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 1,099 records · Page 61Linked to original sources
PubMed1995

The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing.

The origin recognition complex (ORC) is a six protein assembly that binds S. cerevisiae origins of replication and directs DNA replication throughout the genome and transcriptional silencing at the yeast mating-type loci. Here we report the cloning of the genes encoding the 120 kDa (ORC1), 62 kDa (ORC3), and 56 kDa (ORC4) subunits of ORC and the reconstitution of the complete complex after expression of all six subunits in insect cells. Orc1p is related to Cdc6p and Cdc18p, which regulate DNA replication and mitosis, and to Sir3p, a regulator of transcriptional silencing. The N-terminal region of Orc1p is highly related to Sir3p, and studies of Orc1p/Sir3p chimeric proteins indicate that this domain is dedicated to the transcriptional silencing function of ORC.

Amino Acid Sequence↗
PubMed2004

Chemical footprinting of structural and functional elements of dhfr oribeta during the CHOC 400 cell cycle.

Oribeta, an origin of replication 3' to Chinese hamster dihydrofolate reductase (dhfr) gene, contains several sequence elements that function as components of a chromosomal replicator. Here we have examined sensitivity to KMnO(4) in vitro and in living cells of three regions within dhfr oribeta which contribute to replicator function: the origin of bidirectional DNA replication (OBR) that serves as an initiation site for DNA synthesis, a stably bent DNA region that binds activator protein one (AP-1) and RIP60 in vitro, and an AT-rich region that contains a dA/dT(23) dinucleotide repeat that has properties of a DNA unwinding element. The in vitro patterns of KMnO(4) modification in linear plasmid differed from that in supercoiled plasmid most prominently in the dA/dT(23) repeat, with evidence of palindrome extrusion in supercoiled plasmid. Although palindrome extrusion was not detected in genomic DNA during the cell cycle, the pattern of genomic DNA modification within the dA/dT(23) repeat differed substantially from that of either linear or plasmid DNA in vitro. An AT-rich region that borders the dA/dT repeat was also highly sensitive to modification by KMnO(4) in cells. Within the bent DNA region, the patterns of chemical modification of both the AP-1 and RIP60 sites differed between plasmid and genomic DNA, and minor differences in the in vitro and cellular modification patterns also were observed for the OBR. Nonetheless, there was little evidence of cell cycle-specific modifications in any sequence examined. These studies suggest that sequences within dhfr oribeta adopt specific conformations in cells, with the most prominent changes in the AT-rich region associated with the dA/dT(23) repeat and DNA unwinding.

Animals↗
PubMed1977

Physical structure of the replication origin of bacteriophage lambda.

The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined. Three of the ori- mutations are small deletions, and one is a transversion. The sequence of the origin region, defined by these mutations, contains a number of unusual features.

Base Sequence↗
PubMed1993

Large T-antigen and sequences within the regulatory region of JC virus both contribute to the features of JC virus DNA replication.

The requirements for the DNA replication of the human papovavirus JC were analyzed using JC T-antigen as well as the T-antigens of the related viruses SV40 and BK. With all three T-antigens, the boundary of the core origin mapped on the early side to position 5093 of the viral genome. In conjunction with earlier studies, the core origin of DNA replication was therefore defined as a 68-bp region which, similar to the SV40 core origin, contains three major structural elements, early palindrome, T-antigen binding site II, and A/T-rich tract. Replication was stimulated by sequences flanking the core origin on the early side. Specifically, the stimulating sequences on the early side were identified as T-antigen binding site I. The degree to which flanking sequences were able to stimulate viral DNA replication was dependent on the T-antigen used in the experiment, with JC T-antigen relying most and BK T-antigen relying least on the flanking sequences. SV40 T-antigen showed an intermediate dependence. The same hierarchy was observed when replication activities were compared. BK T-antigen was more active in replicating DNA than SV40 T-antigen, which in turn was more effective than JC T-antigen. Dependence on flanking sequences is, thus, inversely correlated to the replicating activity of the respective T-antigen, showing that, in addition to the origin, the T-antigen contributes to the characteristics of JC virus DNA replication.

