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PubMed1976

Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase.

T7 DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) is composed of an 84,000 dalton protein specified by the gene 5 of the phage and a 12,000 dalton protein (TsnC protein) specified by the tsnC gene of E. coli [Modrich, P. & Richardson, C. C. (1975) J. Biol. Chem. 250 5515-5522]. Both proteins are necessary for T7 DNA polymerase activity and for the replication of T7 DNA. The TsnC protein is identical to thioredoxin of E. coli by the following criteria: (1) Homogeneous preparations of both proteins have TsnC and thioredoxin activity. (2) Both proteins show similar stability to heat. (3) They have identical mobilities, corresponding to a molecular weight of 12,000, on polyacrylamide gels containing sodium dodecyl sulfate. (4) Their amino-acid compositions are indistinguishabe. (5) Antibody prepared against thioredoxin inhibits TsnC activity. (6) TsnC protein isolated from purified T7 DNA polymerase has thioredoxin activity. In addition, preparations of T7 DNA polymerase itself exhibit thioredoxin activity and are partially inhibited by antibody to thioredoxin.

Amino Acids↗
PubMed1990

Structure of RNAs replicated by the DNA-dependent T7 RNA polymerase.

The DNA-dependent RNA polymerase of bacteriophage T7 efficiently and specifically replicates two structurally related RNAs, termed X and Y RNAs. Replication of both RNAs involves synthesis of complementary strands initiated with pppC and pppG. RNAs transcribed from DNA template containing the established sequences of X and Y RNAs were efficiently replicated by T7 RNA polymerase. Both RNAs possess palindromic sequences with a dual axis of symmetry, permitting formation of hairpin-, dumbbell-, or cloverleaf-type structures. The template must consist of RNA and not DNA sequence, and the terminal unpaired dinucleotides of the RNA are necessary for replication. Nucleotidyl transferase activity of E. coli adenylates the unpaired CCOH dinucleotide at the 3' end of a C strand of X RNA. This feature, as well as the length (64 nucleotides) and compact structure of X and Y RNAs, suggests that they may resemble tRNA molecules and tRNA-like structures at the 3' termini of many plant viral RNA genomes.

Base Sequence↗
PubMed1998

Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins.

Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.

Bacteriophage M13↗
PubMed2003

Enhanced genetic rescue of negative-strand RNA viruses: use of an MVA-T7 RNA polymerase vector and DNA replication inhibitors.

A modified cDNA rescue system that improves recovery of recombinant nonsegmented, negative-strand RNA viruses from cloned DNAs is described. Rescue systems based on vaccinia virus-T7 RNA polymerase vectors have been used to derive many negative-strand viruses; however, some strains can be recalcitrant to rescue possibly because of the simultaneous replication of the vaccinia virus-T7 vector. Our goal was to engineer a system where replication of the vaccinia virus-T7 vector could be blocked, yet allow for sufficient T7 RNA polymerase expression to enable genetic rescue. To that end, a recombinant modified vaccinia virus Ankara (MVA) was engineered that contained the bacteriophage T7 gene-1 under the control of a strong early promoter that would enable T7 RNA polymerase expression in the absence of MVA DNA replication. The new T7 helper, MVAGKT7, was then utilized successfully for the genetic rescue of a measles virus minigenome and full-length cDNAs, in the presence of DNA synthesis inhibitors. In addition to blocking completely MVAGKT7 replication, AraC treatment was found to enhance minigenome-encoded gene expression and the efficiency of measles virus rescue.

Animals↗
PubMed1987

Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19.

By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer. The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers. Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis. It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis. In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA.

Centrifugation, Zonal↗
PubMed1994

Evolutionary role of abortive transcript as a primer for DNA replication.

Abortive cycling features transcription initiation by RNA polymerase in both prokaryote and eukaryote. It is known that T7 RNA polymerase produces abortive transcripts up to eight ribonucleotides in length depending on the initial sequence of the DNA message. On the other hand, T7 RNA polymerase initiates DNA replication from the T7 primary origin by synthesizing primers. And the shortest primer from the phi l.lB promoter in the primary origin also seems to be eight ribonucleotides in length. Therefore, it is likely that the longest abortive transcript serves as the shortest primer for T7 DNA replication from the primary origin. Considering that promoters often exist in DNA replication origins for example, E. coli oriC and many eukaryotic origins, the early DNA replication system appears to have taken advantage of the abortive cycling of RNA-dependent RNA polymerase that already existed before the emergence of DNA world. The evolutionary primitive RNA polymerase could do both transcription and priming of DNA replication. Accordingly, abortive cycling would play an important role in evolution at the emergence of DNA world. The priming activity of the primitive RNA polymerase would be taken over by primase later, which seems to be a specialized RNA polymerase for abortive cycling.

