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)

Treatment of hamster cells in culture with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces DNA polymerase beta (beta-pol) gene expression and cellular levels of the enzyme. Transcriptional activity of a cloned beta-pol promoter in transient expression assays is also stimulated. Among the requirements for these responses are methylation damage to genomic DNA, cellular cAMP-dependent protein kinase, and the ATF/CREB site of the cloned beta-pol promoter. In the present study, HeLa cell nuclear extract from MNNG-treated cells was much more active in an in vitro transcription assay than nuclear extract from normal cells. By using an oligonucleotide affinity column to deplete the nuclear extract of ATF/CREB, we showed that the difference was due to ATF/CREB activator. Purified ATF/CREB activator from MNNG-treated cells was approximately 10-fold more active than ATF/CREB purified from normal cells as a transcriptional activator for the depleted nuclear extract. ATF/CREB in the extract from normal cells is known to activate in vitro transcription by increasing the rate of promoter clearance [Narayan, S., Widen, S. G., Beard, W. A., & Wilson, S. H. (1994) J. Biol. Chem. 269, 12755-12763]. With ATF/CREB from MNNG-treated cells, the amount of preinitiation complex formed was much greater than with ATF/CREB from normal cells, and the kinetics of both the closed to open preinitiation complex isomerization and promoter clearance were altered. These results indicate that the mechanism of transcriptional activation secondary to DNA alkylation damage is recruitment of more preinitiation complex and alteration of the kinetic scheme of transcription initiation.
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PMID:DNA damage-induced transcriptional activation of a human DNA polymerase beta chimeric promoter: recruitment of preinitiation complex in vitro by ATF/CREB. 781 26

In this report a modification to the in vivo footprinting assay is described. The method includes dimethyl sulfate treatment of whole yeast cells, followed by reiterative primer extension of the methylated genomic DNA using Taq DNA polymerase. Under appropriate reaction conditions chain extension terminates opposite a methylated purine when Taq DNA polymerase encounters a modified adenine or guanine. The procedure was used to examine, in vivo DNA-protein contacts over the upstream activation site (UAS) of the Saccharomyces cerevisiae PYK gene. In vivo analysis, using isogenic strains of yeast and Escherichia coli transformed with plasmid DNAs, confirmed the binding of both the trans-acting factor RAP1 and the transcriptional activator GCR1 to cis-acting recognition sites located within the PYK UAS element.
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PMID:A simple in vivo footprinting method to examine DNA-protein interactions over the yeast PYK UAS element. 819 Jun 36

The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA-binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary-phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary-phase-inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprinting experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.
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PMID:DnaA protein stimulates polA gene expression in Escherichia coli. 910 10

The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the "downstream component"), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various protions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from -588 to -592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF-1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication.
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PMID:The Epstein-Barr virus (EBV) DNA polymerase accessory protein, BMRF1, activates the essential downstream component of the EBV oriLyt. 912 59

Activation of transcription at bacteriophage T4 late promoters and coupling of late transcription to concurrent replication requires a peculiar transcriptional activator, the gp45 sliding clamp of the T4 DNA polymerase. In order to activate transcription, the topologically DNA-linked trimeric gp45 must interact with two T4-encoded RNA polymerase-binding proteins, the gp33 co-activator, and the gp55 late sigma factor. The carboxy termini of gp55 and gp33 share a similar sequence, which has been shown to be required for response of late transcription to activation by gp45. Alanine-scanning mutagenesis of the C terminus of gp55 shows that residues within the short hydrophobic sequence L(D/A)FLYE, are necessary for gp55 to bind to gp45, and to respond maximally to transcriptional activation by gp45. When fused to GST, the peptide SLDFLYE suffices for specific gp45 binding. Thus, it constitutes the main gp55 epitope for gp45 interaction.
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PMID:Activator-sigma interaction: A hydrophobic segment mediates the interaction of a sigma family promoter recognition protein with a sliding clamp transcription activator. 981 12

The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. A new random saturation mutagenesis method was developed to isolate Gcn4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope. This will be used to identify the amino acids of Gcn4p involved in protein-protein interactions. Saturation mutagenesis in the DNA binding domain of Gcn4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area. These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector. The 3' ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites. These molecules were then converted to double stranded DNA upon treatment with DNA polymerase. Here, a library collection of 100,680 in an altered Gcn4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains. The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values. Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library. Thus, among 21 mutants, 16 had one point mutations and 5 had two point mutations. This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules.
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PMID:Random changes of amino acid residues with expected frequency by saturated point mutagenesis. 1085 Jun 67

In early Caenorhabditis elegans embryos, asymmetric cell divisions produce descendants with asynchronous cell cycle times. To investigate the relationship between cell cycle regulation and pattern formation, we have identified a collection of embryonic-lethal mutants in which cell divisions are delayed and cell fate patterns are abnormal. In div (for division delayed) mutant embryos, embryonic cell divisions are delayed but remain asynchronous. Some div mutants produce well-differentiated cell types, but they frequently lack the endodermal and mesodermal cell fates normally specified by a transcriptional activator called SKN-1. We show that mislocalization of PIE-1, a negative regulator of SKN-1, prevents the specification of endoderm and mesoderm in div-1 mutant embryos. In addition to defects in the normally asymmetric distribution of PIE-1, div mutants also exhibit other losses of asymmetry during early embryonic cleavages. The daughters of normally asymmetric divisions are nearly equal in size, and cytoplasmic P-granules are not properly localized to germline precursors in div mutant embryos. Thus the proper timing of cell division appears to be important for multiple aspects of asymmetric cell division. One div gene, div-1, encodes the B subunit of the DNA polymerase alpha-primase complex. Reducing the function of other DNA replication genes also results in a delayed division phenotype and embryonic lethality. Thus the other div genes we have identified are likely to encode additional components of the DNA replication machinery in C. elegans.
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PMID:DNA replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos. 1111 26

