Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “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 1,117 records · Page 62Linked to original sources
PubMed1994

The bovine papillomavirus type 1 genome contains multiple loci of static DNA bending, but bends are absent from the functional origin of replication.

Twenty-four overlapping restriction fragments spanning the entire bovine papillomavirus type 1 (BPV-1) genome were analyzed by electrophoresis to determine the extent of static DNA bending in the BPV-1 genome. Thirteen of 24 fragments contained static bends. Based on known locations of previously mapped bend loci and the overlapping pattern of these 13 fragments, we estimate that there are 8-11 distinct static bend loci in the BPV-1 genome. The bend loci were not uniformly distributed on the genome and with one exception, were clustered from nucleotides 5816 to 2621 on the BPV-1 map. This portion of the BPV-1 genome contains most of the transcriptional regulatory sequences as well as the origin of replication. The concordance between the genomic distribution of DNA bends and cis-active elements is consistent with the possibility that bent sequences may contribute to the function of at least some of these elements. However, unlike SV40, there was no static bend at that functional origin of replication for BPV-1. The nearest bends to the origin were approximately 120 bp to the 5' side and 300 bp to the 3' side. As both of these bends were outside of the sequences required for origin function, it is unlikely that static bending plays a critical role in BPV-1 replication.

Base Sequence↗
PubMed1994

High frequency vector-mediated transformation and gene replacement in Tetrahymena.

Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.

Animals↗
PubMed2001

DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number.

The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.

Bacterial Proteins↗
PubMed1986

Insertion of an R1 plasmid into the origin of replication of the E. coli chromosome: random timing of replication of the hybrid chromosome.

A 16 bp BgI II fragment was deleted in vitro from the minimal origin of replication of the Escherichia coli chromosome, oriC, and was replaced by a 10 kb R1 miniplasmid, pKN1562, containing the basic R1 replicon and a kanamycin resistance gene. The deletion-insertion was transferred by homologous recombination into the chromosome of a dnaA(ts) strain. P1 transduction separated the origin "mutation" from the dnaA46 allele. Integration of mini-R1 into oriC was verified by Southern blotting and by analysis of the R1 incompatibility phenotype. It was possible to isolate normal R1 miniplasmids from the integrated R1. Chromosome replication was initiated at random times after a short delay. The constructed strains grew 20%-30% slower than the wild type and showed more heterogeneous cell sizes.

Chromosomes, Bacterial↗
PubMed2000

Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif.

Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and maintaining the prereplicative complex (pre-RC) by interacting with the origin recognition complex (ORC) and associating with chromatin origins. These interactions are required to load minichromosome maintenance proteins (MCMs) and other initiator proteins onto replication origins. Although the temperature-sensitive cdc6 mutant, cdc6-1, has been widely used for these studies, the molecular mechanism of the cdc6-1 mutation has been unclear. In this study, we have identified a base substitution at Gly260-->Asp, near the CDC-NTP motif. Using a chromatin immunoprecipitation assay (CHIP), we found that cdc6-1 fails to load Mcm5 onto the replication origins. Chromatin fractions were used to study Mcm5 binding in both the wildtype and mutant background. These studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin. Specifically, the cdc6-1 mutation protein, cdc6(G260D), which failed to load Mcm5 onto replication origins, also failed to unload the Mcm5 protein. Furthermore, the overexpression of wildtype CDC6 accelerated the unloading of Mcm5 from chromatin fractions. In the absence of functional Cdc6, the Mcm5 protein showed nonorigin binding to chromatin with the cell cycle arrested at the G1S phase transition. Our results suggested that the cdc6(G260D) mutant protein fails to assemble an operational replicative complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins.

Amino Acid Substitution↗
PubMedDate not supplied

[A preparation of short DNA chains synthesized in vivo after introduction into DNA from live cells of trioxsalen crosslinks is not enriched by replication initiation regions].

