Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.
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PMID:Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid. 17 58

Adenovirus type 5 (Ad5) DNA was degraded with endo R . HpaI; the left-terminal fragment, HpaI-E has recently been shown to be the smallest segment of Ad5 DNA, that can transform non-permissive cells. This fragment was labelled at its termini by limited exonuclease III digestion followed by repair synthesis with DNA polymerase and alpha-32P-labelled deoxynucleoside triphosphates. It was then further digested with each of the restriction endonucleases HpaII, HaeIII, AluI, HinfI and TaqI; the cleavage products thus obtained were ordered into a physical map.
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PMID:Mapping of restriction sites in the transforming HpaI-E fragment of adenovirus type 5 DNA. 71 11

Adenovirus 12 (Ad12) (Huie) (highly oncogenic group A) readily induces tumors in newborn rodents. Since Ad12 is isolated from human fecal samples, we investigated whether it plays a role in the etiology of human gastrointestinal cancer. If Ad12 is a causal agent of human cancer, then human tumors should contain Ad12 transforming genes, as indicated by studies of cells transformed in vitro and in vivo by oncogenic viruses. Ad12 DNA and the Ad12 transforming restriction fragment (EcoRI-C fragment, left 16% of the viral genome) were labeled in vitro to 10(7) to 4 X 10(8) cpm/mug by the nick translation reaction of DNA polymerase of Escherichia coli. The fidelity and sensitivity of these probes were established by (i) analysis of DNA from Ad12-transformed cells and from hamsters with tumors induced by Ad12, (ii) reconstruction experiments with added Ad12 DNA and EcoRI restriction fragments, and (iii) comparison of annealing characteristics with Ad12 probes labeled in vivo. With Ad12 [3H]DNA as probe, no viral DNA sequences were detected in 18 normal gastrointestinal tissues and 34 gastrointestinal tumors, including cancers of the colon, rectum, small intestine, and stomach, under conditions that would detect 0.1 copy of the Ad12 genome per tumor cell. Similar analyses of Ad12-transformed hamster cells and Ad12 primary hamster tumors indicated 6-18 copies per cell of over 90% of the viral genome. With the Ad12 EcoRI-C transforming fragment as probe, no hybridization was detected with 32 human gastrointestinal tumors and five normal tissues; this result excludes 1-2% of the Ad12 genome per tumor cell. Our date are strong evidence that Ad12 is not a major cause of human gastrointestinal cancer. The Ad12 transforming EcoRI-C fragment hybridized (50-68% efficiency) with other Ad12 isolates and with Ad18 and 31 (members of oncogenic group A), but not at all with 28 other human Ad serotypes (manuscript in preparation). Thus other group A members probably are also not involved in human gastrointestinal cancer. No viral DNA sequences were detected in 12 normal lungs and 22 lung tumors, suggesting that respiratory cancer does not involve an Ad12 etiology.
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PMID:Do highly oncogenic group A human adenoviruses cause human cancer? Analysis of human tumors for adenovirus 12 transforming DNA sequences. 107 16

Initiation of Adenovirus DNA replication in vitro requires the presence of three viral proteins (pTP, pol, DBP) and two cellular transcription factors, NFI and Oct-1, that stimulate replication more than 100-fold. NFI assists in binding and positioning of the DNA polymerase in the origin whereas Oct-1 changes the structure of origin DNA. Optimal templates contain, in addition to origin sequences, the covalently bound viral terminal protein (TP). This terminal protein stimulates the template activity over 20 fold compared to protein-free templates. To study the way in which TP exerts its function in vitro we devised a novel method to isolate and label a short origin containing fragment in which the TP was bound in a functional form. This fragment replicated very efficiently and could be used for studying the binding of other replication proteins. Employing alpha-chymotrypsin digestion we show that for enhancement of replication in vitro only a small part of TP is required.
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PMID:Adenovirus DNA replication: the function of the covalently bound terminal protein. 129 Dec 41

POU domain proteins constitute a family of eukaryotic transcription factors that exert critical functions during development. They contain a conserved 160 amino acids DNA binding domain, the POU domain. Genetic data have demonstrated that some POU domain proteins are essential for the proliferation of specific cell types, suggesting a possible role in DNA replication. In addition, the ubiquitous POU transcription factor Oct-1 or its isolated POU domain enhances adenovirus DNA replication. Here we compared the DNA binding specificities of POU domain proteins from different subclasses. They exhibit overlapping, yet distinct binding site preferences. Furthermore, purified Pit-1, Oct-1, Oct-2, Oct-6, Oct-4 and zebrafish POU[C] could all stimulate adenovirus DNA replication in a reconstituted in vitro system. Thus, activation appears to depend on a property common to most POU domain proteins. Adenovirus DNA replication is also stimulated by the transcription factor NFI/CTF. In contrast to NFI, the POU domain did not enhance binding of precursor terminal protein-DNA polymerase to the origin nor did it stabilize the preinitiation complex. These results suggest that the POU domain acts on a rate limiting step after formation of the preinitiation complex.
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PMID:POU domain transcription factors from different subclasses stimulate adenovirus DNA replication. 147 98

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.
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PMID:Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals. 177 81

