Effects of platinum antitumor agents and pyrimidine dimers on the in vitro replication of T7 DNA.
Explore the source record for details and available documents.
Cisplatin↗
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.
Evidence for direct involvement of T7 RNA polymerase bacteriophage DNA replication.
Experiments in a number of different systems have suggested that the initiation of DNA replication is often dependent upon transcription at the origin of replication. During infection with bacteriophage T7, the T7 genome is transcribed first by the bacterial RNA polymerase and then by a phage-coded enzyme, the product of gene 1. The bacterial enzyme does not appear to be directly involved in the initiation of replication as phage DNA synthesis is not inhibited by rifampin. For testing whether the T7 RNA polymerase plays a role in replication, a T7 gene 1 temperature-sensitive mutant was used, and the RNA polymerase was inactivated at intervals after infection by rapidly raising the temperature of the culture. The experiments indicated that the inactivation of the T7 RNA polymerase caused the cessation of phage DNA synthesis, even at later times during infection when the inhibition of protein synthesis with chloramphenicol had no effect on DNA replication. This suggests that in addition to its role in gene expression, the T7 RNA polymerase plays a direct role in T7 DNA replication.
Chloramphenicol↗
Effects of bulky polycyclic aromatic hydrocarbon adducts on DNA replication by exonuclease-deficient T7 and T4 DNA polymerases.
In vitro DNA replication by exonuclease-deficient T7 DNA polymerase (Sequenase) and an exonuclease deficient T4 DNA polymerase was examined on a 244-nucleotide DNA template treated with three electrophilic polycyclic aromatic hydrocarbon (PAH) metabolites: racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE), trans-2,3-dihydroxy-anti-1,10b-epoxy-10b,1,2,3-tetrahydrofluoranthene (FADE), or 3,4-epoxy-3,4-dihydrocyclopenta[cd]pyrene (CPPE). The DNA replication terminated opposite template guanines and, to a lesser extent, at template adenines, as expected, as purines were modified preferentially by the chemical treatments. Analysis of the products synthesized on the damaged templates indicated that bypass replication by Sequenase proceeded in three steps: (1) replication first terminated one base 3' to each adduct; (2) a nucleotide was then incorporated opposite the PAH-modified base; and (3) replication continued at some sites to give full bypass of the lesions. The rate of lesion bypass was affected by the type of chemical adduct, the sequence context of the adduct, and the concentration of deoxynucleoside triphosphates. Short DNA repeats appeared to facilitate translesion replication.
Bacteriophage T4↗
In vitro packaging of bacteriophate T7 DNA synthesized in vitro.
An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.
Coliphages↗
Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits.
The DNA polymerase induced after infection of Escherichia coli by phage T7 has been purified 500-fold to near homogeneity as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme complements extracts of cells infected with a T7 gene 5 mutant to permit cell-free replication of duplex T7 DNA. In contrast, purified T4 DNA polymerase or E. coli DNA polymerase I is unable to do so, thus suggesting a specific requirement for the T7 enzyme in the replication of the viral DNA. E. coli TsnC protein is present in purified T7 DNA polymerase in one-to-one stoichiometry with T7 gene 5 protein, and can be isolated in homogeneous form from heat-denatured enzyme by chromatography on DEAE-cellulose. The inactive form of T7 gene 5 protein that accumulates in tsnC hosts has been partially purified. When partially purified gene 5 protein is mixed with purified TsnC protein, DNA polymerase activity is restored, and formation of a one-to-one complex between the two proteins occurs. These results indicate that the functional form ofT7 DNA polymerase is a complex composed of phage- and host-specified subunits.
Bacterial Proteins↗
In vitro replication of bacteriophage T7 DNA damaged by ultraviolet radiation.
The effect of ultraviolet radiation on DNA replication has been examined with an in vitro system capable of replicating intact chromosomes of T7 DNA from an exogenous template. Exposure of the template DNA to ultraviolet radiation resulted in a sharp drop in the amount of in vitro DNA synthesis. The residual replication detected when irradiated templates were used was found to proceed semiconservatively and to result in the production of pieces of duplex DNA approximately the same size as the average distance between pyrimidine dimers. It was also found that prior irradiation of the template inhibits formation of fast-sedimenting concatemer-like DNA structures normally synthesized in vitro. Hybridization studies demonstrated that the product synthesized in vitro from ultraviolet-irradiated templates includes DNA from both the left and right halves of the T7 chromosome. This may mean that after ultraviolet irradiation more than one origin of replication exists.
Centrifugation, Zonal↗
Marker rescue and partial replication of bacteriophage T7 DNA.
