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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↗
PubMed1994

Enzyme-dependent pausing during in vitro replication of O4-methylthymine in a defined oligonucleotide sequence.

We had previously reported that an oligonucleotide containing a site-specifically incorporated O4-methylthymine (m4T) was replicated under kinetic conditions by the Klenow fragment of E. coli DNA polymerase I (Kf) (Dosanjh et al., 1993). Using other polymerases for complete replication, but with limiting enzyme, a pause site before the m4T was observed. In order to investigate whether such a pause could be due to enzyme dissociation or stalling, trapping experiments were designed to aid in differentiating the two mechanisms. Rather than the generally used heparin or sheared DNA trap, these experiments utilized as the acceptor the same oligonucleotide containing unmodified thymine. It was observed that, under enzyme-limiting conditions, the nature of the enzyme played a major role in replication of m4T. With a running start, Kf and calf-thymus polymerase alpha-primase allowed replication beyond the m4T, while Sequenase and T7 showed a strong pause site at the base before m4T. When the oligonucleotide trap was added after different times of replication, it was found that Sequenase remained bound to the template-primer, regardless of whether T or m4T was present. In contrast, Kf dissociated and re-associated rapidly. Thus, m4T appears to be a strong replication block when using limiting amounts of a highly processive enzyme such as Sequenase or T7. This may imply that such enzymes discriminate against forming a poor basepair but remain bound to the primer-template or become inactivated.

Base Sequence↗
PubMed2002

A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs, both by themselves unable to complete the virus life cycle.

The replication-deficient vaccinia virus (VV) MVA-T7 produces large amounts of T7 RNA polymerase and permits efficient protein expression from cDNA of T7-promoted genes. Yet, unlike recombinant VV vTF7-3, (VV) MVA-T7 produces no cytopathic effect in primate cells, thus allowing the study of processes with slow kinetics. We have applied MVA-T7 to aid genome expression of HAV, a representative of the Picornaviridae family that is well known for its inefficient replication in mammalian cell cultures. After cDNA transfection and MVA-T7 infection, empty capsids and mature HAV particles were formed with different kinetics and were characterized by their morphology, protein content, and infectivity. The data suggests that HAV genome replication is initiated from RNA, which was transcribed in vivo by the MVA-T7-encoded T7 RNA polymerase. HAV genome replication was also demonstrated in a recombination assay. After co-expression of two subgenomic HAV cDNAs, both by themselves unable to complete the viral life cycle, infectious HAV was rescued, indicating that replication-dependent genetic recombination has occurred. We propose that the high-level genome expression mediated in vivo by the VV-encoded T7 RNA polymerase augments the amount of viral RNA, such that replication of viruses poorly replicating in cell cytoplasm is detectable.

Bacteriophage T7↗
PubMed1982

Influence of phage T3 and T7 gene functions on a type III(EcoP1) DNA restriction-modification system in vivo.

The ocr+ gene function (gp 0.3) of bacteriophages T3 and T7 not only counteracts type I (EcoB, EcoK) but also type III restriction endonucleases (EcoP1). Despite the presence of recognition sites, phage DNA as well as simultaneously introduced plasmid DNA are protected by ocr+ expression against both the endonucleolytic and the methylating activities of the EcoP1 enzyme. Nevertheless, the EcoP1 protein causes the exclusion of T3 and T7 in P1-lysogenic cells, apparently by exerting a repressor-like effect on phage gene expression. T3 which induces an S-adenosylmethionine hydrolase is less susceptible to the repressor effect of the SAM-stimulated EcoP1 enzyme. The abundance of EcoP1 recognition sites in the T7 genome is explained by their near identity with the T7 DNA primase recognition site.

DNA Replication↗
PubMed1999

Functional analysis of affinity-purified polyhistidine-tagged DnaA protein.

DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin. We describe a system for efficient production and purification of replicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator. DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with the expected molecular weight. The yield of purified protein was greater than 10 mg per liter of cell culture. The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replication, binding to oriC DNA, binding of allosteric effector adenine nucleotides, and interaction with membrane acidic phospholipids. This system for rapid and high-yield generation of replication-active DnaA protein should facilitate structure-function studies and mutagenic analyses of this initiator protein.

Amino Acid Sequence↗
PubMed1994

The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation.

Lysine 318 in the conserved sequence SXXXGXGKS of bacteriophage T7 gene 4A' protein was mutated to an alanine to understand the effect of this substitution on the helicase and primase activities. The dTTPase activity of 4A'/K318A mutant protein was much lower than that of 4A', and both Km and kcat values were affected. The Km of the mutant protein was 3-5-fold higher, and the kcat was about 100-fold lower, than that of 4A'. The mutation did not affect the ability of 4A'/K318A to assemble into hexamers or bind DNA in the presence of MgdTTP. Interestingly, the mutant protein does not bind DNA in the presence of MgdTMP-PCP. The reduced dTTPase activity, however, decreased the helicase activity of the mutant protein to an undetectable level, whereas its primase activity was only 1.5-2.5-fold lower. When 4A'/K318A mutant protein was mixed with 4A', heterooligomers were formed and the helicase and the DNA-dependent dTTPase activities of 4A' were inhibited, but the DNA-independent activity actually increased. The extent of decrease in activities upon heterooligomer formation depended both on the length of time 4A' and 4A'/K318A proteins were incubated and on the concentration of the mutant protein. In addition, the decrease in the dTTPase activity was observed only when the two proteins were incubated in the absence of MgdTTP and DNA, conditions under which both proteins form unstable hexamers. Even though 4A'/K318A does not bind a 30-mer DNA in the presence of MgdTMP-PCP, heterooligomers were capable of binding DNA with the same stoichiometry as 4A'. Protein-DNA cross-linking experiments with (dT)30 and poly(5-BrdU) showed that DNA interacts with five and perhaps all six subunits of 4A'. Therefore, unless heterooligomer restores the ability of the mutant protein to bind DNA in the presence of MgdTMP-PCP, these results suggest that the DNA can bind 4A' by interacting with a few subunits. However, a fully active hexamer is required for both the helicase and the single-stranded M13 DNA-dependent dTTPase activities.