Antigens, Viral, Tumor↗
PubMed2002

DNA replication joins the revolution: whole-genome views of DNA replication in budding yeast.

Replication origins, which are responsible for initiating the replication of eukaryotic chromosomal DNAs, are spaced at intervals of 40 to 200 kb. Although the sets of proteins that assemble at replication origins during G(1) to form pre-replicative complexes are highly conserved, the structures of replication origins varies from organism to organism. The identification of replication origins has been a labor-intensive task, requiring the analysis of chromosomal DNA replication intermediates. As a result, only a few replication origins have been identified and studied. In a pair of recently published papers, Raghuraman and colleagues and Wyrick, Aparicio and colleagues provide complementary microarray-based approaches to the identification of replication origins. These genome-wide views of DNA replication in Saccharomyces cerevisiae provide new insights into the way that the genome is duplicated and hold promise for the analysis of other genomes.

Biological Evolution↗
PubMed1987

P1 plasmid replication requires methylated DNA.

Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.

Base Sequence↗
PubMed1987

Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin.

In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307:516-520, 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.

Animals↗
PubMed1994

DNA helical instability facilitates initiation at the SV40 replication origin.

Previous analysis of mutations in bacterial and yeast replication origins has identified a genetic component, termed a DNA unwinding element (DUE), whose intrinsic helical instability is essential for origin function. For the SV40 replication origin, we show here that the early palindrome (EP) and A + T-rich (AT) domains both exhibit helical instability, despite their dissimilar A + T compositions. To test the possible contribution of helical instability to SV40 origin function, the relationship between helical stability of mutant origin sequences and their known origin activity in vitro and in vivo was examined. Origin activity correlates inversely with the helical stability of mutations within the EP domain but not the AT domain or the T-antigen binding domain. The quantitative correlation holds for four different measures of origin activity determined in vitro and in vivo. An even better-correlated collection of mutations was found in a specific portion of the EP domain. This specific EP subdomain coincides with the sequence known to be strand-separated after T-antigen binds the origin in vitro and with the origin of bidirectional replication in vivo. Our analysis of origin mutations indicates that the helical instability of the specific EP subdomain is required to facilitate T-antigen-induced melting and the initiation of DNA replication. The sensitivity of the required EP subdomain to mutations that stabilize the DNA helix defines the DUE of the SV40 replication origin.

Adenine↗
PubMed2003

Identification of a cis-element that determines autonomous DNA replication in eukaryotic cells.

A 36-bp human consensus sequence (CCTMDAWKSGBYTSMAAWTWBCMYTTRSCAAATTCC) is capable of supporting autonomous replication of a plasmid after transfection into eukaryotic cells. After transfection and in vitro DNA replication, replicated plasmid DNA containing a mixture of oligonucleotides of this consensus was found to reiterate the consensus. Initiation of DNA replication in vitro occurs within the consensus. One version, A3/4, in pYACneo, could be maintained under selection in HeLa cells, unrearranged and replicating continuously for >170 cell doublings. Stability of plasmid without selection was high (> or =0.9/cell/generation). Homologs of the consensus are found consistently at mammalian chromosomal sites of initiation and within CpG islands. Versions of the consensus function as origins of DNA replication in normal and malignant human cells, immortalized monkey and mouse cells, and normal cow, chicken, and fruit fly cells. Random mutagenesis studies suggest an internal 20-bp consensus sequence of the 36 bp may be sufficient to act as a core origin element. This cis-element consensus sequence is an opportunity for focused analyses of core origin elements and the regulation of initiation of DNA replication.

Amino Acid Sequence↗
PubMed1976

Multiple origins and circular structures in replicating T5 bacteriophage DNA.