That mammalian DNA polymerase-beta (beta-pol) gene transcription is upregulated by activated ras and also by phorbol ester (TPA) treatment suggests the involvement of protein kinase C in the gene expression control for this DNA repair enzyme. Yet, the core promoters of the human, bovine and rodent beta-pol genes do not have a TPA response element or other binding site for the transcriptional activator AP-1. Instead, these beta-pol promoters appear to be regulated mainly by proteins binding to the cAMP response element (CRE) centered within 50 bp 5' of the transcriptional start site. In this study, the CRE in the human beta-pol promoter was found to mediate TPA upregulation of the cloned promoter in HeLa cell transient expression experiments. To further examine the role of this CRE in TPA stimulation, we used several mutated promoters that were either deficient in protein binding to the CRE or contained extra CRE sites arranged as tandem repeats. All constructs with at least one functional CRE were upregulated by TPA, whereas mutants lacking CRE protein-binding function were not TPA upregulated. Analyses of HeLa nuclear extract DNA-binding proteins indicated that the beta-pol CRE was bound by CRE-binding protein (CREB) family members CREB-1 and activating transcription factor-1, but not by AP-1 or complexes containg AP-1 subunits. These results suggest that CREB, rather than AP-1 proteins, are required for the CRE-mediated TPA activation of the beta-pol promoter.
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PMID:Human DNA Polymerase-beta Promoter: Phorbol Ester Activation Is Mediated through the cAMP Response Element and cAMP-Response-Element-Binding Protein. 1238 74

The Epstein-Barr virus (EBV) BMRF1 gene encodes an early lytic protein that functions not only as the viral DNA polymerase processivity factor but also as a transcriptional activator. BMRF1 has been previously shown to activate transcription of an EBV early promoter, BHLF1, though a GC-rich motif which binds to SP1 and ZBP-89, although the exact mechanism for this effect is not known (D. J. Law, S. A. Tarle, and J. L. Merchant, Mamm. Genome 9:165-167, 1998). Here we demonstrate that BMRF1 activates transcription of the cellular gastrin gene in telomerase-immortalized keratinocytes. Furthermore, BMRF1 activated a reporter gene construct driven by the gastrin promoter in a variety of cell types, and this effect was mediated by two SP1/ZBP-89 binding sites in the gastrin promoter. ZBP-89 has been previously shown to negatively regulate the gastrin promoter. However, ZBP-89 can function as either a negative or positive regulator of transcription, depending upon the promoter and perhaps other, as-yet-unidentified factors. BMRF1 increased the binding of ZBP-89 to the gastrin promoter, and a ZBP-89-GAL4 fusion protein was converted into a positive transcriptional regulator by cotransfection with BMRF1. BMRF1 also enhanced the transcriptional activity of an SP1-GAL4 fusion protein. These results suggest that BMRF1 activates target promoters through its effect on both the SP1 and ZBP-89 transcription factors. Furthermore, as the EBV genome is present in up to 10% of gastric cancers, and the different forms of gastrin are growth factors for gastrointestinal epithelium, our results suggest a mechanism by which lytic EBV infection could promote the growth of gastric cells.
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PMID:The Epstein-Barr virus protein BMRF1 activates gastrin transcription. 1561 2

Despite the small size of its genome (3.2 kb) and having only four genes that are encoded within it, the hepatitis B virus (HBV) is one of the most successful viral pathogens in human history. It is estimated that there are about 350-400 million people worldwide who are chronically infected with HBV, and even with the extensive efforts that are being done with preventive vaccination, this malady still remains a clear and present danger to the public health. How is it possible that this small double-stranded DNA virus can escape and outfox the surveillance of the complex human immune system? One explanation is that HBV gene products play multiple roles in infections and throughout the viral life cycle so that the virus can effectively survive under various hostile circumstances. Indeed, the HBV DNA polymerase, for example, exerts several functions such as reverse transcription and RNA degradation, and the HBV X protein not only acts as a transcriptional activator, but it also interferes with the host cells' DNA repair mechanism as well as inducing apoptosis and controlling signal transduction. The HBV surface protein, which is encoded in the env gene, is another intriguing example of such multifunctionality. Thus, our present article overviews and summarizes the multifaceted role of this membrane protein as shown in 1) its role as a structural protein of the virus envelope; 2) its function as the viral ligand for interacting with the viral receptors on host cells; 3) its characteristics as an energy-independent transporter molecule that can mediate the nuclear accumulation of itself and other tagged molecules; 4) its role as a viral transactivator protein that can cause hepatocellular carcinoma; 5) its hypothetical function in viral apoptotic mimicry that results in host anti-inflammatory responses; and last 6) its immunostimulatory property by providing for strong and well-defined B- and T-cell epitopes. Understanding these various functions and the versatility of this single protein will help us decipher and understand the viral- and immuno-pathogenesis of HBV itself.
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PMID:[Hepatitis B virus surface antigen: a multifaceted protein]. 1561


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