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At least 379 records · Page 21Linked to original sources
PubMed2003

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

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

Bacterial Proteins↗
PubMed1985

The herpes simplex virus amplicon: analyses of cis-acting replication functions.

Previous studies have shown that defective virus vectors (amplicons) derived from herpes simplex viruses could be efficiently propagated in virus stocks in the presence of trans-acting helper virus functions. The present study established that two separate cis-acting functions--a DNA replication origin and a cleavage/packaging signal--are required for amplicon propagation. Using deleted derivatives of cloned amplicons, we mapped one of the viral DNA replication origins (ori-2 or oriL) at coordinate 0.422 of the standard HSV-1 genome and at an equivalent position within the HSV-2 genome.

Base Sequence↗
PubMed1994

An asymmetric nucleotide in the parvoviral 3' hairpin directs segregation of a single active origin of DNA replication.

The 3' telomere of the linear single-stranded DNA genome of minute virus of mice (MVM), a murine parvovirus, can assume a complex hairpin structure. This contains a stem in which there is a mismatched 'bubble' sequence where a GA doublet opposes a GAA triplet. During replication, this hairpin is copied to form an imperfect palindrome which bridges adjacent genomes in a dimer duplex intermediate, leaving the two 'bubble' sequences embedded in potential replication origins on either side of the axis of symmetry. Such junctions are resolved asymmetrically in vitro in a DNA synthetic reaction which requires the viral initiator protein NS1. We show that the sequence surrounding the doublet is a potent origin, but the analogous region containing the triplet is completely inactive. The active origin is approximately 50 bp long, extending from an Activated Transcription Factor binding site at one end to a position some 7 bp beyond the major initiation site, to which NS1 ultimately becomes covalently attached. The actual sequence of the GA doublet is unimportant, but insertion of any third nucleotide here inactivates the origin, indicating that it represents a critical spacer element. Segregation of this asymmetry, therefore, allows the virus to confine replication initiation to one particular telomeric configuration.

Base Sequence↗
PubMed1988

Initiation of DNA replication at cloned origins of bacteriophage T7.

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.

Base Sequence↗
PubMed1976

Isolation and propagation of a segment of the simian virus 40 genome containing the origin of DNA replication.

Heteroduplex DNA molecules formed from two DNAs that differ from each other by a deletion can be cleaved at the mismatched region (a deletion loop) with the single-strand-specific S1 endonuclease. A heteroduplex DNA molecule, constructed from the DNA of simian virus 40 (SV40) mutant with a deletion of the map region 0.54-0.55 and the DNA of a second SV40 mutant having a deletion of the map segment 0.70-0.73, is cleaved twice with S1 endonuclease. One of the products is a DNA fragment of about 0.13 the length of SV40 DNA which contains the origin of SV40 DNA replication (0.67 on the SV40 DNA map).

DNA Replication↗
PubMed1989

ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication.

Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.

Adenosine Triphosphate↗
PubMed1993

The DNA binding domain of herpes simplex virus type 1 origin binding protein is a transdominant inhibitor of virus replication.

The origin binding protein (OBP) of herpes simplex virus (HSV) type 1 specifically interacts with two high-affinity sites in each HSV DNA replication origin. The sequence-specific DNA binding activity of OBP maps to the carboxy-terminal one-third of the protein. For a single binding site, recombinantly expressed forms of this DNA binding domain have the same sequence specificity and binding affinity as the full-length OBP. However, unlike the full-length protein, truncated OBP does not bind HSV replication origins in a cooperative manner. To determine if cooperative interactions between DNA-bound OBP molecules are essential for viral DNA replication, the 317-amino-acid carboxy-terminal DNA binding domain of OBP was expressed in chick embryo fibroblasts. Cells were infected with HSV type 1, and viral DNA synthesis and virus production were monitored. We found that cells expressing truncated OBP were severely restricted for virus replication and that HSV DNA synthesis was undetectable. The results demonstrate that the amino-terminal two-thirds of OBP is essential for HSV DNA replication and that the OBP DNA binding domain acts as a transdominant inhibitor of viral DNA replication. The results also suggest that this experimental approach could be used to generate a refined map of essential OBP functions and that the approach may be generally applicable to the analysis of the multifunction HSV DNA replication complex.

