Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “T7 DNA replication”

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 325 records · Page 18Linked to original sources
PubMed1996

Branched structures in the intracellular DNA of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A.R. Morgan, D.R. Tovell, and D.L.J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions.

Animals↗
PubMed1981

Adenovirus DNA replication in vitro: a protein linked to the 5' end of nascent DNA strands.

Soluble nuclear extracts prepared from adenovirus-infected HeLa cells supported adenovirus DNA replication with exogenous DNA-protein complex as template, but protease-treated, phenol-extracted DNA was less active. Replication was enhanced when creatine phosphate and creatine phosphokinase were included in the reaction mixture, rendering the reaction independent of exogenous ATP. Genomic-length, newly synthesized DNA strands were first observed 30 min after initiation of replication and continued to increase in amount for at least 4 h. Thus, the rate of replication is consistent with previous estimates of the rate of replication in vivo. Nascent DNA strands bound to benzoylated, naphthoylated DEAE-cellulose due to their association with protein. The 5' termini of nascent DNA strands were resistant to the 5'- to 3'-specific T7 exonuclease, and the 3' termini of nascent strands were sensitive to the 3'- to 5'-specific exonuclease III. These results suggest that a protein becomes covalently linked to the 5' termini of nascent DNA strands replicated in vitro. Nuclear extracts prepared from adenovirus type 2-infected cells also supported replication of DNA-protein complex prepared from the unrelated type 7 adenovirus. The limited sequence homology between these two viruses at the origin of replication further defines recognition sequences at the origin. These results are discussed in terms of a model for adenovirus DNA replication in which the terminal protein and sequences within the inverted terminal repetition are involved in the formation of an initiation complex that is able to prime DNA replication.

Adenoviruses, Human↗
PubMed2005

Protein--protein interactions in the eubacterial replisome.

Replication of genomic DNA is a universal process that proceeds in distinct stages, from initiation to elongation and finally to termination. Each stage involves multiple stable or transient interactions between protein subunits with functions that are more or less conserved in all organisms. In Escherichia coli, initiation of bidirectional replication at the origin (oriC) occurs through the concerted actions of the DnaA replication initiator protein, the hexameric DnaB helicase, the DnaC?helicase loading partner and the DnaG primase, leading to establishment of two replication forks. Elongation of RNA primers at each fork proceeds simultaneously on both strands by actions of the multimeric replicase, DNA polymerase III holoenzyme. The fork that arrives first in the terminus region is halted by its encounter with a correctly-oriented complex of the Tus replication terminator protein bound at one of several Ter sites, where it is trapped until the other fork arrives. We summarize current understanding of interactions among the various proteins that act in the different stages of replication of the chromosome of E. coli, and make some comparisons with the analogous proteins in Bacillus subtilis and the coliphages T4 and T7.

Adenosine Triphosphatases↗
PubMed1983

The 3'-5' proofreading exonuclease of bacteriophage T4 DNA polymerase is stimulated by other T4 DNA replication proteins.

The bacteriophage T4 DNA polymerase has an intrinsic 3'-5' proofreading exonuclease activity that plays a central role in determining the fidelity of T4 DNA replication. In order to monitor this activity, we have measured the rate at which the polymerase decreases the size of a double-stranded DNA substrate in the absence of deoxyribonucleoside triphosphates. With this assay, we find that the addition of the polymerase accessory proteins, 45 protein and 44/62 protein, increases the rate at which the polymerase-associated exonuclease digests the DNA substrate 3- to 4-fold. This stimulation requires the continuous hydrolysis of ATP catalyzed by the accessory protein complex. When added alone, the T4 helix-destabilizing protein, 32 protein, inhibits the exonuclease rate at high concentrations (greater than 100 micrograms/ml), while stimulating about 3-fold at low concentrations. The 32 protein and the accessory proteins together increase the exonuclease rate 8- to 10-fold above that found for the polymerase alone. The bacteriophage T7 DNA polymerase displays a similar 3'-5' exonuclease activity, but this exonuclease is not stimulated by any of the T4 replication proteins. It therefore appears that specific protein-protein interactions are involved.

Adenosine Triphosphate↗
PubMed1987

Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere.

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.

Base Sequence↗
PubMed2002

Functional studies of potential intrastrand triplex elements in the Escherichia coli genome.