The experiments undertaken in order to verify the trioxsalen-crosslinking method suggested by Russev and Vassilev for isolation of eukaryotic replication origins are described. It was found that the preparation of viral DNA isolated by the above mentioned method from CV-1 cells lytically infected with SV40 was not enriched in sequences including SV40 replication origin. The hybridization pattern of DNA preparation isolated by the trioxsalen-crosslinking procedure from chicken erythroblastosis cells with the cloned fragments of globin gene domain was found to be identical to those of the total DNA probe. The DNA fraction enriched in replication origins was isolated from the same cells with the aid of nascent DNA strand extrusion method by Zannis-Hadjopoulos et al. The hybridization pattern of this DNA fraction with the cloned fragments of chicken alpha-globin gene domain was different from those of total DNA. Taking together, the results of our experiments demonstrate that trioxsalen-crosslinking procedure does not lead to the isolation of replication origins from the objects studied in the present investigation.

Animals↗
PubMed1991

A yeast origin of replication is activated late in S phase.

The mechanism that causes large regions of eukaryotic chromosomes to remain unreplicated until late in S phase is not understood. We have found that 67 kb of telomere-adjacent DNA at the right end of chromosome V in S. cerevisiae is replicated late in S phase. An ARS element in this region, ARS501, was shown by two-dimensional gel analysis to be an active origin of replication. Kinetic analyses indicate that the rate of replication fork movement within this late region is similar to that in early replicating regions. Therefore, the delayed replication of the region is a consequence of late origin activation. The results also support the idea that the pattern of interspersed early and late replication along the chromosomes of higher eukaryotes is a consequence of the temporal regulation of origin activation.

Base Sequence↗
PubMed1994

The genomic instability associated with integrated simian virus 40 DNA is dependent on the origin of replication and early control region.

DNA rearrangements in the form of deletions and duplications are found within and near integrated simian virus 40 (SV40) DNA in nonpermissive cell lines. We have found that rearrangements also occur frequently with integrated pSV2neo plasmid DNA. pSV2neo contains the entire SV40 control region, including the origin of replication, both promoters, and the enhancer sequences. Linearized plasmid DNA was electroporated into X1, an SV40-transformed mouse cell line that expresses SV40 large T antigen (T Ag) and shows very frequent rearrangements at the SV40 locus, and into LMtk-, a spontaneously transformed mouse cell line that contains no SV40 DNA. Stability was analyzed by subcloning G-418-resistant clones and examining specific DNA fragments for alterations in size. Five independent X1 clones containing pSV2neo DNA were unstable at both the neo locus and the T Ag locus. By contrast, four X1 clones containing mutants of pSV2neo with small deletions in the SV40 core origin and three X1 clones containing a different neo plasmid lacking SV40 sequences were stable at the neo locus, although they were still unstable at the T Ag locus. Surprisingly, five independent LMtk- clones containing pSV2neo DNA were unstable at the neo locus. LMtk- clones containing origin deletion mutants were more stable but were not as stable as the X1 clones containing the same plasmid DNA. We conclude that the SV40 origin of replication and early control region are sufficient viral components for the genomic instability at sites of SV40 integration and that SV40 T Ag is not required.

Animals↗
PubMed2006

Cell cycle execution point analysis of ORC function and characterization of the checkpoint response to ORC inactivation in Saccharomyces cerevisiae.