Experiments reported here show that some UV-irradiated wild-type T7 phage markers can be rescued efficiently by coinfection with T7 amber mutant phage in a permissive host. Other results show that the segments of a UV-irradiated genome that replicate efficiently are those that also are rescued efficiently during a marker rescue experiment. At higher doses, fewer markers are rescued efficiently and fewer segments of the genome replicate efficiently. The results clearly indicate that the probability of marker rescue is correlated with the ability of the DNA containing the marker to replicate. Sucrose density gradient analysis shows that UV irradiation does not produce double-strand scissions in T7 DNA at doses used here. Therefore, the partial replication and rescue of markers from the left end of the genome is not due simply to injection of only the left end of the T7 DNA.
Chromosome Mapping↗
Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.
Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.
Bacteriophage T7↗
Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication.
The product of gene 2.5 of bacteriophage T7, a single-stranded DNA binding protein, physically interacts with the phage-encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phages that contain null mutants of gene 2.5 were constructed by homologous recombination. These gene 2.5 null mutants contain either a deletion of gene 2.5 (T7 delta 2.5) or an insertion into gene 2.5 and cannot grow in Escherichia coli (efficiency of plating, < 10(-8)). After infection of E. coli with T7 delta 2.5, host DNA synthesis is shut off, and phage DNA synthesis is reduced to < 1% of phage DNA synthesis in wild-type T7-infected E. coli cells as measured by incorporation of [3H]thymidine. In contrast, RNA synthesis is essentially normal in T7 delta 2.5-infected cells. The defects in growth and DNA replication are overcome by wild-type gene 2.5 protein expressed from a plasmid harboring the T7 gene 2.5.
Bacteriophage T7↗
Bacteriophage T7 Deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity.
In vivo, replication of T7 DNA does not occur after infection of Escherchia coli tsnC mutants (CHAMBERLIN, M. (1974) J. Virol. 14, 509-516). In vitro, extracts of tsnC mutant E. coli infected with T7 hage are incapable of replicating duplex T7 DNA, although extracts of wild type E. coli infected with T7 phage support replication of T7 DNA. In addition, extracts of the infected tsnC mutant are deficient in T7 DNA polymerase activity. Extracts prepared from uninfected E.coli tsnC-+ cells restore the ability of the infected tsnC extracts to replicate duplex T7 DNA, and also restore normal levels of the phage DNA polymerase activity. A 12,000-dalton heat-stable protein responsible for this complementation has been purified to near homogeneity from uninfected tsnC+ extracts and it is designated "TsnC protein."
Bacterial Proteins↗
Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Initiation of bidirectional synthesis.
Replication of bacteriophage T7 DNA initiates in vivo at an origin located 15% of the distance from the genetic left end of the chromosome. Bidirectional DNA synthesis from this site results in complete replication of the chromosome. The combination of T7 RNA polymerase, T7 DNA polymerase, and T7 gene 4 protein initiates DNA synthesis in vitro within the cloned origin sequence (Fuller, C. W., and Richardson, C. C. (1985) J. Biol. Chem. 260: 3185-3196). DNA synthesis is primed by T7 RNA polymerase transcripts, and proceeds in the same direction (rightward) as transcription to yield partially replicated Y-form DNA molecules. The DNA product of in vitro synthesis (Y-form DNA) has been characterized by electron microscopic, sedimentation, and gel electrophoretic analyses. These studies show that Y-form DNA is the product of unidirectional replication of both leading and lagging strands from the origin to the right-hand end of the template. The inclusion of either Escherichia coli single-stranded DNA-binding protein or the functionally similar T7 gene 2.5 protein results in marked stimulation of bidirectional synthesis. Studies using purified Y-form DNA provide direct evidence that this species is an intermediate in the complete replication of the linear template. Purified Y-form DNA is converted to linear DNA in a reaction catalyzed by T7 DNA polymerase, T7 gene 4 protein, and single-stranded DNA-binding protein. Y-form DNA is a competent, transient intermediate during the bidirectional replication of linear DNA molecules and DNA-binding protein is essential to initiate leftward synthesis.
Centrifugation, Density Gradient↗
Role of the acidic carboxyl-terminal domain of the single-stranded DNA-binding protein of bacteriophage T7 in specific protein-protein interactions.