Amino Acid Sequence↗
PubMedDate not supplied

[Protein factor from regenerating rat liver destabilizing secondary DNA structure].

In DNA preparations isolated from regenerating rat liver 22 hours after partial hepatectomy, i.e. at the period of the most intensive DNA synthesis a "Denaturating Protein Factor" (DPF) tightly bound to DNA was found. Isolated protein fraction with a molecular weight of 6500 dalton was found to be homogenous upon SDS-polyacrylamide electrophoresis. The degree of destabilisation of DNA was estimated by its reaction with water-soluble [14C]CME-carbodiimide which modifies selectively guanine and thymine residues only in the denatured DNA regions. Pronase treated DPF loses its DNA-denaturing capacity. Pronase treatment of DNA--DPF complex restores native DNA structure. DPF from rat liver was able to denature DNA from calf thymus and bacteriophage T7 DNA. A hypothesis is proposed that the DPF is responsible for the destabilization of DNA secondary structure in the process of replication.

Animals↗
PubMed1996

Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame.

The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.

Animals↗
PubMed1986

Unusual occurrence of EcoP1 and EcoP15 recognition sites and counterselection of type II methylation and restriction sequences in bacteriophage T7 DNA.

Selected and counterselected oligodeoxynucleotide sequences were identified in the total sequence of bacteriophage T7 DNA using a statistical criterion derived for a probability model of the Markov chain type. All extremely rare tetra- and pentadeoxynucleotides are (or contain) recognition sequences for the Escherichia coli DNA methylases dam or dcm. Most of the 37 hexadeoxynucleotides absent from T7 DNA are recognition sequences for type II modification/restriction enzymes of E. coli or related species. In contrast to most restriction sites counterselected during evolution, the EcoP1 site GGTCT occurs 126 times in the T7 genome, and phage T7 replication is severely repressed in P1-lysogenic host cells. We demonstrate that the frequency of the EcoP1 site is determined by that of the overlapping recognition sites for T7 primase, an essential phage enzyme. The recognition site of a type III enzyme, EcoP15, is also not counterselected. In T7 DNA all 36 EcoP15 sites are arranged in such a manner that the sequence CAGCAG is confined to the H strand, the complementary sequence CTGCTG to the L strand. This "strand bias" is highly significant and, therefore, very probably selected. A functional relation between this strand bias and the refractive behaviour of phage T7 to EcoP15 restriction is suspected.

Base Sequence↗
PubMed1986

Primary structural relationships may reflect similar DNA replication strategies.

The primary structures of several proteins of bacterial and viral origin involved in the initiation of DNA synthesis and its subsequent elongation were compared. It was found that the known sequences of DNA polymerases and the single-stranded DNA binding proteins from phage T7 and Escherichia coli aligned well. Furthermore, segmental homologies were found in the phage phi 29 and adenovirus polymerases as well as in their DNA binding proteins. These results suggest similar mechanisms of DNA synthesis for E. coli and T7 on the one hand and for phi 29 and adenovirus on the other.

Adenoviruses, Human↗
PubMed2005

Human protein tau represses DNA replication in vitro.

Here, in the experiments of both PCR and real-time PCR, a repression of DNA amplification was observed in the presence of protein tau. Furthermore, a strong repression appeared when an in vitro DNA replication assay was performed at the physiological temperature (37 degrees C). The incorporation of dNTP was markedly decreased to approximately 12% of control by the presence of tau23 and to approximately 15% by tau40. In the competitive experiments, the PCR product could be restored when the competitor DNA was added, indicating that the association of tau with the template gave rise to the repression. However, tau did not repress the yield of RNA in transcription, suggesting that tau was replaced or ejected from the template by the elongating T7 RNA polymerase.

DNA↗
PubMed1983

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase.

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.

Base Sequence↗
PubMed2002

Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases.

Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase. We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon). Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report. Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively. On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities. RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM. The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases. As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM. The cells were halted at G1 phase in the cell cycle by RAL.

Antineoplastic Agents↗
PubMed1984

Plasmid-phage recombination in T7 infected Escherichia coli.

Recombination between genetically marked T7 bacteriophage and plasmids containing inserts of T7 DNA has been studied in order to gain some insight into the phage recombination process. The results suggest that plasmid-phage recombination requires the products of T7 genes 3 (endonuclease), 4 (DNA primase), 5 (DNA polymerase), and 6 (exonuclease), as has been demonstrated previously for phage-phage recombination. Plasmid replication does not compensate for a complete block in phage polymerase synthesis, suggesting a direct role for this enzyme in recombination, rather than an indirect role, by means of producing replicative structures that are recombinogenic. In most respects, plasmid-phage recombination appears to be similar to phage-phage recombination. The participation of two autonomous, structurally dissimilar, homologues, however, might render certain aspects of the recombination process more amenable to analysis. As examples, the characterization of an apparent marker effect and the demonstration of genetic heterozygotes among the products of plasmid-phage recombination are presented.

DNA Primase↗