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 505 records · Page 28Linked to original sources
PubMed1990

The DNA unwinding element in a yeast replication origin functions independently of easily unwound sequences present elsewhere on a plasmid.

We have previously identified a DNA unwinding element (DUE) in autonomously replicating sequences (ARSs) and demonstrated a correlation between single-strand-specific nuclease hypersensitivity of the DUE and ARS-mediated plasmid replication in yeast. The DUE in the H4 ARS is the most easily unwound sequence in a supercoiled DNA molecule, in the context of the Ylp5 plasmid. To determine whether sequences which are more readily unwound than the ARS can influence replication activity, we have inserted such sequences, called 'torsional sinks', into the plasmids at a site distal to the ARS. We show that the torsional sink sequences effect reduction or elimination of the nuclease hypersensitivity of a variety of H4 ARS derivatives. However, we detect no difference in the in vivo replication activity of an individual ARS plasmid with or without a torsional sink. Thus, the function of the DUE in a yeast replication origin is unaffected by easily unwound sequences present elsewhere on the same plasmid.

Base Sequence↗
PubMed1993

Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.

Adenosine Deaminase↗
PubMed2005

An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin.

In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.

Adenosine Triphosphate↗
PubMed1999

Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors.

Lactobacillus reuteri L1 and N16 strains contain a 7.0-kb plasmid (pTE80) and a 15-kb plasmid (pTE15), respectively, encoding resistance to erythromycin (Em(r)). Physical maps of both plasmids were established. Nucleotide sequences of the genetic determinants encoding Em(r) on pTE80 and pTE15 revealed the existence of a very similar (ca. 99% nucleotide sequence and ca. 98% amino acid sequence identity) open reading frame for an Em(r) transmethylase gene (erm) in both plasmids. These structural erm genes, 753 and 750 bp in length, respectively, were highly related (ca. 98% nucleotide sequence and ca. 97% amino acid sequence identity) to the erm gene of L. fermentum plasmid pLEM3. Sequence analysis showed that these two erm genes from pTE80 and pTE15 could be categorized under the ermB (ermAM) class. These are the first members of the ermB (ermAM) class of Em(r) determinant from L. reuteri to be characterized at the nucleotide sequence level. The Em(r) gene from pTE80 (erm80) was then ligated into pUC18/19 to construct replication origin (RO)-screening vectors pUE80(+) and pUE80(-) (pUE80(+/-)). These plasmids contain the pUC18/19-derived multiple cloning site, ampicillin-resistance trait, and the LacZ' gene, which enable direct screening for recombinants in Escherichia coli. Once the recombinant contains a RO from L. reuteri, the Em(r) trait of erm80 is used as a selection marker for the replication of the chimeric plasmid as it is transformed into L. reuteri using the cloned RO as a replicon. Replication regions from pTE80 and pTE15 were successfully cloned into the constructed vector pUE80(-). The RO cloned from pTE80 was further identified as being highly stable in L. reuteri and also bearing a relatively narrow host range compared with that of pTE15. The Em(r) determinant (erm80) and RO cloned from pTE80 could be used in the future construction of derivatives of cloning vectors for this microbe. Moreover, the pUE80(+/-) and pTE80-RO constructed in this study have the potential to be developed as a suicide vector and an E. coli-L. reuteri shuttle vector, respectively.

Amino Acid Sequence↗
PubMed1996

Activation of replication origins in phi29-related phages requires the recognition of initiation proteins to specific nucleoprotein complexes.

Protein p6 of Bacillus subtilis phage phi29 activates the initiation of viral DNA replication by forming a multimeric nucleoprotein complex at the origins of replication, located at both ends of the linear genome. This activation requires a precise positioning of the protein p6 array with respect to the initiation site. To investigate this activation mechanism, we have purified the phi29 protein p6 counterparts from the related phages Nf and GA-1 and analyzed the formation of complexes with DNA. In the homologous protein p6-DNA complexes the phi29 and Nf protein arrays showed an identical positioning, different than that of the GA-1 protein array. In contrast, in the heterologous complexes the protein showed a different arrangement except in the case of the Nf protein-phi29 DNA complex. We have also purified the proteins involved in the initiation of replication (terminal protein and DNA polymerase) from phages Nf and GA-1 and measured the ability of the different p6 proteins to activate homologous and heterologous replication origins. The results obtained indicate that the activation requires not only the formation of a specific nucleoprotein complex but also its specific recognition by the proteins involved in the initiation of DNA replication.

Bacillus Phages↗
PubMed2004

Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA-ATP.

Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5'TGTGNAT/AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5'TG/TGGATCAG/A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation.

Adenosine Diphosphate↗
PubMed2002

The human cruciform-binding protein, CBP, is involved in DNA replication and associates in vivo with mammalian replication origins.