Biological Evolution↗
PubMed1972

Initiation and reinitiation of DNA synthesis during replication of bacteriophage T7.

In its first round of replication, the T7 chromosome follows a simple pattern, as viewed in the electron microscope. The iniation of DNA synthesis occurs about 17% from the genetic left end of the viral DNA rod. Bidirectional DNA synthesis from this origin then generates a replicating intermediate that we call an "eye form." In the eye form, when synthesis in the leftward direction reaches the left end of the viral chromosome, the molecule is converted into a Y-shaped replicating rod. The remaining growing point continues synthesis rightward, until presumably it runs off the right end of the DNA rod, thus terminating replication. Numerous T7 chromosomes were found in which a second round of replication had begun before the first round had finished. Analysis of these reinitiated DNA molecules showed that the second round of replication, like the first, began 17% from the end of the chromosome and involved bidirectional DNA synthesis.

Centrifugation, Density Gradient↗
PubMed1985

In vitro concatemerization of bacteriophage T7 DNA: role of DNA synthesis and gene 6 exonuclease.

The replication of bacteriophage T7 DNA in vivo proceeds via the synthesis of complex concatemeric intermediates which are joined via the 160 base pair terminal redundancies at either end of the phage chromosome. To gain some insight into the mode of generation of these structures, we have examined the role of DNA synthesis in the formation of concatemeric bacteriophage T7 DNA in vitro. Incubation of mature T7 DNA with T7-infected cell extracts and a deoxynucleoside [32P]triphosphate resulted in the incorporation of significant radioactivity into the DNA. Highest levels of incorporation were at the termini of the DNA and decreased toward the middle of the molecule. Incorporation was dependent upon the presence of the activity of the gene 6 exonuclease and correlated with the generation of concatemeric DNA. A model explaining the role of exonucleolytic degradation and DNA synthesis in the generation of concatemeric DNA is presented.

Carbon Radioisotopes↗
PubMed1994

Blocking of in vitro DNA replication by deoxycytidine adducts of the mutagen and clastogen 2-bromoacrolein.

Calf thymus single-stranded DNA was modified with 2-bromoacrolein (2BA), a genotoxic metabolite of tris(2,3-dibromopropyl)phosphate. This DNA was used as a template for in vitro DNA replication by T7-polymerase and Klenow fragment of Escherichia coli DNA polymerase I. Increasing levels of 2BA modification led to decreased DNA synthesis as measured by [methyl-3H]dTTP incorporation. M13 mp19 single-stranded DNA template modified with 2BA was used to determine the sites of termination of DNA replication by T7 polymerase and Klenow fragment of Escherichia coli DNA polymerase I. It was found that DNA replication stopped frequently before and occasionally opposite deoxycytidine nucleotides. These results indicated that an as yet unidentified deoxycytidine:2BA adduct may have been formed in the reaction of 2BA with M13 DNA. To investigate if such adducts were formed, we reacted 2BA with deoxycytidine in vitro at pH 4.4, and putative deoxycytidine:2BA adducts were isolated by high-performance liquid chromatography. They were characterized by 1H and 13C nuclear magnetic resonance and with fast atom bombardment mass spectrometry as two diastereomeric 3-bromo-7-(beta-D-deoxyribofuranosyl)- 3,4-dihydro-2-hydroxy-(2H,7H)[1,6-a]pyrimidin-6-one adducts and a 3-bromo-7-(beta-deoxyribofuranosyl)-(4H,7H)-pyrimido[1,6-a]pyrimidin-6 -one adduct. Only the latter adduct, however, was formed in the reaction of 2BA with calf thymus single-stranded DNA in vitro. Tris(2,3-dibromopropyl)phosphate is clastogenic. Because clastogenicity may result from DNA adducts that block replication, the role of the presently identified deoxycytidine adducts of the reaction metabolite 2BA in the clastogenicity of tris(2,3-dibromopropyl)phosphate is discussed.