Animals↗
PubMed1978

Definition of the boundaries of the origin of DNA replication in simian virus 40.

We have determined by use of DNA sequencing techniques the exact location of the deletion in d1 892, a viable deletion mutant of Simian virus 40 (SV40) reported to map very near the unique replication origin or SV40. With the help of this localization we have narrowed down the boundaries of the replication origin to 85 nucleotides within the sequence of SV40.

Base Sequence↗
PubMed1999

Initiation of DNA replication in eukaryotes: questioning the origin.

Although proteins involved in DNA replication in yeast have counterparts in multicellular organisms, the definition of an origin of DNA replication and its control in higher eukaryotes might obey to different rules. Origins of DNA replication that are site-specific have been found, supporting the notion that specific DNA regions are used to initiate DNA synthesis along metazoan chromosomes. However, the notion that specific sequences will define origins is still being debated. The variety and complexity of transcriptional programs that have to be regulated in multicellular organisms may impose a plasticity that would not be compatible with a fixed origin simply defined at the sequence level. Such a plasticity would be essential to developmental programs where the control of DNA replication could be more integrated to the control of gene expression than in unicellular eukaryotes.

Animals↗
PubMed1994

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.

Bacillus Phages↗
PubMed1984

Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication.

Base substitution of the ori region of simian virus 40 leads to plaque morphology mutants with markedly decreased DNA replication. Second-site mutations within the simian virus 40 T antigen gene suppress the plaque phenotype and replication defect of base-substituted ori mutants. Two second-site mutations have been mapped to a small segment of the T antigen gene, just beyond the distal splice junction. DNA sequence analysis revealed a single missense change in this segment of the T antigen gene of each of these second-site revertants, leading to a change in codon 157 in one case and codon 166 in the other. The mutant T antigens displayed relaxed specificity for the ori signal, i.e., they can function with several variously modified ori sequences, including those with small nucleotide deletions or insertions that are inactive for replication when coupled with wild-type T antigen. Thus a region of T antigen has been identified that appears to be intimately involved in vivo in binding to the ori sequence to initiate viral DNA replication.

Animals↗
PubMed2003

[Initiation of DNA replication in higher eukaryotes].

In this review, of problems concerning initiation of DNA replication in higher eukaryotes is discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.

Animals↗
PubMed1996

Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria.

We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6K gamma DNA replication origin (oriR(R6K gamma) so they replicate only in bacteria supplying the pi replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of pi. They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria. Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA. Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker. One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants. Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones. Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker. Plasmid-free segregants with an allele replacement can be subsequently selected as TetS or SucR recombinants. A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.

Alleles↗
PubMed1988

Interaction between the octamer-binding protein nuclear factor III and the adenovirus origin of DNA replication.

Nuclear factor III (NFIII) is a HeLa sequence-specific DNA-binding protein that stimulates initiation of adenovirus DNA replication in vitro and may be involved in regulation of transcription of several cellular and viral genes. We have studied the interaction between NFIII and the binding site in the adenovirus type 2 (Ad2) origin in detail by methidiumpropyl-EDTA.iron(II) and hydroxyl radical footprinting and by alkylation interference experiments. Our results indicate that (i) the core of the recognition sequence is 5'-TATGATAAT-3'; (ii) both major and minor groove base contacts are detected, and all base pairs in the core are involved in binding; (iii) many backbone contacts are observed divided into a large domain coinciding with the core and a small domain; (iv) contact points are not confined to one side of the DNA helix in contrast to the nuclear factor I (NFI)-binding site; (v) the binding site overlaps the NFI-binding site for at least one nucleotide. A number of Ad2 mutants as well as related binding sites in the origins of other adenovirus serotypes were systematically compared for binding with NFIII. The results are in good agreement with the contact point studies and show that at least one AT base pair is commonly required by NFI and NFIII for optimal binding. The strongest binding site, which contains the octamer/decanucleotide motif (ATGCAAAT[NA]), was found in the Ad4 origin, which lacks an NFI-binding site. Stimulation of in vitro DNA replication of Ad2, Ad4, and Ad12 by NFIII showed that the maximal level of stimulation is dependent on the affinity of NFIII for the origin.