The gene 2.5 single-stranded DNA (ssDNA) binding protein of bacteriophage T7 is essential for T7 DNA replication and recombination. Earlier studies have shown that the COOH-terminal 21 amino acids of the gene 2.5 protein are essential for specific protein-protein interaction with T7 DNA polymerase and T7 DNA helicase/primase. A truncated gene 2.5 protein, in which the acidic COOH-terminal 21 amino acid residues are deleted no longer supports T7 growth, forms dimers, or interacts with either T7 DNA polymerase or T7 helicase/primase in vitro. The single-stranded DNA-binding protein encoded by Escherichia coli (SSB protein) and phage T4 (gene 32 protein) also have acidic COOH-terminal domains, but neither protein can substitute for T7 gene 2.5 protein in vivo. To determine if the specificity for the protein-protein interaction involving gene 2.5 protein resides in its COOH terminus, we replaced the COOH-terminal region of the gene 2.5 protein with the COOH-terminal region from either E. coli SSB protein or T4 gene 32 protein. Both of the two chimeric proteins can substitute for T7 gene 2.5 protein to support the growth of phage T7. The two chimeric proteins, like gene 2.5 protein, form dimers and interact with T7 DNA polymerase and helicase/primase to stimulate their activities. In contrast, chimeric proteins in which the COOH terminus of T7 gene 2.5 protein replaced the COOH terminus of E. coli SSB protein or T4 gene 32 protein cannot support the growth of phage T7. We conclude that an acidic COOH terminus of the gene 2.5 protein is essential for protein-protein interaction, but it alone cannot account for the specificity of the interaction.
Amino Acid Sequence↗
Gene 4 protein of bacteriophage T7. Purification physical properties, and stimulation of T7 DNA polymerase during the elongation of polynucleotide chains.
With the use of an in vitro complementation assay to measure activity, the gene 4 protein of bacteriophage T7 has been purified 1000-fold to yield a nearly homogeneous protein. The purified gene 4 protein is a single polypeptide having a molecular weight of 58,000. In addition to being essential for T7 DNA replication in vivo and in vitro, the gene 4 protein is required for DNA synthesis by the purified T7 DNA polymerase on duplex T7 DNA templates. In the absence of ribonucleoside 5'-triphosphates, DNA synthesis by the gene 4 protein and the T7 DNA polymerase is dependent on phosphodiester bond interruptions containing 3'-hydroxyl groups (nicks) in the duplex DNA. The reaction is specific for the T7 DNA polymerase, but any duplex DNA containing nicks can serve as template. The Km for nicks in the reaction is 3 x 10(-10) M.
Coliphages↗
Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection.
Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.
Base Sequence↗
Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA.
Growth of bacteriophage T7 is inhibited by the antibiotic coumermycin A1, an inhibitor of the Escherichia coli DNA gyrase. Since growth of the phage is insensitive to the antibiotic in strains containing a coumermycin-resistant DNA gyrase, this enzyme appears to be required for phage growth. We have investigated the effect of coumermycin on the kinetics of DNA, RNA, and protein synthesis during T7 infection. DNA synthesis is completely inhibited by the antibiotic. In addition, coumermycin significantly inhibits transcription of late but not early genes. Thus, E. coli DNA gyrase may play an important role in transcription as well as in replication of T7 DNA.
Anti-Bacterial Agents↗
A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase.
Thioredoxin was purified to homogeneity from the Escherichia coli mutant tsnC 7007 that is defective in phage T7 DNA replication and previously shown to contain a missense thioredoxin. Tryptic peptide maps of reduced and carboxymethylated 7007 thioredoxin combined with amino acid sequence analysis revealed one amino acid substitution; Gly-92 in thioredoxin is exchanged to an aspartic acid residue in the 7007 protein. The missense thioredoxin gave no activity with the gene 5 protein of phage T7 in the complementation to active T7 DNA polymerase. It competitively inhibited the complementation of wild type thioredoxin and gene 5 protein and formed a complex with the gene 5 protein that was retained by antithioredoxin Sepharose. The 7007 thioredoxin has reduced catalytic activity with thioredoxin reductase, ribonucleotide reductase, or as a protein disulfide reductase. The apparent Km value of 7007 thioredoxin as a substrate for thioredoxin reductase was increased 3-fold relative to normal thioredoxin, and the Vmax value was decreased 7-fold. The position of GLy-92 in the known three-dimensional structure of thioredoxin-S2 is correlated with the changed functional properties of the substituted mutant protein.
Amino Acid Sequence↗
Repair of double-strand breaks by incorporation of a molecule of homologous DNA.
An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome. The efficiency of double-strand break repair was markedly increased by DNA molecules ('donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending approximately 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32P, repaired genomes incorporated label only near the site of the double-strand break. When repair was carried out with unlabelled donor DNA and [32P]-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break. These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.
Bacteriophage T7↗
Mutant bacteriophage T7 RNA polymerases with altered termination properties.
We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (T(phi)) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.
Amino Acid Sequence↗