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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↗
PubMed2005

Bacteriophage origins of mitochondrial replication and transcription proteins.

Mounting evidence suggests that key components of the mitochondrial transcription and replication apparatus are derived from the T-odd lineage of bacteriophage rather than from an alpha-Proteobacterium, as the endosymbiont hypothesis would predict. We propose that several mitochondrial replication genes were acquired together from an ancestor of T-odd phage early in the evolution of the eukaryotic cell, at the time of the mitochondrial endosymbiosis. We further propose that at a later stage the single-subunit RNA polymerase, originally acquired for mitochondrial DNA replication, was co-opted to serve in mitochondrial transcription.

Bacteriophage T7↗
PubMed2004

Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis.

The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G --> T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations.

2-Acetylaminofluorene↗
PubMed1972

Regions of single-stranded DNA in the growing points of replicating bacteriophage T7 chromosomes.

In partially replicated T7 chromosomes, the points where parental strands are separating and new DNA is being synthesized can be seen in the electron microscope to contain regions of single-stranded template DNA. The single-stranded regions are located on only one of the two daughter arms of the replicating chromosome. Inman and Schnös observed such single-stranded regions in 50% of the growing points of replicating lambda DNA, and, as reported in this paper, we find them in about 85% of the growing points of T7 DNA. Both studies support the conclusion that DNA synthesis involves the direct elongation of one daughter strand in the growing point. Evidently, this elongation is accompanied by the unwinding of the parental double helix to expose a region of single-stranded DNA which is then converted to the duplex state by a discontinuous mechanism involving the synthesis of DNA fragments.

Centrifugation, Density Gradient↗
PubMed1978

T7 gene 6 exonuclease has an RNase H activity.

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A) . poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a ribonucleoside 5'-monophosphate product. When the RNA strand of a 0X174 RNA . DNA hybrid molecule synthesized with E. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed.

Coliphages↗
PubMed1987

Gene 18 protein of bacteriophage T7. Overproduction, purification, and characterization.

Genetic and physical analyses indicate that gene 18 protein of bacteriophage T7 is essential for packaging of T7 DNA. T7 DNA is replicated via linear intermediates, culminating in the formation of concatemers many genomes in length which are then packaged into capsids. In infections with phage carrying amber mutations in gene 18, development is blocked at the concatemer stage. Biochemical studies on the role of gene 18 protein in concatemer processing and DNA packaging have been hampered by its low level of expression of gene 18 during T7 infections. We have cloned gene 18 on a plasmid downstream from the bacteriophage lambda PL promoter controlled by the temperature-sensitive lambda repressor encoded by c 1857. Thermal induction leads to the expression of the 10,000-Da gene 18 protein to the extent of approximately 10% of the total protein after 2 h. The overexpressed gene 18 protein is susceptible to proteolytic degradation, a condition that can be alleviated by expression in an Escherichia coli strain carrying the lon100 deletion which reduces production of protease La. Extracts of induced cells will complement an extract of T7-infected cells lacking gene 18 protein for packaging of exogenous T7 DNA. The assay has been used to monitor the purification of gene 18 protein to essential homogeneity. The identity of the purified protein has been confirmed by sequencing of the N terminus. Gel filtration analysis suggests that the native protein is an octomer. Treatment of gene 18 protein with 3 M guanidine hydrochloride denatures it to a monomer. Removal of the denaturing agent by dialysis regenerates the octomeric structure and the ability to complement packaging extracts.

Chromatography, Gel↗
PubMed1994

Error-prone replication of repeated DNA sequences by T7 DNA polymerase in the absence of its processivity subunit.

