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)

Two types of DNA ligase, I and II, have been purified approximately 4,000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNA ligase I and II consisted of single polypeptides with molecular weights of 95,000 and 65,000, respectively, according to the estimation by SDS-polyacrylamide gel electrophoresis and the AMP-binding assay. Ligase activities were higher in premeiotic spermatogonia and spermatocytes than those in liver and bone marrow cells. Moreover, DNA ligase II showed rapid increase during meiotic prophase and a decrease in round spermatids. Since this behavior of DNA ligase II is consistent with that of m-rec and DNA polymerase beta, both of which have been shown to be involved in DNA recombination in meiotic cells, DNA ligase II might be an enzyme which works at the final step of meiotic recombination reaction.
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PMID:Purification of DNA ligases from mouse testis and their behavior during meiosis. 234 May 90

Effects of ATP and some other nucleotides (AMP, ADP, CTP, GTP, UTP and dATP) on reparative DNA synthesis and repair patch ligation in bleomycin-pretreated permeable mouse sarcoma cells were studied. Reparative DNA synthesis was significantly stimulated by 2.5 mM ATP, ADP or dATP. The stimulation was observed on both DNA polymerase alpha- and beta-dependent reparative DNA synthesis. ATP concentration required for repair patch ligation was much lower than that required for the stimulation of reparative DNA synthesis. An apparent Km value for ATP of the repair patch ligation was about 40 microM. ADP supported repair patch ligation after being converted into ATP by adenylate kinase in permeable cells.
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PMID:Effects of ATP and other nucleotides on DNA repair synthesis in bleomycin-pretreated permeable mouse sarcoma cells. 244 62

A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody. 244 48

The synthesis of specific protein has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr approximately 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid-G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell-cycle-dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr approximately 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase-delta. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S-phase. These data support the view that the cAMP-mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1-phase.
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PMID:Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways. 264 71

The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.
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PMID:Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor. 266 51

Human terminal deoxyribonucleotidyltransferase (nucleoside-triphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. "TATA" and "CAAT" sequences, respectively, were found to start at nucleotides -185 and -204, -328, and 465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements.
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PMID:Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region. 283 41

Terminal deoxynucleotidyl transferase (Tdt), a unique DNA polymerase found only in lymphoid cells, may be involved in the generation of immunoglobulin-combining site diversity. To study the actual metabolic function of the enzyme, we developed a system in which Tdt expression is induced under defined culture conditions. We found that pharmacologic agents that raise intracellular cyclic AMP levels, such as caffeine, induce a three- to 10-fold increase in enzyme biosynthesis rate and activity. This phenomenon is observed only in pre-B cell lines of human or murine origin.
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PMID:Cyclic AMP induces terminal deoxynucleotidyl transferase in immature B cell leukemia lines. 298 68

Six mutations, impairing DNA polymerase of E. coli in combination with the wild type gene for rho factor or ts-mutation rho 15 have been studied in relation to the expression of seven operons having different types of regulation. The expression of genes for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase is shown to be constitutive and resistant to mutationally altered RNA polymerase and rho factor. The expression of genes for adenine phosphoribosyltransferase and of deo operon is regulated by rho dependent attenuators with attenuation being lifted incomplete medium. Mutation rho 15 decreases the level of enzymes of thr and lac operons independent of mRNA levels of these operons. Mutation rho 15 effect on posttranscriptional level is modified by mutations damaging RNA polymerase. The data obtained suppose RNA polymerase to affect all stages of realization of genetic information, beginning with promoter recognition and RNA synthesis and including the protein synthesis on mRNA.
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PMID:[Effect of mutation changes in RNA-polymerase and transcription termination factor rho on expression of various operons in E. coli]. 302 82

Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.
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PMID:The enzymatic basis of cyclophosphamide specificity. 302 54

A DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40. Contributions to these products by contaminating DNA polymerase gamma were eliminated by the addition of dideoxy-ATP. Addition of 50 microM dATP to the primase reaction caused a 50% inhibition of AMP incorporation as compared to reactions containing low levels of dATP present only as a contaminant of the ATP added. The inhibition was due primarily to a reduction of new chain initiations. The dATP did not "lock" the primase reaction into the DNA mode of synthesis since the proportion of internal and 3'-terminal RNA segments was little affected. However, the addition of both 50 microM dATP and exogenous DNA polymerase to the primase reaction greatly reduced the amount of internal and 3'-terminal RNA segments, presumably due to the displacement of primase by DNA polymerase. Our data are consistent with the hypothesis (Hu, S.-Z., Wang, T.S.-F., and Korn, D. (1984) J. Biol. Chem. 259, 2602-2609) that the physiologically significant primer is a mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide and that the formation of the RNA to DNA junction is inherently a primase function.
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PMID:Characterization of a DNA primase from rat liver mitochondria. 370 Apr 9


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