Chromosomal replication initiates through the assembly of a prereplicative complex (pre-RC) at individual replication origins in the G1-phase, followed by activation of these complexes in the S-phase. In Saccharomyces cerevisiae, the origin recognition complex (ORC) binds replication origins throughout the cell cycle and participates in pre-RC assembly. Whether the ORC plays an additional role subsequent to pre-RC assembly in replication initiation or any other essential cell cycle process is not clear. To study the function of the ORC during defined cell cycle periods, we performed cell cycle execution point analyses with strains containing a conditional mutation in the ORC1, ORC2 or ORC5 subunit of ORC. We found that the ORC is essential for replication initiation, but is dispensable for replication elongation or later cell cycle events. Defective initiation in ORC mutant cells results in incomplete replication and mitotic arrest enforced by the DNA damage and spindle assembly checkpoint pathways. The involvement of the spindle assembly checkpoint implies a defect in kinetochore-spindle attachment or sister chromatid cohesion due to incomplete replication and/or DNA damage. Remarkably, under semipermissive conditions for ORC1 function, the spindle checkpoint alone suffices to block proliferation, suggesting this checkpoint is highly sensitive to replication initiation defects. We discuss the potential significance of these overlapping checkpoints and the impact of our findings on previously postulated role(s) of ORCs in other cell cycle functions.

Alleles↗
PubMed1993

DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication.

The ATP-dependent binding of simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication is an essential step in the initiation of SV40 DNA synthesis. Previous studies indicated that the ATP-dependent complex consists of a double hexamer of T antigen at the origin. The binding reaction and the subsequent unwinding of the duplex DNA from the origin were examined using a gel mobility shift assay. T antigen bound to the core origin cooperatively in the presence of ATP. In the presence of human single-stranded DNA-binding protein (HSSB), T antigen, complexed to the core origin, started the unwinding of duplex DNA. At low concentrations of T antigen and in the presence of ATP, DNA polymerase alpha (pol alpha) stimulated the binding of T antigen to the core origin, while HSSB did not. This stimulation resulted in an increase in the subsequent unwinding reaction in the presence of HSSB. Primase alone did not affect the binding reaction and was not required for the stimulation by pol alpha. The stimulation required the hydrolysis of ATP and the AT tract domain of the core origin. Kinetic studies showed that while pol alpha stimulated the binding of T antigen to the core origin, it did not stabilize the complex. Pol alpha also stimulated the formation of the ATP-dependent T antigen-site I complex, a region that also contains an AT-rich sequence. These observations imply a regulatory role for pol alpha in the initiation of SV40 DNA replication.

Adenosine Triphosphate↗
PubMed1983

DNA sequences which support activities of the bacteriophage phi X174 gene A protein.

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.

Bacteriophage phi X 174↗
PubMed2005

Plasmid host-range: restrictions to F replication in Pseudomonas.

Host-range, a fundamental property of a bacterial plasmid, is primarily determined by the plasmid replication system. To investigate the basis of the restricted host-range of the well-studied F-plasmid of Escherichia coli, we characterized in vitro the interactions of the host DnaA initiation protein and DnaB helicase from Pseudomonas aeruginosa and Pseudomonas putida with the replication origin, oriS, and initiation protein, RepE, of the RepFIA replicon. The results presented here show that a pre-priming complex can form at the F-origin with the replication proteins from the non-native hosts in the presence of RepE. However, RepE cannot form a stable complex with DnaB of P. aeruginosa or P. putida but does stably interact with E. coli DnaB. This unstable association may affect the ability of F to replicate in Pseudomonas. In addition, replication studies in vivo suggest that inefficient expression of the RepE initiation protein from its native promoter in Pseudomonas is a factor in restricting its host-range. This, however, is not the only barrier to F replication, as mini-F derivatives with an alternative promoter for RepE expression do not replicate in P. putida and are not stably maintained in P. aeruginosa.

Adenosine Triphosphatases↗
PubMed2001

Feedback controls restrain the initiation of Escherichia coli chromosomal replication.

In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA. After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation. Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback-type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process. The loading of the ring-shaped beta-subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein. Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation. Titration of large amounts of DnaA protein to a non-oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells. All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles. The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.

Bacterial Proteins↗
PubMed1981

In situ photochemical crosslinking of HeLa cell mitochondrial DNA by a psoralen derivative reveals a protected region near the origin of replication.

