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 polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3'-end-group-labelled DNA was similar to that of a heat-denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3' leads to 5' direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50%, 29% and 13% of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U. maydis DNA polymerase is similar to the bacteriophage T4-induced enzyme.
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PMID:A DNA polymerase from Ustilago maydis. 2. Properties of the associated deoxyribonuclease activity. 124 76

Nuclear extracts, prepared from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells during a time course of infection, were analyzed for activation of early gene transcription and for late gene transcription. The templates used in the in vitro transcription assays contained promoters for baculovirus genes that have been classified as immediate early, delayed early, and late. The promoters were derived from the baculovirus 39K, p26, gp64, and DNA polymerase genes. In addition, the adenovirus major late promoter was included in these studies. We found that transcription from promoters classified as immediate early or delayed early was accurately initiated by using extracts from uninfected cells. Furthermore, transcription from all early promoters tested was found to be transactivated by nuclear extracts prepared at 4 and 8 h postinfection. However, baculovirus enhancer-dependent transcriptional activation was not observed in tests with templates containing the hr5 enhancer sequence. Transcription from baculovirus late promoters was also not observed. A decline in transcription by nuclear extracts prepared from cells late in infection was associated with the presence of DNase activity.
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PMID:In vitro transactivation of baculovirus early genes by nuclear extracts from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells. 131 63

DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.
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PMID:Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease. 132 12

The myb proto oncogene product (c-Myb) is a transcriptional regulator and its expression and function are tightly linked to the cellular entry into S phase and DNA synthesis. It has been shown [Venturelli, D., Travali, S. & Calabretta, B. (1990). Proc. Natl. Acad. Sci. USA, 87, 5963-5967] that inhibition of T-cell proliferation by a myb antisense oligomer is accompanied by down-regulation of DNA polymerase alpha expression. To examine whether the transcription of the DNA polymerase alpha gene is directly regulated by c-Myb, we have identified and characterized the 5' regulatory region of the human DNA polymerase alpha gene. Two major and several minor transcription start sites were identified by nuclease S1 mapping. DNA sequence analysis showed that the promoter region contains no TATA box, one CCAAT box and putative Ap-1, AP-2 and E2F binding sites. In DNAase I footprinting, the bacterially expressed c-Myb protected six sites in the 5' flanking region of the human DNA polymerase alpha gene. However, c-Myb did not activate the DNA polymerase alpha gene promoter in a co-transfection assay. Our results suggest that an unknown factor(s) is required for the c-Myb-induced activation of the DNA polymerase alpha gene promoter, or c-Myb does not directly activate this promoter.
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PMID:The c-myb proto-oncogene product binds to but does not activate the promoter of the DNA polymerase alpha gene. 140 40

After high dose methotrexate (CAS 59-05-2) therapy of children with non-metastatic osteosarcoma the neutral DNase activity is missing in lymphocytes and in phytohemagglutinin (PHA)-stimulated lymphocytes. The neutral DNase activity reappeared in lymphocytes 14 days and in PHA-stimulated lymphocytes 10 days after the end of therapy. The DNA polymerase activity is low when neutral DNase is missing and increases when neutral DNase activity reappeared. The neutral DNase activity in lymphocytes and in PHA-stimulated lymphocytes is probably identical with DNase I. Drug induced changes in DNA conformation can enhance DNase I cleavage rate. It is assumed, that high dose methotrexate alters DNA conformation and therefore binds DNase I; after this, free DNase I is no more detectable.
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PMID:Missing neutral DNase activity in lymphocytes and phytohemagglutinin-stimulated lymphocytes after high dose methotrexate therapy. 141 65