We previously used a pattern recognition program for nucleic acids to detect sequences with the potential to form intrastrand triplexes. Potential intrastrand triplex (PIT) element families were found in Escherichia coli, Synechocystis sp. and Haemophilus influenza. We were particularly intrigued with the family found in E. coli, which contained 25 dispersed copies of a particular PIT sequence corresponding to the purine triplex motif. E. coli PIT elements appear to occur exclusively in non-coding regions. We now report biochemical experiments testing the interaction of E. coli PIT elements with polymerases and single-stranded DNA-binding protein (SSB). The elements were also tested in genetic experiments as promoters, transcription terminators, or replication pause sites in E. coli. We show that PIT elements display provocative characteristics in certain biochemical assays. When appropriately oriented, the elements block elongation by Taq DNA polymerase at 72 degrees C, but not elongation by T7 DNA polymerase at 37 degrees C. The G-rich strand of the E. coli PIT sequence folds into a form with reduced affinity for SSB. On the other hand, in vivo studies did not detect replication delays for conjugal transfer of episomes containing PIT elements. These sequences were shown not to act as promoters, but the presence of PIT elements in RNA leaders upstream of a coding region could strongly influence expression of the downstream gene. These effects were shown to be post-transcriptional and were solely dependent on the Watson-Crick stem-loop structure within the PIT element. Thus, although PIT element DNA displays unusual biochemical properties, it remains unknown how these elements arose, and why they persist in the E. coli genome.

Base Sequence↗
PubMed1998

The mechanism of action of T7 DNA polymerase.

The recent crystal structure determination of T7 DNA polymerase complexed to a deoxynucleoside triphosphate and primer-template DNA has provided the first glimpse of a replicative DNA polymerase in a catalytic complex. The structure complements many functional and structural studies of this and other DNA polymerases, allowing a detailed evaluation of proposals for the mechanism of nucleotidyl transfer and the exploration of the basis for the high fidelity of template-directed DNA synthesis.

Binding Sites↗
PubMed1991

Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases.

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.

Base Sequence↗
PubMed2001

Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The absence of culture systems permissive for HCV replication has presented a major bottleneck to antiviral development. We sought to recapitulate the early steps in the life cycle of HCV by means of DNA-based expression of viral genomic sequences. Here we report expression of replicating HCV RNA by using a, to our knowledge, novel binary expression system in which cells were transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis Delta ribozyme to control 3' cleavage, and infected with vaccinia-T7 polymerase. HCV genomic and replicative strand synthesis, in addition to protein synthesis, was detectable and depended on full-length HCV sequences. Moreover, the system was capable of generating HCV RNA quasispecies, consistent with the action of the low-fidelity HCV NS5B RNA polymerase. IFN-alpha, but not ribavirin, directly inhibited the viral replicative cycle in these cells, identifying the virus itself and not solely the immune system as a direct target of IFN action. The availability of a cell-based test for viral replication will facilitate screening of inhibitory compounds, analysis of IFN-resistance mechanisms, and analysis of virus-host cell interactions.

Amantadine↗
PubMed1995

Rescue of measles viruses from cloned DNA.

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.

Base Sequence↗
PubMed1990

cis-acting elements involved in replication of alfalfa mosaic virus RNAs in vitro.

A DNA copy of alfalfa mosaic virus (AIMV) RNA3 was transcribed in vitro in two different orientations with T7 RNA polymerase and the transcripts were used as templates for a virus-specific RNA-dependent RNA polymerase (RdRp) purified from AIMV-infected bean plants. Minus-stranded templates were transcribed by the RdRp into subgenomic plus-stranded RNA4. A deletion analysis showed that a sequence in minus-strand RNA3, located between nucleotides -8 and -55 upstream of the initiation site for RNA4 synthesis, was sufficient for subgenomic promoter activity in vitro. Plus-stranded templates were transcribed by the RdRp into full-length minus-stranded copies. A deletion analysis indicated that a sequence located between nucleotides 133 and 163 from the 3'-end of AIMV RNA3 was sufficient to direct the synthesis of minus-stranded products by the RdRp. Thus, the 3'-terminal region of the AIMV RNAs, which contains the binding sites with a high affinity for coat protein, appears not to be involved in recognition of the RNAs by the RdRp in vitro.

Cloning, Molecular↗
PubMed1995

dUTPase from the retrovirus equine infectious anemia virus: high-level expression in Escherichia coli and purification.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21 (DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein. This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter-1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (pI) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.6 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence.

Amino Acid Sequence↗