We have examined the effect of thioredoxin, an accessory protein that confers high processivity to bacteriophage T7 DNA polymerase, on the fidelity of DNA synthesis. In the presence of thioredoxin, exonuclease-proficient T7 DNA polymerase is highly accurate. In fidelity assays that score errors that revert M13mp2 lacZ alpha-complementation mutants, error rates are < or = 2.2 x 10(-6) for base substitution and < or = 3.7 x 10(-7) and < or = 4.5 x 10(-7) for frameshifts that revert mutations in the +1 and -1 reading frames, respectively. Rates are more than 10-fold higher during synthesis by polymerase.thioredoxin complex lacking 3'-->5' exonuclease activity, demonstrating that frameshift as well as substitution errors are subject to proofreading. The contribution of thioredoxin to accuracy has been examined by comparing the fidelity of the exonuclease-deficient polymerase in the presence or absence of the accessory protein. Thioredoxin either enhances or reduces fidelity, depending on the type of error considered. In the absence of thioredoxin, T7 DNA polymerase is 3-fold more accurate for base substitutions and > or = 27-fold and 9-fold more accurate, respectively, for 1- and 2-nt deletion errors at nonreiterated nucleotide sequences. Higher fidelity for all three errors may reflect the inability of the polymerase to continue synthesis from the premutational intermediates in the absence of the accessory protein. In marked contrast, the rate for frameshift errors wherein one or more nucleotides has been added to a repeated DNA sequence increases 46-fold when thioredoxin is absent from the polymerization reaction. The error rate increases as the length of the repeated sequence increases, consistent with a model where strand slippage creates misaligned template-primers. Thus, replicative expansion of repetitive sequences occurs in the absence of a replication accessory protein.

Base Sequence↗
PubMed1989

Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein.

Leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 DNA requires the helicase and primase activities of the gene 4 protein. Gene 4 protein consists of two colinear polypeptides of 56- and 63-kDa molecular mass. We have demonstrated previously that the 56-kDa protein possesses helicase but lacks primase activity (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 396-400). The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells. The preparation contains 5-10% contaminating 56-kDa protein, as shown by Western analysis using polyclonal antibodies to the purified 56-kDa protein. The 63-kDa protein catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-kDa protein. The 63-kDa protein efficiently synthesizes sequence-specific di-, tri-, and tetraribonucleotides and stimulates the elongation of tetraribonucleotides by T7 DNA polymerase. Although the 56-kDa protein alone lacks primase activity, it enhances the primase activity of the 63-kDa protein 4-fold. This stimulation can be accounted for by a similar increase in the amount of primers synthesized by the 63-kDa protein in the presence of the 56-kDa protein.

Blotting, Western↗
PubMed1999

Increased synthesis of an Escherichia coli membrane protein suppresses F exclusion of bacteriophage T7.

Increased synthesis of the protein FxsA alleviates the exclusion of T7 in cells harboring the F plasmid. In contrast to wild-type or cells defective in fxsA, overexpression of fxsA+ allows T7 to form plaques at normal efficiency even though the burst size is reduced to about half that obtained on the isogenic F- strain. No defect in DNA synthesis was observed but late protein synthesis remains partially inhibited and a reduced level of cell leakiness, a prominent feature of F+ cells abortively infected by T7, persists. The FxsA protein is shown to be a cytoplasmic membrane protein. How T7 avoids exclusion by F in cells that exhibit increased levels of FxsA is discussed in terms of its membrane localization.

Amino Acid Sequence↗
PubMed1976

Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione.

E. coli B tsnC 7004, an E. coli B/1 mutant with normal phenotype unable to replicate phage T7 DNA [Chamberlin, M. (1974)J. Virol. 14,509-516], contained no detectable level of thioredoxin when assayed with ribonucleotide reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Gently lysed E. coli tsnC 7004 cell extracts reduced CDP when supplemented with NADPH as efficiently as the parent strain E. coli B/1 despite the lack of thioredoxin, indicating the presence of another hydrogen transport system. This could be divided into two parts by heat treatment at 85degrees; one heat-stable fraction, which was active in the presence of dithiothreitol or glutathione, and one heat-labile fraction. Addition of yeast glutathione reductase [NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2] to the heated extracts restored full activity. The results demonstrate a novel hydrogen transport system in E. coli consisting of NADPH, glutathione, glutathione reductase, and a heat-stable enzyme called "glutaredoxin". Reduced glutathione at physiological concentrations functions as hydrogen donor for ribonucleotide reduction only in the presence of glutaredoxin. Glutaredoxin was not reduced by E. coli thioredoxin reductase (NADPH:oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) and showed no crossreaction with antibodies against thioredoxin. These results demonstrate the existence of two different electron transfer systems from NADPH to deoxyribonucleotides and provide a function for glutathione in DNA synthesis.

