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)

The 140,000-Da adenovirus-encoded DNA polymerase (Ad Pol) is required for viral DNA replication both in vitro and in vivo. The polymerase co-purifies in a complex with the 80,000-Da precursor (pTP) of the terminal protein (TP) found covalently attached to the 5' ends of adenovirus DNA. To better understand their function in DNA replication, we have examined the properties of the Ad Pol and the pTP X Ad Pol complex on natural and synthetic DNA templates. The pTP X Ad Pol complex utilizes a variety of homopolymer template-primer combinations including poly(dC) X oligo(dG), poly(dA) X oligo(dT), poly(dT) X oligo(dA), and poly(dT) X oligo(rA). With poly(dT) as template and oligo(rA) or oligo(dA) as primer, DNA synthesis by the pTP X Ad Pol complex is stimulated as much as 100-fold by the 59,000-Da adenovirus DNA-binding protein (Ad DBP). ATP (4 mM) can further increase the rate of DNA synthesis 3- to 10-fold. The Ad DBP does not stimulate the activity of host (HeLa cell) DNA polymerase alpha with poly(dT) X oligo(dA) (or oligo(rA)) as the template-primer, and Escherichia coli single-stranded DNA binding protein cannot substitute for the Ad DBP in the stimulation of the Ad Pol activity. Under optimal conditions, poly(dA) chains 30,000 nucleotides in length are formed indicating that the Ad Pol can be a highly processive enzyme. An exonuclease activity co-sediments with the pTP X Ad Pol complex during glycerol gradient centrifugation, and co-purifies with the 140,000-Da Ad Pol after dissociation of the pTP X Ad Pol complex with urea. The Ad Pol-associated nuclease hydrolyzes single-stranded DNA in a 3'----5' direction and is at least 10-fold more active on single-stranded DNA than on duplex DNA. The Ad Pol has no detectable endonuclease activity on single-stranded DNA or duplex circular DNA. Analysis of the products of the nuclease activity showed that 5'-deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The Ad DBP inhibits the hydrolysis of DNA by the polymerase-associated nuclease activity.
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PMID:Properties of the adenovirus DNA polymerase. 654 Feb 63

Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, ribonuclease-sensitive fraction from the cytosol of uninfected cells. This fraction stimulated the initiation about 3-fold and the replication of origin fragments 5-10-fold. Sedimentation analysis indicated the presence of a fast-sedimenting and a slow-sedimenting component which complemented each other. At least part of the stimulation was caused by low-molecular-mass RNA.
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PMID:Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells. 672 75

At least 12 virus-induced DNA-binding proteins with mol. wt. ranging from 14 X 10(3) have been isolated from frog virus (FV 3)-infected fathead minnow cells by DNA affinity chromatography. Two enzymic activities, DNA-dependent DNA polymerase and endodeoxyribonuclease, were present in the DNA-binding proteins; these enzymic activities were similar to those induced by FV 3 in infected cells. A single species of DNA-binding proteins with a mol. wt of 36 000 had very high affinity for single-stranded DNA, but a low one for double-stranded DNA. Proteins with such characteristic affinity for single-stranded DNA destabilize the DNA helix and are essential for viral DNA replication. Thus, the 36 000 mol. wt. DNA-binding protein is a candidate for such a role in FV 3 DNA replication.
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PMID:DNA-binding proteins in frog virus 3-infected cells. 689 83

Bacteriophage T4 DNA polymerase, product of phage gene 43 (gp43), is a multifunctional DNA-binding protein and a key component of the phage DNA replicase. It is also an RNA-binding protein that selectively recognizes a site on its mRNA (the translational operator) and represses its own translation. We examined the ability of the protein to discriminate between DNA and RNA by using a gel mobility shift assay with defined RNA and DNA substrates. A higher affinity to RNA as compared with DNA (about 100-fold) was observed in assays that utilized synthetic DNA and in vitro transcribed RNA substrates bearing the T4 gene 43 translational operator sequence. The replacement of thymine with uracil in the synthetic DNA did not improve binding. The results suggest that the protein's selectivity for RNA is based in structure (intramolecular interactions) specific to the ribonucleotide sequence of the operator. Competition studies suggest that the protein determinants for RNA and DNA recognition are only partially overlapping.
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PMID:Binding specificity of T4 DNA polymerase to RNA. 751 97

The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.
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PMID:Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha. 764 8

The mechanisms governing the function of cellular USF and herpesvirus immediate-early transcription factors are subjects of considerable interest. In this regard, we identified a novel form of coordinate gene regulation involving a cooperative interplay between cellular USF and the varicella-zoster virus immediate-early protein 62 (IE 62). A single USF-binding site defines the potential level of IE 62-dependent activation of a bidirectional viral early promoter of the DNA polymerase and major DNA-binding protein genes. We also report a dominant negative USF-2 mutant lacking the DNA-binding domain that permits the delineation of the biological role of both USF-1 and USF-2 in this activation process. The symmetrical stimulation of the bidirectional viral promoter by IE 62 is achieved at concentrations of USF-1 (43 kDa) or USF-2 (44 kDa) already existing in cells. Our observations support the notion that cellular USF can intervene in and possibly target promoters for activation by a herpesvirus immediate-early protein.
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PMID:The cellular transcription factor USF cooperates with varicella-zoster virus immediate-early protein 62 to symmetrically activate a bidirectional viral promoter. 793 7

Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.
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PMID:Replication factor A is required in vivo for DNA replication, repair, and recombination. 796 28

By using appropriate amounts of four bacteriophage phi 29 DNA replication proteins--terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation at 30 degrees C. Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) phi 29 DNA, measured as the ability to produce phage particles, was identical to that of the natural phi 29 DNA obtained from virions. The results presented in this paper establish some of the requisites for the development of isothermal DNA amplification strategies based on the bacteriophage phi 29 DNA replication machinery that are suitable for the amplification of very large (> 70 kb) segments of DNA.
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PMID:Terminal protein-primed DNA amplification. 799 6

We have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon-gamma (IFN-gamma). We now demonstrate that IFN-gamma and tumour necrosis factor alpha (TNF-alpha) display synergism in their antiviral activity. As little as 2 ng/ml of IFN-gamma and TNF-alpha reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, E1 and those encoding the major DNA-binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18.5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses.
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PMID:Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor. 811 18

Meiotic cells of Coprinus contain a protein that can be identified by its ability to enhance activity of the meiotic DNA polymerase reported previously (8). Its activity is found only during the prophase stages in meiotic cells, and is accompanied by the meiotic polymerase. The protein, which was purified to near homogeneity, is a single polypeptide with a molecular mass of 30k, and shows no activities of nuclease, ATPase, or DNA-binding protein. The protein could enhance the meiotic polymerase activity by at least 5 fold. The other Coprinus polymerases were not influenced by the protein. The protein could increase Vmax value of the meiotic polymerase, but not the Km. The significance of this protein to meiotic DNA synthesis is considered in relation to other biochemical properties of meiotic cells.
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PMID:A new meiotic protein factor which enhances activity of meiotic DNA polymerase from Coprinus cinereus. 811 80


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