Adenoviruses, Human↗
PubMed1980

Recombinant plasmids carrying promoters, genes and the origin of DNA replication of the early region of bacteriophage T7.

Two full-length contiguous HpaI fragments of the 0 to 18.2% region of T7 H DNA (HpF-H and HpG) were inserted into plasmids pHV14 or pC194 using oligo(dG . dC) connectors or synthetic HindIII adaptors. Amplification of the two early T7 fragments was achieved by transforming lysostaphin-treated S. aureus W57 with the hybrid plasmids. Experimental evidence is presented suggesting that neither of these T7 segments can be cloned in an intact form in E. coli. One of the hybrids, pHV14-HpF-H, proved to be unstable even in B. subtilis 168. The supercoiled recombinant plasmids were tested for their capacity to support RNA synthesis by purified E. coli or T7 RNA polymerases and to serve as templates in a cell-free T7 DNA replication system. The results of these in vitro studies indicate the presence of active "early" promoters in the cloned fragment HpF-H and active "late" promoters, as well as a functional origin of replication in the cloned fragment HpG.

Cloning, Molecular↗
PubMed1995

The Saccharomyces Ty5 retrotransposon family is associated with origins of DNA replication at the telomeres and the silent mating locus HMR.

We have characterized the genomic organization of the Ty5 retrotransposons among diverse strains of Saccharomyces cerevisiae and the related species Saccharomyces paradoxus. The S. cerevisiae strain S288C (or its derivatives) carries eight Ty5 insertions. Six of these are located near the telomeres, and five are found within 500 bp of autonomously replicating sequences present in the type X subtelomeric repeat. The remaining two S. cerevisiae elements are adjacent to the silent mating locus HMR and are located within 500 bp of the origin of replication present in the transcriptional silencer HMR-E. Although the S. cerevisiae Ty5 elements no longer appear capable of transposition, some strains of S. paradoxus have numerous Ty5 insertions, suggesting that transposition is occurring in this species. Most of these elements are adjacent to type X telomeric repeats, and regions flanking four of five characterized S. paradoxus insertions carry autonomously replicating sequences. The genomic organization of the Ty5 elements is in marked contrast to the other S. cerevisiae retrotransposon families (Ty1-4), which are typically located within 500 bp of tRNA genes. For Ty3, this association reflects an interaction between Ty3 and the RNA polymerase III transcription complex, which appears to direct integration [Chalker, D. L. & Sandmeyer, S. B. (1992) Genes Dev. 6, 117-128]. By analogy to Ty3, we predict that Ty5 target choice is specified by interactions with factors present at both the telomeres and HMR that are involved in DNA replication, transcription silencing, or the maintenance of the unique chromatin structure at these sites.

Base Sequence↗
PubMed1993

Searching for replication origins in mammalian DNA.

The attempts at identifying precise replication origins (ori) in mammalian DNA have been pursued mainly through physico-chemical and biochemical approaches, in view of the essential failure of the search for autonomously replicating sequences in cultured cells. These approaches involve the mapping of short stretches of nascent DNA, the identification of the regions where either leading or lagging strands switch polarity, or the localization of replication intermediates by two-dimensional gel electrophoresis. Due to the complexity of animal cell genomes, most of these studies have been performed on amplified domains and with the use of synchronization procedures. The results obtained have been controversial. In order to avoid the use of experimental procedures potentially affecting the physiological mechanism of DNA replication, we have developed a method for the localization of ori in single-copy loci in exponentially growing cells. This method entails the absolute quantification of the abundance of selected DNA fragments along a genomic region within samples of newly synthesized DNA by competitive polymerase chain reaction (PCR); the latter is immune to all the uncontrollable variables which severely affect the reproducibility of conventional PCR. The application of this method to SV40 ori-driven plasmid replication precisely identifies the known ori localization. Using the same approach, we have mapped an ori for bi-directional DNA replication in a 13.7-kb locus of human chromosome 19 encoding lamin B2.

Animals↗