Dithiothreitol↗
PubMed1974

T7 exonuclease (gene 6) is necessary for molecular recombination of bacteriophage T7.

The role of T7-induced exonuclease (gene 6) in molecular recombination was studied by examining the fate of parental DNA during parental-to-progeny recombination. The method used was to compare infections with T7(+), T7am-6-233 (am gene 6), or T7ts6-136 (ts gene 6) under permissive and nonpermissive conditions. CsCl density gradient analysis of replicative DNA indicated that T7 exonuclease is necessary for recombination to occur, i.e., in the absence of the exonuclease the parental DNA replicated continuously as a hybrid molecule and did not recombine. Further studies under conditions where replicative DNA was denatured and analyzed by CsCl density gradient centrifugation indicated that the exonuclease is also needed for a limited amount of covalent repair of recombinants. A repair function for the T7-induced exonuclease is also suggested by results obtained from alkaline sucrose gradient analysis of replicative DNA. Under conditions nonpermissive for the exonuclease, discontinuities in the DNA accumulated during infection by T7am6-233 or by T7ts6-136.

Bromodeoxyuridine↗
PubMed1981

Initiation sites for discontinuous DNA synthesis of bacteriophage T7.

We have previously shown that the discontinuous replication of bacteriophage T7 DNA is primed by tetraribonucleotides (major component) or pentaribonucleotides. Both tetramers and pentamers start with pppA-C and are rich in A and C at the third and fourth nucleotides. In this study, the sites of transition from primer RNA to DNA in vivo have been located on a 340-nucleotide segment of the H strand of the T7 genome by 32P-labeling in vitro of the 5'-hydroxyl ends of DNA resulting from alkaline hydrolysis of RNA-linked T7 DNA fragments. Five strong transition sites were detected with a common sequence 5'-G-A-C-N1-N2-N3-N4-3', in which N1 was either C or A, N2 ws A, C, or G, and either N3 or N4 was the nucleotide for the switchover to DNA synthesis. We conclude that the complementary sequence 3'-C-T-G-G/T-N'2-(N'3)-5' in the template strand is the most frequently used signal for synthesis of primer RNA. Whereas primer-RNA synthesis starts at a precisely defined nucleotide, the transition to DNA synthesis varies within two nucleotides. Because the observed signal sequence would be present on a statistical basis once per 128 nucleotides, only about 10% of the existing signals are used for primer synthesis in each round of replication so that nascent fragments 1000-2000 long result. This provides an unexpected flexibility for RNA priming of DNA synthesis.

Base Sequence↗
PubMed1975

Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components.

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.

Chromosome Mapping↗
PubMed1986

Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7.

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.

DNA Repair↗
PubMed1978

Regulation of transcription of the late genes of bacteriophage T7.

The transcription program of bacteriophage T7 in vivo was analyzed by hybridizing T7 mRNAs, labeled at intervals after infection, to Hpa I restriction fragments of T7 DNA. Transcripcion of the late genes is temporally regulated: class II genes are transcribed between 4 and 16 min after infection; most class III genes are transcribed from 8 min after infection until lysis. Genes 8--10 are transcribed as both class II and class III genes. The rate of T7 RNA synthesis decreases sharply at 10 min after infection. The rapid decrease in the rate of T7 RNA synthesis and the shutoff of class II RNA synthesis were not observed in cells infected with phage defective in gene 3.5 (lysozyme). Although the decrease in the rate of T7 RNA synthesis is independent of DNA replication, the failure to shut off class II RNA synthesis normally in 3.5-- -infected cells may reflect the role of T7 lysozyme in DNA replication. In vitro, the regions of T7 DNA transcribed by the phage RNA polymerase were found to be dependent upon ionic conditions.

Coliphages↗