We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with these origins was found to be approximately 9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with ors8 and ors12 was found at the G(1)/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G(1)/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3beta, -epsilon, -gamma, and -zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3epsilon, -gamma, and -zeta antibodies alone or in combination inhibited p186 replication by approximately 50-80%, while anti-14-3-3beta antibodies had a lesser effect ( approximately 25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.

Base Sequence↗
PubMed1998

Regulation of mammalian replication origin usage in Xenopus egg extract.

Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.

Animals↗
PubMed1992

Replication origins and pause sites in sea urchin mitochondrial DNA.

We have used a combination of one- and two-dimensional agarose gel electrophoresis, and solution hybridization to strand-specific probes, to map the replication origin of sea urchin mitochondrial DNA and to investigate the structure of replication intermediates. These assays are consistent with replication initiating unidirectionally from the D-loop region by D-loop expansion, as in vertebrates. A prominent site of initiation of lagging-strand synthesis lies at, or near to, the boundary between the genes for ATPase 6 and COIII, which is also close to a pause site for leading-strand synthesis. These findings suggest a role for pause sites in the regulation of mitochondrial transcription and replication, possibly involving template-binding proteins.

Animals↗
PubMed1986

Mechanism of autonomous control of the Escherichia coli F plasmid: different complexes of the initiator/repressor protein are bound to its operator and to an F plasmid replication origin.

E protein, the 29 kd product of the F plasmid repE gene, plays both positive and negative roles in the autoregulation of F replication. We have cloned and expressed the repE gene in an inducible ATG-fusion vector and have detected specific binding of E protein to the repE operator and to four 19-base pair direct repeats (incB) within the F plasmid replication origin ori2. Binding of E protein at the repE operator occurs with higher affinity than at ori2(incB) and gives almost complete protection to at least 30 base pairs, whereas binding of E protein to the direct repeats in the ori2 region shows an alternating pattern of enhanced and reduced sensitivity to DNAase cleavage consistent with a protein-induced folding of the DNA. These results provide direct biochemical support for a model of F plasmid replication in which the E protein serves both as an initiator of replication and as an autorepressor of its own synthesis.

Base Sequence↗
PubMed2005

Functional analysis of two putative chromosomal replication origins from Pseudomonas aeruginosa.

Two autonomously replicating elements previously isolated from Pseudomonas aeruginosa were characterized in vitro for pre-priming complex formation using combinations of replication proteins from P. aeruginosa and Escherichia coli. The results of these studies showed that the P. aeruginosa DnaA and DnaB proteins could form a pre-priming complex on plasmid templates containing either of the two autonomously replicating elements of P. aeruginosa, pYJ50 (containing oriCI), and pYJ52 (containing oriCII), or the E. coli chromosomal origin (plasmid pYJ2). The E. coli DnaA, DnaB, and DnaC proteins were also able to form a pre-priming complex on pYJ2, pYJ50, and pYJ52. Neither pYJ50 nor pYJ52 could be established in E. coli, suggesting a block in steps subsequent to the formation of the pre-priming complex. Similarly, pYJ2 could not be established in P. aeruginosa. Since pYJ50 and pYJ52 could be established in P. aeruginosa and both putative origins form a pre-priming complex in vitro, attempts were made to delete each of these two putative origins. The results indicate that the oriCI sequence is essential for cell viability under typical laboratory growth conditions but that oriCII is not.

Adenosine Triphosphatases↗
PubMed1992

Physical characterization of the replication origin of the cryptic plasmid pCB101 isolated from Clostridium butyricum NCIB 7423.

The complete nucleotide sequence of a 3484-bp Sau3A fragment, previously shown to carry the replication origin of the Clostridium butyricum NCIB 7423 plasmid pCB101 (6.05 kb), has been determined. Of the four open reading frames (ORF A-D) identified within this fragment, two (B and C) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that both polypeptides are required for autonomous replication of the plasmid in Bacillus subtilis. ORF C is immediately preceded by a small ORF (C') that encodes a relatively small polypeptide (50 amino acids) that demonstrates significant homology with RepA of plasmid pLS1. Whereas the ORF C polypeptide (27,100 Da) exhibits no homology to any known protein, that encoded by ORF B (RepB, 43,039 Da) exhibits significant homology with the Rep proteins of the pC194/pUB110 subfamily of single-strand (ss) DNA plasmids, which are widely distributed in gram-positive bacteria. Conserved amino acids include the presumed active site of topoisomerase activity and four cysteine residues in the N-terminus of all Rep proteins compared. The repB gene is preceded by a sequence motif exhibiting substantial homology to the "plus" origins of this family of ss DNA plasmids and was shown to act as a "hot spot" for deletion formation in certain plasmid chimaeras. The compelling suggestion that pCB101 replicates via a rolling circle mechanism was substantiated by the demonstration of ss DNA replication intermediates in B. subtilis cells carrying a pCB101-derived plasmid.

Amino Acid Sequence↗
PubMed2004

Discovery of a major D-loop replication origin reveals two modes of human mtDNA synthesis.