Replicating T5 phage DNA was gently isolated using NaI density gradient centrifugation and examined by electron microscopy. At the beginning of phage DNA synthesis, linear unit-length T5 DNA molecules containing from one to four replicating "eye-loops" were consistently observed. Replication in these molecules was found to proceed bidirectionally from multiple, internal origins. A primary origin of replication is located near the center of the T5 genome, which does not coincide with the location of any of the nicks (single-strand breaks) found in mature T5 DNA. The initiation of replication at the various origins within an individual molecule does not appear to follow any definite temporal sequence. At later times in the infection, we have observed a significant number of circular T5 DNA molecules-both replicating and nonreplicating-whose average circumference is approximately the length of mature T5 DNA minus the terminal redundancy. The replicating circular molecules appear to be either in a theta configuration, a sigma configuration with the tails all being less than the length of the circle, or a combination of theta and sigma forms.

Coliphages↗
PubMed1986

A new membrane-associated DNA replication protein, the gene 69 product of bacteriophage T4, shares a patch of homology with the Escherichia coli dnaA protein.

A new phage T4 DNA replication protein, gp69, is found to be associated with membrane fractions, as predicted by the translated base sequence of gene 69. In addition, gp69 shares a patch of homology with a segment of the Escherichia coli dnaA initiation protein. The patchy homology of dnaA protein and gp69 suggests that they may serve some similar functions, such as interactions with the same E. coli components in bacterial and viral DNA replication. We have shown before that gene 69 spans an origin of T4 DNA replication, and that this origin is preferentially associated with membrane fractions. We suggest the possibility that gp69 is involved in the attachment of this origin to the bacterial envelope.

Amino Acid Sequence↗
PubMed1988

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. II. Effects of DNA replication of mutations in the 30-nucleotide icosahedral bacteriophage origin.

phi X174 viral strand circular DNA can be synthesized in vitro from phi X174 replicative form I (RFI) DNA in the presence of the phi X A protein, the Escherichia coli DNA polymerase III elongation system, the E. coli rep helicase, and the E. coli single-stranded DNA binding protein. M13mp9 or pBR322 RFI DNAs containing a 30-base pair sequence found at the phi X origin of replication supported phi X A protein synthesis as well as the phi X template, giving rise to a net molar excess of deoxynucleotide incorporation. In this paper, we show that mutations in positions 1-3 of the 30-nucleotide origin replicated at a lower efficiency than plasmids containing the wild-type origin, because of a deficiency in the reinitiation reaction. Mutations in positions 4-7, upstream of the phi X A protein cleavage site, failed to support replication because of their inability to support nicking. An origin containing a mutation at the residue to which the phi X A protein is covalently linked to the DNA was an active template that supported a net molar excess of incorporation. Mutations at the 3' end of the origin region, retaining only the first 21-25 nucleotides of the 30-base pair origin, failed to support replication because of impaired binding of the phi X A protein to the template and consequently poor nicking. A construct bearing the first 28 nucleotides of the origin supported wild-type replication, as did a plasmid containing a 28-mer origin with a point mutation at position 26, but this latter construct also appeared to be partially deficient in phi X A protein binding activity. These results are consistent with the presence of a phi X A protein binding domain at the 3' end of the origin.

Bacteriophage phi X 174↗
PubMed2004

Bending of adenovirus origin DNA by nuclear factor I as shown by scanning force microscopy is required for optimal DNA replication.

Nuclear factor I (NFI) is a transcription factor that binds to the adenovirus type 5 (Ad5) origin of replication and recruits the adenovirus DNA polymerase, thereby stimulating initiation of DNA replication in vitro. Using scanning force microscopy, we demonstrate that NFI induces a 60 degrees bend upon binding to the origin. The A/T-rich region preceding the core recognition sequence of NFI influences the DNA bend angle, since substitution of A/T base pairs by G/C base pairs severely decreases bending. Mutations in the A/T-rich region do not affect binding of NFI to DNA. However, mutations that reduce the protein-induced bend lead to a loss of NFI-stimulated replication, indicating that DNA bending is functionally important. In contrast, basal initiation or DNA binding of the polymerase is not impaired by these origin mutations. We conclude that binding of NFI to the Ad5 origin causes structural changes in DNA that are essential for the stimulatory function of NFI in replication. We propose that NFI-induced origin bending facilitates the assembly of a functional initiation complex.

Adenoviruses, Human↗
PubMed1990

Structural modifications induced by the mtDBP-C protein in the replication origin of Xenopus laevis mitochondrial DNA.

