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 1,189 records · Page 66Linked to original sources
PubMed1995

Intrachromosomal recombination mediated by the polyomavirus large T antigen.

We used a spleen necrosis virus-based retroviral vector to introduce the polyomavirus replication origin into rat cells and developed a system to analyze homologous recombination events that do not reconstitute a selectable marker. Introduction of the gene coding for the polyomavirus large T antigen into the cell lines by DNA transfection promoted high-frequency recombination between the two retroviral LTRs, leading to amplification and excision of DNA sequences. To analyze homology requirements, we constructed cell lines carrying only the replication origin without exogenous repeats. Most of the cell lines sustained high-frequency recombination, presumably by undergoing homologous recombination between repetitive DNA lying in the vicinity of the integrated origin. Our results indicate that homologous recombination promoted by large T antigen does not require recombination hot spots in the viral genome other than the replication origin and they explain the cytotoxicity observed in some cell types when large T antigen is expressed in the presence of a functional origin.

Animals↗
PubMed1992

A single-stranded DNA-binding protein from Crithidia fasciculata recognizes the nucleotide sequence at the origin of replication of kinetoplast DNA minicircles.

A sequence-specific single-stranded DNA-binding protein from the trypanosomatid protozoan Crithidia fasciculata binds to a sequence of 12 nucleotides located at the origin of replication of kinetoplast DNA minicircles. This sequence, termed the universal minicircle sequence (UMS), is conserved in the kinetoplast DNA minicircles among species of the family Trypanosomatidae. The purified protein binds specifically to the heavy strand of the DNA at this site, which consists of the sequence 5'-GGGGTTGGTGTA-3'. Binding analyses using mutated UMS dodecamers have revealed the significant contribution of each of the individual residues at the binding site, with the exception of the 3'-terminal adenine residue, to the generation of specific protein-DNA complexes. The possible role of this sequence-specific single-stranded DNA-binding protein in replication of kinetoplast DNA minicircles and the relation of the UMS to chromosomal telomeric sequences are discussed.

Animals↗
PubMed1994

Induction of structural changes in the bovine papillomavirus type 1 origin of replication by the viral E1 and E2 proteins.

Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 E1 and E2 proteins with the viral origin of replication (ori). E1 was found to generate significant distortions to the structure of ori, as assayed by KMnO4 oxidation of DNA. The primary site of ori distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of E1 required for ori distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for E1 binding to ori, as assayed by nuclease protection. Under these conditions, E1 bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of E1 were required for binding to the adjacent ori AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of E1 to ori.

Adenosine Triphosphate↗
PubMed1992

Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication.

We have localized a cis-acting sequence that promotes initiation of lytic-phase DNA replication (oriLyt) within the HindIII D fragment of the human cytomegalovirus (HCMV) AD169 genome and investigated its sequence requirements by testing the ability of plasmid constructs to mediate DNA replication in a transient transfection-plus-infection assay. Replication of plasmids containing HCMV oriLyt required at least the virus-specified DNA polymerase activity supplied by HCMV infection of transfected cells and was autonomous in that it did not result from recombination with the virus genome. Progeny molecules in the transient assay were high-molecular-weight tandem oligomers, which is consistent with predictions of a rolling-circle model. Experiments testing subclones of HindIII-D defined a core 2.4-kbp region containing elements required for oriLyt function that extended rightward from around 1.0 kbp upstream of UL57 near the middle of the long unique component of the virus genome. Sequences flanking this core also were needed for full activity. The defined region contains at least four clustered sets of repeated sequence elements identical to or candidate counterparts of elements present in the corresponding cytomegalovirus Colburn lytic-phase replication origin. These elements are novel in that they apparently do not correspond to previously characterized motifs. Also present are multiple copies of elements similar to known binding sites for the transcription factors ATF/CREB, MLTF/USF, and Sp1. Preliminary deletion analysis suggests that multiple components within the boundaries of oriLyt cooperate to enable initiation of HCMV lytic-phase DNA synthesis.

