Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA replication origin”

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

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

At least 361 records · Page 20Linked to original sources
PubMedDate not supplied

Mouse p53 inhibits SV40 origin-dependent DNA replication.

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.

Animals↗
PubMed1977

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.

Base Sequence↗
PubMed1983

Polyomavirus origin for DNA replication comprises multiple genetic elements.

To define the minimal cis-acting sequences required for polyomavirus DNA replication (ori), we constructed a number of polyomavirus-plasmid recombinants and measured their replicative capacity after transfection of a permissive mouse cell line capable of providing polyomavirus large T antigen in trans (MOP cells). Recombinant plasmids containing a 251-base-pair fragment of noncoding viral DNA replicate efficiently in MOP cells. Mutational analyses of these viral sequences revealed that they can be physically separated into two genetic elements. One of these elements, termed the core, contains an adenine-thymine-rich area, a 32-base-pair guanine-cytosine-rich palindrome, and a large T antigen binding site, and likely includes the site from which bidirectional DNA replication initiates. The other, termed beta, is located adjacent to the core near the late region and is devoid of outstanding sequence features. Surprisingly, another sequence element named alpha, located adjacent to beta but outside the borders of the 251-base-pair fragment, can functionally substitute for beta. This sequence too contains no readily recognized sequence features and possesses no obvious homology to the beta element. The three elements together occupy a contiguous noncoding stretch of DNA no more than 345 base pairs in length in the order alpha, beta, and core. These results indicate that the polyomavirus origin for DNA replication comprises multiple genetic elements.

Animals↗
PubMed1977

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. IV. Localization of the SV40 DNA complementary to the 5' ends of viral mRNA.

Cytoplasmic mRNA isolated from cells infected with SV40 was isolated by passage over oligo(dT)-cellulose columns. This RNA was annealed to SV40 DNA fragments produced by cleavage with EcoRII endonuclease. The RNA resistant to RNase digestion was analyzed by digestion with ribonucleases and oligonucleotide mapping. The results were compared with oligonucleotides from in vitro transcripts of the fragments and with whole genome SV40 cRNA which had been fractionated by hybridization to the fragments. The 5' ends of "early" and the large "late" SV40 mRNA, transcribed from opposite DNA strands, overlap for a region of 60 to 100 nucleotides. The region of overlap includes a portion of the segment of DNA containing the origin of DNA replication.

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↗
PubMed1995

Expression of human polyomavirus JC T antigen by an adenovirus hybrid vector and its binding to DNA sequences encompassing the JC virus origin of DNA replication.

In the search for factors that influence the outcome of human polyomavirus JC (JCV) infection, the roles not only of host-related immunological control but also of virus-dependent regulatory steps have to be taken into account. Besides cell-specific control of early expression of the multifunctional virus protein large tumour antigen (T Ag), control mechanisms involve individual steps of the DNA replication process. For the analysis of T Ag DNA binding, the protein was expressed by an adenovirus hybrid vector in the 293 cell line to provide saturating amounts of JCV T Ag. After determination of the size and immunoreactivity, functional activity was analysed by specific DNA binding. To avoid the interference of cellular proteins, T Ag was immunoprecipitated prior to the reaction. Binding to T Ag-binding sites I and II within a 141 bp DNA segment in the control region was analysed using deletion mutants of a JCV subtype from brain tissue of a patient with fatal central nervous system disease. The specificity of the binding was confirmed by recombinant T Ag binding to origin of DNA replication (ori) sequences of wild-type JCV genomes. These data document that recombinant T Ag overexpressed by the adenovirus vector in eukaryotic cells was JCV-specific, had the expected length and exhibited specific ori-binding activity, thus providing the essential tool for future analysis of virus-host interactions at the level of viral DNA replication.

Adenoviridae↗
PubMedDate not supplied

[Construction of hybrid plasmids containing the yeast replicator].

