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)

We have localized a cis-acting sequence that promotes initiation of lytic-phase DNA replication (oriLyt) within the HindIII D fragment of the human cytomegalovirus (HCMV) AD169 genome and investigated its sequence requirements by testing the ability of plasmid constructs to mediate DNA replication in a transient transfection-plus-infection assay. Replication of plasmids containing HCMV oriLyt required at least the virus-specified DNA polymerase activity supplied by HCMV infection of transfected cells and was autonomous in that it did not result from recombination with the virus genome. Progeny molecules in the transient assay were high-molecular-weight tandem oligomers, which is consistent with predictions of a rolling-circle model. Experiments testing subclones of HindIII-D defined a core 2.4-kbp region containing elements required for oriLyt function that extended rightward from around 1.0 kbp upstream of UL57 near the middle of the long unique component of the virus genome. Sequences flanking this core also were needed for full activity. The defined region contains at least four clustered sets of repeated sequence elements identical to or candidate counterparts of elements present in the corresponding cytomegalovirus Colburn lytic-phase replication origin. These elements are novel in that they apparently do not correspond to previously characterized motifs. Also present are multiple copies of elements similar to known binding sites for the transcription factors ATF/CREB, MLTF/USF, and Sp1. Preliminary deletion analysis suggests that multiple components within the boundaries of oriLyt cooperate to enable initiation of HCMV lytic-phase DNA synthesis.
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PMID:Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. 131 54

We have investigated the DNA polymerase alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNase I digestion by partially purified nuclear proteins. The DNA polymerase alpha promoter can confer upon the reporter an appropriate, late response to serum addition. No single sequence element could be shown to confer serum inducibility. Rather, multiple sequence elements appear to mediate the full serum response.
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PMID:Human DNA polymerase alpha gene: sequences controlling expression in cycling and serum-stimulated cells. 200 99

The 5'-flanking region of the human poly(ADP-ribose) polymerase gene was isolated and characterized. The nucleotide sequence of a part of the poly(ADP-ribose) polymerase gene completely matched that of the cDNA. The transcriptional initiation sites (cap sites) of this gene, located about 166-bp upstream from the translational initiation site, were identified by S1 mapping analysis. Neither CAAT box nor TATA box was found within 500-bp upstream from the cap sites of poly(ADP-ribose) polymerase gene. The 200-bp immediately upstream of the cap site had a high G+C content (76.5%) and contained double repeats of the sequence CCGCCC, putative Sp1 binding sites, and a palindromic structure. The 5'-flanking region of poly(ADP-ribose) polymerase gene also showed promoter activity in chloramphenicol acetyltransferase assay and structural similarity to that of DNA polymerase beta gene.
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PMID:Characterization of a putative promoter region of the human poly(ADP-ribose) polymerase gene: structural similarity to that of the DNA polymerase beta gene. 210 70

The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein.
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PMID:Protein binding elements in the human beta-polymerase promoter. 231 44

The promoter of mouse DNA polymerase beta gene was analyzed by combining 5'-upstream region of this gene with chloramphenicol acetyltransferase (CAT) gene and by introduction of the recombinant plasmid DNA into mouse NIH/3T3 cells. Serial deletion of the mouse DNA sequence revealed that the promoter function resides within a 33 base pair region from the nucleotide position -48 to -15 with respect to the transcription initiation site, and is highly active without enhancer sequence. The promoter region was separated into two subregions: one (-48 to -35) contains a GC-box and the other contains a 10 base pair palindrome, whose sequence is similar to one of promoter consensus sequences found in a number of promoters including adenovirus promoters. The DNA polymerase beta promoter-directed CAT expression was competitively inhibited by the simultaneous transfection of plasmid DNA containing SV40 early promoter sequence. The viral sequences which are competitive to the GC-box of DNA polymerase beta gene promoter were the GC-boxes of SV40 promoter. Therefore, it is concluded that transcription of mouse DNA polymerase beta gene is regulated by mouse trans-acting factors equivalent to human Sp1 which is known to be trans-acting protein factor acting on SV40 GC-box sequences.
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PMID:Mouse DNA polymerase beta gene promoter: fine mapping and involvement of Sp1-like mouse transcription factor in its function. 284 59

DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.
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PMID:Human beta-polymerase gene. Structure of the 5'-flanking region and active promoter. 318 28

The core promoter of the human DNA polymerase beta (beta Pol)-encoding gene (POL beta) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755-12763]. To study the homologous promoter (ppol beta) in a bovine system, we cloned and characterized the 5'-flanking region of the bovine gene (pol beta). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5'-flanking region was isolated from a testis library in bacteriophage lambda EMBL3. S1 nuclease mapping and primer extension analysis of the 5'-end of the pol beta mRNA identified the major transcription start point (tsp), which is located 142-bp 5' of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5'-deletion mutants demonstrated that a fragment of only 91-bp 5' of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOL beta). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Sp1-binding sites, and the spacing separating them.
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PMID:The bovine DNA polymerase beta promoter: cloning, characterization and comparison with the human core promoter. 759 Mar 51

We identified cis elements in the 5'-flanking region of rat Na,K-ATPase alpha 2 subunit gene (Atp1a2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5'-deletion mutation analysis, the region between nucleotide positions -175 and -108 was identified as a positive regulatory region. In the region, the distal E box (nucleotides -144 to -139) acts as a negative regulatory element, and the Sp1 consensus sequence (nucleotides -123 to -118) and the GGGAGG sequence (nucleotides -114 to -109) act as positive regulatory elements. Gel-retardation analysis revealed that binding factors are an E-box-binding protein and Sp1. DNase I footprinting and methylation-interference analyses revealed that Sp1 binds to the region from nucleotides -122 to -101 and the E-box-binding protein to the region from nucleotides -144 to -136. T4 DNA polymerase footprinting revealed that there are three Sp1-binding sites in the region and that Sp1 binds to one of the three sites in a mutually exclusive manner. The mechanism by which Sp1 activates the Atp1a2 promoter is discussed.
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PMID:Anomalous interaction of Sp1 and specific binding of an E-box-binding protein with the regulatory elements of the Na,K-ATPase alpha 2 subunit gene promoter. 824 64

The major late promoter (MLP) of the subgroup C human adenoviruses is a preeminent model for the study of the mechanisms of basal and activated transcription, both in vivo and in vitro. However, while the structure and function of the human virus MLP has been the subject of extensive investigation, the conservation of the various promoter elements among the adenoviruses from different species has not been examined. Conservation of specific elements would strongly suggest the importance and universality of their function. To address this issue, sequences were obtained from cloned DNAs of several representative Mastadenoviridae, mouse adenovirus type 1 (MAV-1), Tupaia adenovirus type 1 (TAV-1), and two bovine adenoviruses of two distinct subgroups, BAV-3 and BAV-7. The results of the sequencing studies showed that the TATA box and an upstream inverted CAAT box are conserved in all species and that the binding site for transcription factor USF is present in all except MAV-1, in which a sequence similar to an Sp1-binding site is present at a similar position. The initiator element (INR) sequence is not well conserved, and only one or other of the two downstream activating elements, DE1 and DE2, is predicted to be present in the nonprimate virus MLP regions. Ribonuclease protection assays on RNA isolated from MAV-1-infected cells late in infection indicated that the predicted MLP is functional, and transcription initiation and splice donor sites were identified. The human virus MLP is embedded in the essential DNA polymerase sequence on the opposite DNA strand. The primary amino acid sequences of the C-terminal regions of the predicted DNA polymerases show strong conservation of sequence motifs observed in replicative polymerases ranging from prokaryotes to mammals, and additional regions of strong conservation among the adenovirus polymerases. Pairwise comparisons between the newly sequenced regions of the polymerases and previously published sequences show that BAV-7 is most dissimilar to all others, while TAV-1 has a greater similarity to the primate sequences than to the others. The sequence data from both strands were also used to construct phylogenetic trees, based on BAV-7 as the outgroup. The trees constructed from the two sets of sequences are broadly similar, showing close relationships between primate viruses, but differing in the order of divergence of TAV-1 and MAV-1 branches.
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PMID:Conservation of DNA sequence in the predicted major late promoter regions of selected mastadenoviruses. 866 90

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


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