Mammalian mitochondrial DNA (mtDNA) replication has long been considered to occur by asymmetric synthesis of the two strands, starting at the multiple origins of the strand-displacement loop (D-loop). We report the discovery of a major replication origin at position 57 in the D-loop of several human cell lines (HeLa, A549, and 143B.TK-) and immortalized lymphocytes. The nascent chains starting at this origin, in contrast to those initiated at the previously described origins, do not terminate prematurely at the 3' end of the D-loop but proceed well beyond this control point, behaving as "true" replicating strands. This origin is mainly responsible for mtDNA maintenance under steady-state conditions, whereas mtDNA synthesis from the formerly identified D-loop origins may be more important for recovery after mtDNA depletion and for accelerating mtDNA replication in response to physiological demands.

Cell Line↗
PubMed1991

Structure and function of DnaA and the DnaA-box in eubacteria: evolutionary relationships of bacterial replication origins.

DnaA protein (a trans-acting element) and its binding sequence, DnaA-box: (a cis-acting element) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other enteric bacteria. Recently these two elements have been found to be conserved in three Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus and Mycoplasma capricolum) as well as in Gram-negative pseudomonads. DnaA protein was also found to be essential in the initiation of the replication of the B. subtilis chromosome, and regions containing multiple repeats of DnaA-box (DnaA-box region) are found to be active as autonomously replicating elements both in B. subtilis and pseudomonads. In this MicroReview we compare first the structures of these DnaA-box regions and their locations on the chromosome and then functional aspects of DnaA protein and DnaA-box regions in the initiation and regulation of chromosomal replication. From these observations we propose evolutionary relationships between replication origins of eubacteria.

Amino Acid Sequence↗
PubMed1989

Nucleoprotein structures at positively regulated bacterial promoters: homology with replication origins and some hypotheses on the quaternary structure of the activator proteins in these complexes.

The quaternary structure of regulatory proteins undoubtedly plays an important role in the initiation of transcription and DNA replication. To date, the best-characterized regulatory proteins are oligomers in which promoters are bound together by isologous interactions. From the examples presented in this article, it appears that the formation of certain nucleoprotein complexes implicated in transcription initiation might involve heterologous rather than isologous interactions, allowing differentiation between two classes of transcription activators. Nucleoprotein structures present at the oriC replication origin and at malB promoters show striking homologies.

Bacteria↗
PubMed2006

Broadening of DNA replication origin usage during metazoan cell differentiation.

We have examined whether replication of the chicken beta-globin locus changes during differentiation of primary erythroid progenitors into erythrocytes. In undifferentiated progenitors, four principal initiation sites and a replication fork pausing region (RFP) were observed. Forty-eight hours after induction of differentiation, the principal sites were maintained, even in the activated beta(A)-globin gene, some minor sites were enhanced, three new sites appeared and the RFP disappeared. One of the activated origins showed increased histone H3 K9K14 diacetylation, but the others did not. These results demonstrate a broadening of DNA replication origin usage during differentiation of untransformed metazoan cells and indicate that histone H3 diacetylation, other histone modifications so far reported and transcription are not crucial determinants of origin selection in this system.

Acetylation↗
PubMed1983

Binding of simian virus 40 large T antigen from virus-infected monkey cells to wild-type and mutant viral replication origins.

The binding of purified simian virus 40 (SV40) large T antigen (T) from monkey cells infected with wild-type SV40 virus to viral replication origin-containing DNA fragments was studied by DNase footprinting and restriction endonuclease protection methods. A strong affinity binding site (site 1) of 30 base-pairs and a second, adjacent 40 base-pair lower affinity binding site (site 2), which includes the origin of replication, were detected in these assays. These sites appear identical to those previously noted in similar assays performed with the Ad2 + D2 (D2) T protein. Heating T prior to incubation with DNA significantly increased the binding to these two sites, and the order of binding did not change. Moreover, protection of sequences was observed on both strands in these two sites suggesting that both strands can participate in binding of T to these two sites. Studies with DNAs from two internal site 2 deletion mutants as well as with a DNA fragment lacking the distal 13 base-pairs of site 2 revealed that sequences in the "early" portion of site 2 are sufficient for T binding to the intact site. Furthermore, use of a new assay that measures protection of DNA sequences from specific restriction enzyme cleavage revealed that site 2 can be subdivided into two subsites, 2A and 2B, where 2A corresponds to the above-noted early segment of this locus. In titration experiments, the affinity of 2A for T was greater than that of 2B. Hence, binding to a major portion of the replication initiation sequence (i.e. site 2) is the product of at least two interactions. Finally, analyses performed with DNA from a site 1 deletion mutant, cs1085, revealed that prior binding of T to this locus did not facilitate its binding to site 2. The opposite effect was observed when D2T was employed in these assays. Thus, although similar in many respects, these proteins display a detectable difference in their DNA binding mechanisms.

Animals↗