In Saccharomyces cerevisiae strain 6-1G-P188 about 10 per cent of rRNA genes exist as extrachromosomal copies of rDNA repeating units. These extrachromosomal copies can be isolated as covalently closed molecules with lengths around 3mu. We have constructed a set of hybrid plasmids containing the bacterial vector pBR325, the LEU2 gene of yeast encoding beta-isopropylmalatedehydrogenase and various EcoRI restriction fragments of the 3mu DNA. We have tested the ability of our hybrid plasmids to transform LEU2 strain DC5 to leucine prototrophy. One of the plasmids Rcp21/11 transforms DC5 at the frequency comparable with that obtained with YEp13, containing the 2mu DNA replication origin. The 2400 bp EcoRI-B fragment of the 3mu DNA in Rcp21/11 carries a gene for 5S rRNA and two spacers. Our results on transformation experiments allow un to suggest that this EcoRI fragment also carries the 3mu DNA replication origin. Yeast transformants containing this plasmid are highly unstable but during the prolonged growth in selective conditions the stabilization of the LEU+ phenotype is observed being most likely a result of integration of Rcp21/11 into the yeast chromosome.

Cloning, Molecular↗
PubMed1993

Sequences at the C-terminus of the herpes simplex virus type 1 UL30 protein are dispensable for DNA polymerase activity but not for viral origin-dependent DNA replication.

The UL30 protein of herpes simplex virus type 1 (HSV-1) is a catalytically active DNA polymerase which is present in virus infected cells in a heterodimeric complex with an accessory subunit, the UL42 polypeptide. Both proteins are essential for viral DNA synthesis but because the UL42 protein is much more abundant it has been difficult to determine whether its role is related to, or independent of, its interaction with the UL30 protein in vivo. Since the C-terminal region of UL30 has been shown to be important for interaction with the UL42 protein but dispensable for DNA polymerase activity, a recombinant baculovirus which overexpresses a UL30 protein truncated by 27 amino acids at its C-terminus was constructed and used to assess the significance of the protein-protein interaction. The mutated protein was as active as wildtype (wt) UL30 in a DNA polymerase assay in which activated calf thymus DNA was used as template. However, in contrast to the wt protein, the activity of the truncated polymerase on this template was not stimulated by addition of purified UL42. A monoclonal antibody against the UL42 protein co-precipitated the full length but not truncated polymerase from extracts of cells which had been co-infected with a UL42-expressing recombinant baculovirus. Finally, the truncated protein was not active in a transient assay for HSV-1 origin-dependent DNA replication performed in insect cells in tissue culture. These results indicate that sequences at the C-terminus of the UL30 protein which are dispensable for DNA polymerase activity play essential roles both in viral DNA replication and interaction with the UL42 protein, and strongly suggest that the interaction between the proteins is important in vivo.

Amino Acid Sequence↗
PubMed1980

Cloning of an origin of DNA replication of Xenopus laevis.

DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Escherichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3H]-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of the segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. The DNA synthesis in pSW14 was shown to be replication rather than repair synthesis, based on a buoyant density shift of the product when iododeoxyuridine was used for labeling. To determine the number of replications of pSW14, a novel method was employed. Because pSW14 is a head-to-head dimer of the vector with the Xenopus fragment inserted at an EcoRI site, the plasmid has three methylatable sites--two bracketing the Xenopus fragment and one opposite the fragment. By cotransformation of E. coli with pSW14 and pBR322 containing the EcoRI methylase gene, supercoiled pSW14 was methylated and injected into eggs with [3H]thymidine. Disappearance of modified EcoRI sites by semiconservative replication was followed by measuring the sensitivity to EcoRI endonuclease over time. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated [3H]thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude that pSW14 has a functional origin in the Xenopus DNA segment.

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↗
PubMed1991

Factors contributing to the restricted DNA replicating activity of JC virus.

The basis for the restricted host range behavior of JC virus (JCV) in vitro was investigated by focusing on its DNA replicating activity and comparing it to that of simian virus 40 (SV40). Prototype, mutant, and hybrid JCV and SV40 DNAs were tested for their replicating activity in cells permissive for one or both of the viruses. Results from these experiments indicated that, relative to its SV40 counterpart, the JCV T antigen functioned less efficiently and was more specific in its interactions with polyomavirus DNA replication origins. The JCV T antigen exhibited a lower specific DNA binding activity than did the SV40 T antigen, which might contribute to this virus' reduced DNA replicating activity. However, the JCV protein did bind to both the JCV and SV40 replication origins with similar efficiency, indicating that the ability of the JCV T antigen to discriminate between the JCV and SV40 origins involved a step subsequent to specific DNA binding. The results also suggested that the failure of JCV to replicate to detectable levels in monkey kidney cells was due to the inefficient interactions of its T protein with the viral origin and the host replication machinery. The inability of the JCV T antigen to carry out one or more of these DNA replication functions efficiently contributes to the restricted lytic behavior of this virus.

