eukaryotic replication origins.

A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far. We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin. In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin. We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen. DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase. DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A. A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B. Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system.

Adenosine Triphosphatases↗
PubMed1998

Association of autonomous replication activity with replication origins in a human chromosome.

A systematic analysis of the correlation of autonomous replication activity with initiation of replication in a human chromosome was performed. The temporal order of replication of segments in a pericentric 320-kb MEN203 locus on human chromosome 10 (10q11.2) was determined by pulse-labeling of cells with 5-bromodeoxyuridine after synchronization with aphidicolin. The entire MEN203 locus replicated during the late S phase. Two distinct segments replicated earlier than the others in the locus, indicating that replication was initiated within or near these segments. Two other segments also showed an earlier response than the respective neighboring regions. These results suggest that the MEN203 locus contains two distinct replication origins and two possible origins that may be used less frequently. The results were essentially confirmed by synchronization of the cell cycle with mimosine. Analysis of autonomous replication activity of 10-kb long chromosome fragments covering the 320-kb region showed that certain fragments replicated two or three times more efficiently than others. The results are consistent with our previous observations with randomly cloned human chromosome fragments. The replication origins colocalized with fragments exhibiting relatively high autonomous replication activity. Thus, the capacity for autonomous replication of chromosome fragments might be prerequisite for the initiation of chromosomal replication.

Aphidicolin↗
PubMed2002

Site-specific ORC binding, pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins.

Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Delta3 site, weakly to the Delta6 site and not at all to the remaining sequences. In situ, the footprint over Delta3 is extended during G(1) phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Delta2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre-replication complex and initiates DNA synthesis, plus an additional novel origin element (Delta9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa.

Base Composition↗
PubMed2001

In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin.

Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.

Base Sequence↗
PubMed1992

The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.

Adenosine Triphosphate↗
PubMed1997

Differential DNA replication origin activities in human normal skin fibroblast and HeLa cell lines.

A modification of the extrusion method for the isolation of nascent DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was firstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin fibroblast (NSF) and HeLa cells, abundance of DNA sequences in the regions of five of these origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification of origins which may be differentially active in normal cells and transformed cells or in different cell types.

Cell Line, Transformed↗
PubMed1996

Large, complex modular structure of a fission yeast DNA replication origin.

BACKGROUND: In the budding yeast, Saccharomyces cerevisiae, each DNA replication origin is associated with an autonomously replicating sequence (ARS) element. Each element contains several modules, including an essential close match to the 11 base-pair (bp) ARS consensus sequence (ACS) and two or three short (< 20 bp) stimulatory motifs, within a stretch of approximately 150 bp or less. To determine whether a similar origin structure exists in the evolutionarily distant fission yeast, Schizosaccharomyces pombe, we used deletion and linker substitution scanning to identify the sequences important for the function of ars3002, a chromosomal replication origin. RESULTS: We detected two large (30-55 bp) essential regions and several additional stimulatory sequences within a 600 bp stretch of a restriction fragment containing ars3002. The two essential regions are similar to each other, and sequences similar to them are found in all known S. pombe ARS elements, suggesting that one or both of them may represent the S. pombe equivalent of the S. cerevisiae ACS. CONCLUSIONS: Like S. cerevisiae origins, the S. pombe origin, ars3002, possesses a modular structure, but the number and size of modules is greater for ars3002, and ars3002 is larger than S. cerevisiae origins. These observations suggest that origin function in S. pombe requires more protein-DNA interactions than in S. cerevisiae.

Base Sequence↗
PubMed1986

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins.

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

DNA Replication↗