Acrolein↗
PubMed1980

The role of bacteriophage T7 gene 2 protein in DNA replication.

The in vivo function of the gene 2 protein of bacteriophage T7 has been examined. The gene 2 protein appears to modulate the activity of the gene 3 endonuclease in order to prevent the premature degradation of any newly-formed DNA concatemers. This modulation is not however a direct interacton between the two proteins. In single-burst experiments rifamycin can substitute for the gene 2 protein, allowing formation of fast-sedimenting replicative DNA intermediates and progeny phage production. This suggests that the sole function of the gene 2 protein is inhibition of the host RNA polymerase and that the latter enzyme directs or promotes the endonucleolytic action of the gene 3 protein.

DNA Replication↗
PubMed1997

Defects in concatemer processing of bacteriophage T7 DNA deleted in the M-hairpin region.

The intracellular replicating form of T7 DNA is a concatemer in which linear genomes are joined head to tail by sharing 160-bp terminally repeated sequences. A unique hairpin (M-hairpin) end generated on the left side of the TR was proposed to be responsible for the duplication of the concatemer junction for efficient packaging. We characterized the defects caused by loss of the M-hairpin by constructing a recombinant T7 (T7Deltam) deleted in the m region. Initially, the intracellular growth rate of progeny phage was normal in T7Deltam infection. However, the titer of progeny phage was eventually reduced by two- to threefold and lysis was significantly delayed. The restriction fragment, LEDelta160, generated simultaneously with the double-strand cleavage at the onset of packaging reaction was found more or less at the same intensity in both T7(+) and T7Deltam infection at the beginning but preferentially in T7Deltam infection during the later phase of infection. These observations suggest that the DNA packaging of T7 proceeds on the intact concatemer junctions during the early stage of infection while the duplication of the concatemer junction by the M-hairpin seemed to be important during the later phase, presumably due to reduced replication. While the generation of the M-hairpin involves DNA replication, the loss of m did not reduce DNA synthesis, suggesting that the role of the M-hairpin as an origin of replication is minimal.

Bacteriophage T7↗
PubMed2001

Initiation of DNA replication by DNA polymerases from primers forming a triple helix.

Despite extensive studies on oligonucleotide-forming triple helices, which were discovered in 1957, their possible relevance in the initiation of DNA replication remains unknown. Using sequences forming triple helices, we have developed a DNA polymerisation assay by using hairpin DNA templates with a 3' dideoxynucleotide end and an unpaired 5'-end extension to be replicated. The T7 DNA polymerase successfully elongated nucleotides to the expected size of the template from the primers forming triple helices composed of 9-14 deoxyguanosine-rich residues. The triple helix-forming primer required for this reaction has to be oriented parallel to the homologous sequence of the hairpin DNA template. Substitution of the deoxyguanosine residues by N7 deazadeoxyguanosines in the hairpin of the template prevented primer elongation, suggesting that the formation of a triple helix is a prerequisite for primer elongation. Furthermore, DNA sequencing could be achieved with the hairpin template through partial elongation of the third DNA strand forming primer. The T4 DNA polymerase and the Klenow fragment of DNA polymerase I provided similar DNA elongation to the T7 polymerase-thioredoxin complex. On the basis of published crystallographic data, we show that the third DNA strand primer fits within the catalytic centre of the T7 DNA polymerase, thus underlying this new property of several DNA polymerases which may be relevant to genome rearrangements and to the evolution of the genetic apparatus, namely the DNA structure and replication processes.

Bacteriophage T7↗
PubMed1982

Mutagenesis of bacteriophage T7 in vitro by incorporation of O6-methylguanine during DNA synthesis.

An in vitro system in which bacteriophage T7 DNA is replicated and efficiently packaged into procapsids to form viable phage has been used to examine mutagenesis. The fidelity of replication was assayed both by measuring reversion of an amber mutation in an essential gene and by generation of temperature-sensitive mutants among the phage produced in vitro. Under standard reaction conditions, the fidelity of DNA replication is about equal to that normally found in vivo. However, when O6-methyldeoxyguanosine triphosphate is included in the reaction, O6-methylguanine is incorporated into newly synthesized DNA and the mutation frequencies increase 10- to 70-fold over the control. These experiments demonstrate in vitro mutagenesis with the T7 DNA replication-packaging system and provide more direct evidence for the premutagenic role of O6-methylguanine.