TuMV particles were purified from artificially infected leaves of Brassica juneaen, and TuMV-RNA was extracted from these particles. The virus thus purified showed typical nucleoprotein absorbent peak under UV light scanning, and the ratio of A260/A280 was 1.21. The RNA thus prepared showed typical ribonucleic acid absorbent peak, and the ratio of A260/A280 was 2.2. The RNA was then used as template of oligo (dT) 12-18 together with the end product of DNase digested calf thymus DNA were used as primer for the synthesis of cDNA. The length of double stranded cDNAs synthesized were distributed continuously with the sizes between 500-4300 bp. The ds-cDNA repaired by Klenow fragment was inserted into the Small site of pUC19 by blunt-end ligation. Then the recombinant molecules were used to transform E. coli strain DH5 alpha. Rapid electrophoresis of plasmid prepared by alkali method, EcoRI-HindIII digestion and in situ hybridization with 32P labeled TuMV-RNA showed that inserted fragments were with various sizes and complement to TuMV-RNA.
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PMID:[Synthesis and molecular cloning of the cDNA of TuMV-RNA]. 176 57

Treatment of L929 cells with TNF alpha initiates apoptosis and subsequent cell death. The authors have visualized sites of DNA damage in situ by using DNA polymerase to synthesize new strands from the DNA strands breaks as starting point. Biotin-dUTP was incorporated into the newly synthesized strand and visualized by immunocytochemistry. DNA strand breaks were first observed 3 to 4 hours after contact with TNF alpha and preceded cell death. Limiting doses of TNF alpha caused DNA strand breaks only in a subpopulation of L929 cells. At a low dose, TNF alpha led to DNA damage without any subsequent loss of cell viability. The new assay also detects DNase-induced single strand breaks and thus is able to visualize apoptotic as well as non-apoptotic types of DNA damage.
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PMID:Analysis of TNF alpha-induced DNA strand breaks at the single cell level. 186 16

A quantitative and efficient assay was developed to measure the 3'-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein. A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3'-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment. The 32P-labeled nucleotide was located at the penultimate position. The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts. The structure of the released product was characterized on 23% sequencing gels. Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus.
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PMID:Development of an acid-soluble assay for measuring retrovirus integrase 3'-OH terminal nuclease activity. 188 32

A high molecular weight mitochondrial DNA (mtDNA) replication complex, associated with the mitochondrial membrane, was isolated by sucrose gradient centrifugation from purified wheat embryo mitochondria. This complex comprised the mtDNA as well as enzyme activities involved in the replication and transcription of the organelle genome, such as DNA polymerase, RNA polymerase and topoisomerase type I. The isolated complex is active in mtDNA and mtRNA synthesis in vitro. Electron microscopy and lipid analysis confirmed the membrane origin of this complex. Enzyme activities are resistant to physiological ionic strengths, 0.1-0.2 M KC1, while the membrane-mtDNA association is resistant up to 1 M KC1. DNase treatment of the complex released the DNA polymerase activity while protease treatment solubilized mtDNA, suggesting the direct interaction of mtDNA with membrane protein(s). The use of a novel approach to detect mtDNA fragments specifically retained by the mitochondrial membranes after Sal I digestion of the complex suggests that specific mtDNA sequences anchor mtDNA to mitochondrial membranes.
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PMID:Isolation from wheat mitochondria of a membrane-associated high molecular weight complex involved in DNA synthesis. 189 1

An Epstein-Barr virus (EBV) specific monoclonal antibody (MAb), designated 55H3, was produced after immunizing BALB/c mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) activated B95-8 cells. It was possible to demonstrate that this MAb detected an EBV-specific antigen(s) in the EBV genome-positive producer cell lines B95-8 and M-ABA by immunofluorescence. Immunoglobulin prepared from ascites fluid neutralized the activity of the EBV-encoded DNA polymerase, but not the alkaline DNase. By Western blotting, the 55H3 MAb reacts with an 85/80-KD polypeptide. The 55H3 should be useful in examining the role of EBV DNA polymerase in viral replication.
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PMID:A monoclonal antibody that neutralizes Epstein-Barr virus DNA polymerase activity. 216 46


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