Animals↗
PubMed2005

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction.

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.

Animals↗
PubMed1992

Protein-DNA interactions at a yeast replication origin.

An understanding of the protein-DNA interactions in vivo at origins of DNA replication in eukaryotes is essential to delineate the mechanism of initiation of DNA synthesis and its control in the cell cycle. In the yeast Saccharomyces cerevisiae, a family of sequences known as autonomously replicating sequences (ARSs) function as origins of bidirectional DNA replication on plasmids and, in several instances, also in their normal chromosomal location. Here we use nucleotide resolution genomic footprinting to investigate the association of proteins with ARS1. Nuclease protection patterns indicate that at least two different cellular factors interact with functional elements in ARS1. The first seems to be ARS-binding factor 1. The second seems to be a novel protein that generates extensive protection over the essential ARS consensus sequence and phased DNaseI-sensitive sites across a functionally important flanking sequence. Hypersensitivity of this region to cleavage by copper phenanthroline indicates that it is under torsional strain, analogous to that produced at transcriptional start sites by assembly of an initiation complex. The protection in situ is similar to that generated by the origin recognition complex (ORC) protein.

Base Sequence↗
PubMed1991

Initiation of bacteriophage PRD1 DNA replication on single-stranded templates.

In vitro studies have demonstrated that single-stranded DNA molecules containing the 3' terminal nucleotides of the PRD1 DNA replication origin can support initiation by a protein-primed mechanism. We have determined the minimal requirements for priming by analyzing the template activity of various deletion derivatives. Our results showed that the 3'-terminal 15 nucleotides of the replication origin are sufficient for priming. The finding that the requirements for recognition of replication origin are different from those for priming suggests that there are two distinct steps during initiation of PRD1 DNA replication: first, recognition of the replication origin on double-stranded DNA and second, the priming event on single-stranded DNA. In addition our results showed that additional bases at the 3' end of templates did not affect priming activity, suggesting that the priming site is searched for from inside the template.

Bacteriophages↗
PubMed1988

Herpes simplex virus induces the replication of foreign DNA.

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.

Animals↗
PubMed1991

Pseudomonad replication origins: a paradigm for bacterial origins?

Structural features of three analysed bacterial DNA replication origin classes (six enteric origins, three pseudomonad origins, and the Bacillus subtilis origin region) are compared in order to deduce characteristics common to all bacterial origins and characteristics that distinguish the three origin classes. The two Pseudomonas aeruginosa origins are shown to map within 10 kb of each other, and correlations are drawn with four potential origin regions in B. subtilis. The enteric origin class is further distinguished from the other two classes by its genetic organization, the presence of GATC sites, and the role of Dam methylation in enteric initiation. The pseudomonad origin class has the most features that are common to all of these bacterial origins, and hence may be the paradigm bacterial origin class.

Amino Acid Sequence↗
PubMed2002

The role of the carboxyterminal domain of RNA polymerase II in regulating origins of DNA replication in Saccharomyces cerevisiae.

MCM (minichromosome maintenance) proteins function as a replication licensing factor (RLF-M), which contributes to limiting initiation of DNA replication to once per cell cycle. In the present study we show that a truncation of the pol II CTD in a S. cerevisiae strain harboring a mutation in mcm5 partially reverses its ts phenotype and improves maintenance of CEN/ARS minichromosomes. We correlate this phenotype to effects on DNA replication rather than to effects on transcription or specific gene expression. We also demonstrate that a similar truncation of the CTD reduces minichromosome stability and impairs stimulation of DNA replication by trans-activators and that tethering of recombinant pol II CTD to an origin of replication has a significant stimulatory effect on minichromosome stability. Furthermore, we show that pol II is recruited to ARS1. We propose that in S. cerevisiae a mechanism of coordinating pol II transcription and DNA replication is mediated by the CTD of pol II.

Cell Cycle Proteins↗
PubMed2003

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication.

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.

Bacterial Proteins↗