Alkylation↗
PubMed1990

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA.

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.

Centrifugation, Density Gradient↗
PubMed1983

A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein.

This paper describes the construction of a DNA molecule containing a topologically stable structure that simulates a replication fork. This preformed DNA molecule is a circular duplex of 7.2 X 10(3) base pairs (M13mp6 DNA) from which arises, at a unique BamHI recognition site, a noncomplementary 5'-phosphoryl-terminated single strand of 237 nucleotides (SV40 DNA). This structure has two experimental attributes. 1) Templates for both leading and lagging strand synthesis exist as stable structures prior to any DNA synthesis. 2) DNA synthesis creates a cleavage site for the restriction endonuclease BamHI. Form I of T7 DNA polymerase, alone, catalyzes limited DNA synthesis at the preformed replication fork whereas Form II, alone, polymerizes less than 5 nucleotides. However, when T7 gene 4 protein is present, Form II of T7 DNA polymerase catalyzes rapid and extensive synthesis via a rolling circle mode. Kinetic analysis of this synthesis reveals that the fork moves at a rate of 300 bases/s at 30 degrees C. We conclude that the T7 gene 4 protein requires a single-stranded DNA binding site from which point it translocates to the replication fork where it functions as a helicase. The phage T4 DNA polymerase catalyzes DNA synthesis at this preformed replication fork in the presence of gene 4 protein, but the amount of DNA synthesized is less that 3% of the amount synthesized by the combination of Form II of T7 DNA polymerase and gene 4 protein. We conclude that T7 DNA polymerase and T7 gene 4 protein interact specifically during DNA synthesis at a replication fork.

DNA Replication↗
PubMed1977

Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates.

Homogeneous preparations of phage T7 gene 4 protein catalyze the hydrolysis of dNTPs and rNTPs to NDPs and Pi in the presence of single-stranded DNA. Synthesis on single-stranded DNA by T7 DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) does not affect the hydrolysis of NTPs by the gene 4 protein. Gene 4 protein does not catalyze the hydrolysis of NTPs in the presence of duplex DNA, nor can T7 DNA polymerase use duplex DNA as a template. However, the two proteins together can replicate duplex DNA and, under these conditions, synthesis is accompanied by hydrolysis of NTPs. During synthesis on duplex templates in the presence of T7 DNA polymerase, gene 4 protein, dNTPs, and rNTPs, 4.2 NTPs are hydrolyzed for each dNMP polymerized. 2'3'-Dideoxy-TTP, an inhibitor of DNA synthesis, inhibits hydrolysis by the gene 4 protein during synthesis on duplex DNA, and beta, gamma-methylene-dTTP, an inhibitor of hydrolysis by the gene 4 protein, stops DNA synthesis on duplex DNA. The multiple activities of gene 4 protein are shown to reside in a single protein molecule.

Coliphages↗
PubMed1996

Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.

A large variety of different RNA species that are replicated by DNA-dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120. The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA-dependent RNA polymerases (replicases): only single-stranded templates are accepted and complementary replica strands are synthesized. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini. Consensus sequences were not found among the sequences of the replicating RNA species. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active. Template-free production of RNA was completely suppressed by addition of DNA to the incubation mixture. When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template. No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase.

Bacteriophage T7↗
PubMed1977

RNA-linked nascent DNA pieces in T7 phage-infected Escherichia coli cells. I. Role of gene 6 exonuclease in removal of the linked RNA.

The presence of RNA-linked nascent DNA pieces in T7 phage-infected Escherichia coli cells has been shown by the selective degradation of the 5'-hydroxyl-terminated nascent DNA, produced by alkali or RNase treatment, with spleen exonuclease. At 43 degrees C, the proportion of RNA-linked DNA pieces in nascent short dna is 50 to 60% in T7 ts136 (ts mutant of gene 6) phage-infected E. coli, whereas that in T7 wild-type phage-infected cells is less than 6%. Joining of the nascent pieces is greatly retarded in T7 ts136-infected E. coli temperature sensitive polA mutants at 43 degrees C. These results suggest that gene 6 exonuclease plays a role in removal of the linked RNA during the discontinuous replication of T7 DNA.

Animals↗