The in vivo association with proteins of HeLa cell mitochondrial DNA (mtDNA) has been investigated by analyzing the pattern of in situ crosslinking of the DNA by 4'-hydroxymethyl-4, 5',8-trimethylpsoralen (HMT). Either isolated mitochondria or whole cells were irradiated with long wavelength UV light in the presence of ths psoralen derivative, and the mtDNA was then isolated and analyzed in the electron microscope under totally denaturing conditions. No evidence of nucleosomal structure was found. The great majority of the molecules (approximately 90%) had a double-stranded DNA appearance over most of their contour length, with one to several bubbles occupying the rest of the contour, while the remaining 10% of the molecules appeared to be double-stranded over their entire length. Analysis of restriction fragments indicated the presence, in approximately 80% of the molecules, of a protected segment (300 to 1500 bp long) in a region which was centered asymmetrically around the origin of replication so as to overlap extensively the D-loop. Control experiments showed that at most 30% of the bubbles found near the origin could represent D-loops or expanded D-loops: furthermore, it could be excluded that some sequence peculiarity would account for the preferential location of bubbles near the origin of replication. The data have been interpreted to indicate that, in at least 55% of HeLa cell mtDNA molecules, the region around the origin is protected from in situ psoralen crosslinking by proteins or protein complexes which are associated in vivo with the DNA.

Chemical Phenomena↗
PubMed1991

DNA looping between the origin of replication of Epstein-Barr virus and its enhancer site: stabilization of an origin complex with Epstein-Barr nuclear antigen 1.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support replication of Epstein-Barr virus during the latent phase of its life cycle. The DNA segment required for latent replication, oriP, contains two essential binding regions for EBNA-1, termed FR and DS, that are separated by 1 kilobase pair. The FR site appears to function as a replicational enhancer providing for the start of replication at the DS site. We have used electron microscopy to visualize the interaction of EBNA-1 with its binding sites and to study the mechanism for communication between the FR and DS sites. We have found that DNA-bound EBNA-1 forms a DNA loop between the FR and DS sites. From these results, we suggest that EBNA-1 bound to the replicational enhancer acts by a DNA-looping mechanism to facilitate the initiation of DNA replication. Occupancy of the DS site alone is highly sensitive to competition with nonspecific DNA. In contrast, occupancy of the DS site by looping from FR is largely resistant to the competitor DNA. These experiments support the concept that enhancers act in cis from nearby sites to provide a high local concentration of regulatory proteins at their target sites and to stabilize regulatory interactions.

Animals↗
PubMed1987

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.

Adenoviruses, Human↗
PubMed1988

Protein-induced bending of the simian virus 40 origin of replication.

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.

Antigens, Viral, Tumor↗
PubMed1984

Sequence of the putative origin of replication in the UL region of herpes simplex virus type 1 ANG DNA.

Interest has been stimulated concerning the region mapping between 0.38 and 0.42 on the prototype configuration of the herpes simplex virus type 1 (HSV-1) genome due to the high probability of the presence there of a second origin of DNA replication. A 960 bp restriction fragment (HinfI E) of a class II defective HSV-1 ANG DNA has been sequenced using the viral DNA rather than molecularly cloned DNA. This fragment includes the BamHI U/R cleavage site, mapping at approximately 0.4. Part of the sequence derived in this study displays homology with the origins of DNA replication contained in TRS/IRS of HSV-1 and HSV-2 DNA. The homologous region comprising 76 bp occurs as two copies, each of which contains two palindromically arranged copies of an 8 bp sequence identical to the 'consensus' sequence reported to be part of the origin of DNA replication at the terminus of the mammalian adenoviruses. It can be deduced from a comparison of this structure to the TRS/IRS origin of HSV-1 and HSV-2 that there are two origins of replication in the UL region of HSV-1 ANG DNA. Assuming that the orientation of the consensus sequence is relevant to the direction of DNA replication, one can conclude that the UL origin(s) of HSV-1 ANG is (are) bidirectional. It has not yet been possible to clone DNA fragments molecularly which include the region spanning the UL origin(s) of HSV-1 DNA.

Base Sequence↗