The structure of the non-coding region of Xenopus laevis mitochondrial DNA has been studied by electron microscopy analysis of DNA molecules end-labelled with streptavidin-ferritin. We have shown that the effect of a protein modifying the shape of the DNA double-helix can be studied and precisely located by this method. It was found that the non-coding region contains curved segments and that the mitochondrial protein mtDBP-C preferentially enhances the curvature of the promoters-replication origin region.

Animals↗
PubMed1994

Two steps in the assembly of complexes at yeast replication origins in vivo.

The integration of chromosomal DNA replication into the eukaryotic cell cycle might involve temporal regulation of interactions between cellular factors and replication origins. We show here that yeast replication origins exist in two chromatin states during the cell cycle. In the postreplicative state, genomic footprints closely resemble those produced in vitro by the purified ORC and ABF1 proteins, indicating that the binding of these proteins to replication origins is not sufficient to drive the initiation of DNA replication. The prereplicative state is characterized by an additional region of protection overlapping the ORC footprint. This prereplicative complex appears near the end of mitosis and persists through G1. After entry into S phase, origins return to the postreplicative state. Similarities in temporal regulation of the prereplicative state and the Xenopus licensing factor suggest that mechanisms limiting DNA replication to once per cell cycle may be conserved among eukaryotes.

Base Sequence↗
PubMed1989

Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin.

We isolated revertants of a polyomavirus whose origin of DNA replication contains a point mutation in the palindrome to which large T antigen binds. Four independent second-site revertants contain an Asp-286----Asn-286 substitution in large T antigen. This mutant large T antigen activates replication of DNAs containing the mutant polyomavirus origin as well as replication of DNAs containing the wild-type origin; however, replication of DNAs with enhancer mutations is not activated by this large T antigen. The Asn-286 mutation occurs in a positively charge region of large T antigen near the location of several mutations which inactivate DNA replication. We suggest that this region of large T antigen is responsible for recognition of specific DNA sequences at the origin and that ionic forces are important for this interaction.

Amino Acid Sequence↗
PubMed1999

Replication origins of mammalian chromosomes: the happy few.

Replication of eukaryotic cell genomes is a tightly controlled process occurring once and only once per cell cycle. Replication initiates at several thousand origins, whose cis-acting sequences and trans-acting proteins have been partially characterized in the yeast S. cerevisiae in the last few years. In contrast, identification of origins of DNA replication in mammalian cells have proven much more difficult. Currently, less then 20 bona fide mammalian origins have been identified, of which only few characterized in detail. Here we discuss the available methods for origin identification in mammalian DNA and the main results, sometimes controversial, so far generated by their application. In particular, we review the currently available information concerning the three best characterized origins, namely those in the lamin B2 and b-globin gene domains in human cells and the one located downstream of the dihydrofolate reductase gene in hamster cells.

Animals↗
PubMed1974

Polyoma DNA: a physical map.

The action of restriction enzymes on polyoma DNA was studied with uniformly (32)P-labeled viral DNA obtained from either infected 3T6 or secondary mouse-embryo cells. Three restriction enzymes were used to construct a physical map of the polyoma genome. An enzyme from Hemophilus parainfluenzae, Hpa(II), cleaved polyma DNA into eight unique fragments (Hpa(II)-1 to Hpa(II)-8), ranging in size from 27.3 to 1.8% of the genome. An enzyme from Hemophilus influenzae, Hin(III), gave two fragments (56 and 44%); and a third enzyme from Escherichia coli, EcoR(I), cut at a single unique site. The physical map of the polyma genome was constructed from methods involving: (1) further digestion of the fragments produced by enzymes EcoR(I) and Hin(III) with Hpa(II), and (2) analysis of the products of partial digestion with Hpa(II). Analysis by electron microscopy of replicating DNA molecules (less than 50% replicated) cut with the Hin(III) enzyme, in combination with other studies, has indicated that the origin of DNA replication is located at 71 +/- 3 map units from the EcoR(I) cleavage site, probably in Hpa(II)-5.

Adenine↗