Base Sequence↗
PubMed2001

Completion of replication map of Saccharomyces cerevisiae chromosome III.

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of approximately 40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements on S. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in < or = 10% of cells in the population and two ARS elements active in > or = 90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Chromosome Mapping↗
PubMed1988

Complete enzymatic synthesis of DNA containing the SV40 origin of replication.

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.

Antigens, Polyomavirus Transforming↗
PubMed1998

Functional domains of Rts1 and P1 RepA proteins for initiation of replication.

Rts1 RepA and P1 RepA are trans-acting proteins essential for initiation of replication of Rts1 and P1 plasmids, respectively. We recently found that P1 RepA bound in vitro to the Rts1 replication origin as strongly as Rts1 RepA and activated the origin in vivo. However, the ori activation was quite inefficient. This study shows that by replacing a small region of P1 RepA with the corresponding region of Rts1 RepA, the efficiency of Rts1 ori activation increased markedly. Interestingly, the same subregion of P1 RepA was found to be important for in vivo activation of the P1 origin. Thus, a region essential for efficient activation of the replication origin was assigned to the P1 RepA molecule as well as to the Rts1 RepA molecule. The region was distinct from a domain necessary for in vitro binding to the origin, although both regions were required for in vivo activation of the respective origin.

Bacteriophage P1↗
PubMed1991

A limited number of Bacillus subtilis strains carry a tetracycline-resistance determinant at a site close to the origin of replication.

Several strains of Bacillus subtilis, e.g., 168 derivatives and R, were found to carry a single copy of a tetracycline-resistance (TcR) determinant (named tetBS908) at a site close to the origin of replication on the chromosome. This gene is highly homologous (80% identical) to the TcR determinant of plasmids widely dispersed among aerobic spore-forming bacilli. B. subtilis RM125 (168 strain) transformants which carry a varying number of tetBS908 sequences in a tandem array on the chromosome were constructed and examined for their TcR level. A nearly proportional relationship between the TcR level and copy number of tetBS908 existed.

Bacillus subtilis↗
PubMed2003

aadA confers streptomycin resistance in Borrelia burgdorferi.

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.

Anti-Bacterial Agents↗
PubMed2001

Functionally distinct, sequence-specific replicator and origin elements are required for Drosophila chorion gene amplification.

To meet the demand for the rapid synthesis of chorion (eggshell) proteins, Drosophila ovarian follicle cells amplify the chromosomal loci containing the chorion gene clusters up to 60-fold. Amplification occurs by repeated firing of one or more origins located within each gene cluster. Deletion analyses of transgenic constructs derived from the third chromosome cluster have identified a 320-bp amplification control element (ACE3) required for amplification, as well as several stimulatory amplification enhancing regions (AERs). Two-dimensional (2D) gel analyses have identified multiple DNA replication initiation sites (origins) that partially overlap in location with ACE3 and the AERs. To further study sequence requirements for amplification, a vector was used in which transgenic constructs are protected from chromosomal position effects by flanking insulator elements, the suppressor Hairy-wing protein binding site (SHWBS). Using the buffered vector, the 320-bp ACE3 and an 884-bp element designated ori-beta were found to be necessary and sufficient for amplification. Two-dimensional gels revealed that ori-beta was acting as the origin. In contrast, origin activity could not be detected for ACE3. An insulator placed between ACE3 and ori-beta inhibited amplification, indicating that ACE3 activates ori-beta in cis. The results suggest that ACE3 acts as a replicator and support and extend the replicator model for the organization of metazoan chromosomal replicons.

Animals↗
PubMed1978

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.

Bacillus subtilis↗
PubMed1979

Deoxyribonucleic acid-membrane interactions near the origin of replication and initiation of deoxyribonucleic acid synthesis in Escherichia coli.

A previously reported salt-sensitive binding of deoxyribonucleic acid (DNA) to the cell envelope in Escherichia coli, involving approximately one site per chromosome near the origin of DNA replication, is rapidly disrupted in vivo by rifampin or chloramphenicol treatment and by amino acid starvation. DNA replication still initiates with this origin-specific binding disrupted, even when the disruption extends over the period of obligatory protein and ribonucleic acid synthesis that must precede initiation after release of cells from amino acid starvation. Thus the origin-associated membrane-DNA interaction is not necessary either for the initiation event itself or for the maturation of a putative initiation apparatus in E. coli.