An extract from Adenovirus type 4 infected HeLa cells was fractionated by ion-exchange and DNA affinity chromatography. One fraction, which bound tightly to single stranded DNA, contained predominantly a protein of apparent molecular weight 65,000 and three less abundant proteins. Immunological cross-reactivity with adenovirus type 2 proteins confirmed the presence of preterminal protein and indicated that the abundant species was the virus coded DNA binding protein. This fraction contained an aphidicolin resistant DNA polymerase activity and in the presence of a linearised plasmid containing the adenovirus type 4 origin of DNA replication efficient transfer of dCMP onto preterminal protein, indicative of initiation, was observed. Furthermore, addition of all four deoxyribonucleotide triphosphates and an ATP regenerating system resulted in the elongation of initiated molecules to generate plasmid molecules covalently attached to preterminal protein. Adenovirus type 4 DNA binding protein was extensively purified from crude adenovirus-4 infected HeLa extract by immunoaffinity chromatography using a monoclonal antibody raised against adenovirus type 2 DNA binding protein. A low level of initiation of DNA replication was detected in the fraction depleted of DNA binding protein but activity was restored by addition of purified DNA binding protein. DNA binding protein therefore plays an important role in the initiation of Ad4 DNA replication.
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PMID:Replication of adenovirus type 4 DNA by a purified fraction from infected cells. 182 16

Adenovirus (Ad) DNA polymerase (AdPol) and the preterminal protein (pTP) form a complex that is involved in the in vitro initiation of Ad DNA replication. Recombinant vaccinia viruses (vv) were constructed in which the genes encoding AdPol and pTP were cloned into a vaccinia/T7 hybrid expression-based vector downstream from the T7 promoter (pT7)/encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR). HeLa cells infected with the recombinant vv-AdPol or vv-pTP or a mixture of both, together with the vv expressing T7 RNA polymerase produced significant levels of pTP and AdPol which were biologically active in the in vitro initiation of Ad DNA replication. These amounts of pTP and AdPol were only about two-fold less than the levels produced in insect cells infected with the recombinant baculovirus constructs expressing AdPol and pTP.
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PMID:Overproduction of adenovirus DNA polymerase and preterminal protein in HeLa cells. 193 14

Adenovirus type 2 cores can function effectively as templates in an in vitro replication system. Viral DNA replication assays using cores as templates do not differ in their requirements to the well characterized assays using DNA-complex templates, i.e. there is a dependence on terminal protein precursor (pTP), DNA polymerase and DNA binding protein and the assay is greatly stimulated by certain host transcription factors. The products of initiation and limited elongation are easily distinguishable and, in the system described, there is specific proteolysis of the pTP adducts as a function of the adenovirus-coded protease, present in the nuclear extracts from infected cells, or the core templates. Substitution of Mn2+ ions for Mg2+ ions in the replication assay has a dramatic effect on the nature of the replication events, in most cases resulting in the stimulation of initiation without elongation. Similar results can be achieved by utilizing subviral particles as templates, obtained by dialysis of purified adenovirus in a hypotonic buffer at pH 6.4. Restriction enzyme analysis of the replicated products confirmed that DNA synthesis proceeds from the adenovirus termini using both the core and subviral templates. By adding an ATP-regenerating system elongation can be further stimulated, particularly in the case of the subviral templates. Quantification of nucleotide incorporation into the appropriate restriction fragments indicates that for the subviral templates replication can proceed for at least 2000 to 3000 bases from either terminus. These results suggest that the adenovirus genome is packaged in the virion in a conformation readily available for at least the initial replication events. Such a conformation might also be appropriate for early transcription.
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PMID:Adenovirus subviral particles and cores can support limited DNA replication. 260 37

Adenovirus (Ad) virions contain a 55-kDa terminal protein covalently linked to both 5'-ends of the linear duplex DNA genome. The origin of DNA replication is contained within the terminal 50 base pair of the inverted terminal repeats. In the accompanying paper (Kenny, M. K., Balogh, L. A., and Hurwitz, J. (1988) J. Biol. Chem. 263, 9801-9808), it was demonstrated that synthetic oligonucleotide templates which contain the Ad origin, but lack the 55-kDa terminal protein, can serve as templates for the initiation of Ad DNA replication. Partially duplex oligonucleotides that lacked up to 14 nucleotides from the 5'-end of the nontemplate (displaced) strand supported initiation as much as 20-fold more efficiently than fully duplex oligonucleotides. The removal of 18 nucleotides or more from the 5'-end of the displaced strand resulted in a sharp decrease in the ability of the DNA templates to support initiation. The poor template efficiency of certain DNAs could be explained by their inability to bind nuclear factor I. The initiation efficiency observed with other DNAs correlated with their ability to bind the preterminal protein-Ad DNA polymerase complex. At low concentrations of the Ad DNA-binding protein, protein-primed initiation was also observed on single-stranded DNAs. The single-stranded template strand of the Ad origin was at least 5-20-fold better at supporting initiation than other single-stranded DNAs. These findings suggest a model in which the 3'-end of the template strand is rendered single-stranded as a prerequisite for initiation of Ad DNA replication.
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PMID:Initiation of adenovirus DNA replication. II. Structural requirements using synthetic oligonucleotide adenovirus templates. 283 80


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