Amino Acids↗
PubMed1990

Bending of the origin of replication of E. coli by binding of IHF at a specific site.

In studies of DNA replication in Escherichia coli, an important question concerns the role of the initiator protein DnaA. This protein is known to bind to a specific 9-bp sequence in the origin of replication, but it is not understood how it can recognize another, relatively distant, 13-bp sequence that has no homology to the binding site but is where the DnaA protein serves its catalytic function in the initiation of DNA replication. This effect of DnaA might be achieved by bending of DNA in this region. I have searched for putative binding sites for integration host factor (IHF), a protein known to bend DNA. Here I report the finding of an IHF binding site in the E. coli origin and present direct evidence that IHF binds and causes DNA bending in this region. On the basis of these results I propose a model wherein formation of a higher-order nucleoprotein structure would facilitate the action of DnaA protein in the initiation events.

Bacterial Proteins↗
PubMed1994

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.

Base Sequence↗
PubMed1999

Identification and characterization of MmORC4 and MmORC5, two subunits of the mouse origin of replication recognition complex.

Two new members of the mouse origin recognition complex (ORC) have been cloned that are closely related to Saccharomyces cerevisiae ORC4 and ORC5 as well as to their human homolog. Both MmORC4p and MmORC5p have a putative nucleotide triphosphate binding motif. Transcription of MmORC4 and MmORC5 is not suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. The transcription levels of both ORC genes are constantly high in all phases of the cell cycle. A screen based on the two-hybrid approach suggests that the product of the ORC4 gene interacts with the ORC2, but not with the ORC1 protein. The conservation of structure among members of the ORC4- and ORC5-related family of proteins suggests that these proteins play a key role in the initiation of DNA replication in all eukaryotes.

3T3 Cells↗
PubMed2007

Developmentally regulated histone modifications in Drosophila follicle cells: initiation of gene amplification is associated with histone H3 and H4 hyperacetylation and H1 phosphorylation.

We have used gene amplification in Drosophila follicle cells as a model of metazoan DNA replication to address whether changes in histone modifications are associated with replication origin activation. We observe that replication initiation is associated with distinct histone modifications. Acetylated lysines K5, K8, and K12 on histone H4 and K14 on histone H3 are specifically enriched during replication initiation at the amplification origins. Strikingly, H4 acetylation persists at an amplification origin well after replication forks have progressed significantly outward from the origin, indicating that H4 acetylation is associated with origin regulation and not histone deposition at the replication forks. Origin recognition complex subunit 2 (orc2) mutants with severe amplification defects do not abolish H4 acetylation, whereas the dup/cdt1 mutant delays the appearance of acetylation foci, and mutants in rbf result in temporal persistence. These data indicate that core histone acetylation is associated with origin activity. Furthermore, follicle cells undergoing gene amplification exhibit high levels of histone H1 phosphorylation. The patterns of H1 phosphorylation provide insights into cell cycle states during amplification, as H1 kinase activity in follicle cells is responsive to high Cyclin E activity, and it can be abolished by overexpressing the retinoblastoma homolog, Rbf, that represses Cyclin E. These data suggest that amplification origins are able to initiate when the cells are in a late S-phase, when the genome is normally not licensed for replication.

Acetylation↗
PubMed1990

RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.

Animals↗
PubMed2006

Application of artificial zinc-finger proteins to inhibition of DNA replication of human papillomavirus.

Recently, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to inhibition of replication of a mammalian DNA virus, human papillomavirus (HPV) type 18. Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed to block binding of the HPV-18 E2 replication protein to the replication origin. Both the designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited the viral DNA replication: AZP(HPV)-2, especially, reduced the replication level to approximately 10%. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses, but also to mammalian DNA viruses.

Binding Sites↗