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)

A DIRECT APPROACH IS DESCRIBED TO THE QUESTION: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Escherichia coli. One-third to one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas an equivalent mixture of uncomplexed enzymes required nearly 20 min before dTTP synthesis reached its maximal rate. The effect of aggregation is evidently to decrease the volume into which intermediates are free to diffuse. Because there is reason to believe that intracellular concentration gradients of DNA precursors exist, the properties of this enzyme aggregate in vitro may help to explain how such gradients are maintained.
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PMID:Enzyme associations in T4 phage DNA precursor synthesis. 19 73

DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.
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PMID:Distinctive properties of mammalian DNA polymerases. 28 7

DNA polymerases alpha and beta (EC 2.7.7.7.) from calf thymus could utilize dUTP as a substrate for DNA synthesis as well as DNA polymerase I of Escherichia coli. Deoxyuridylate was incorporated into DNA by replacing deoxythymidylate and supported the further elongation of DNA chains on activated DNA or on the intiated homopolymers, poly(dA) . (dT)10 and poly(rA) . (dT)10. The rate of the incorporation of deoxyuridylate into DNA varied from 50 to 160% of that of deoxythymidylate, depending on the nature of the template primers and the species of DNA polymerase used. The apparent Km values for dUTP were very similar to those for dTTP. Uracil DNA-glycosylase excised efficiently the uracil residues in products of DNA polymerase reactions with either activated calf thymus DNA or initiated homopolymers.
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PMID:Utilization in vitro of deoxyuridine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus. 42 63

[3H]dUMP was incorporated into DNA of isolated S-phase HeLa S3 cell nuclei during DNA synthesis. The incorporated radioactivity was made acid soluble during a chase with excess TTP. A partially purified DNA polymerase alpha incorporated [3H]dUMP into activated salmon sperm DNA. The incorporation rate was equal to the incorporation of [3H]TMP, and the radioactivity incorporated was not made acid soluble during a chase. The nuclei thus have the ability to remove misincorporated uracil. From cytosol we have partially purified an enzyme (80 times purification) that splits the N-glycosidic bond between uracil and deoxyribose in dUMP-containing DNA. This uracil-N-glycosidase has a molecular weight of about 50 000. It does not accept dUTP or RNA as substrates. Pulse labelling of isolated nuclei with radioactive deoxyribonucleoside triphosphates in the presence of dUTP lead to a large accumulation of label in small DNA fragments. The size of these fragments was about 80 nucleotides in a 60 s pulse and no increase in size was observed with increasing pulse length. The corresponding value for control experiments with no dUTP, was 200 nucleotides and the fragments increased in size with increasing pulse length. About 90% of the radioactivity was found in the small fragments after a 3 min pulse when the concentration of dUTP in the test mixture was 100 micrometer and no exogenous TTP was present. In control experiments with no dUTP present, only 14% of the radioactivity was found in small DNA pieces. When test mixture containing dUTP was preincubated with cytosol for 60 s before adding the isolated nuclei, the small fragments increased in size to that of DNA fragments found in control incubations; also the relative amount of label bound to the fragments returned to the levels found in the controls. Increasing the TTP concentration from 5 micrometer to 1.88 mM in the absence of exogenous dUTP had no effect on the size of the DNA fragments.
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PMID:Accumulation of small fragments of DNA in isolated HeLa cell nuclei due to transient incorporation of dUMP. 70 36

Extracts of Bacillus subtilis contain a deoxyuridinetriphosphatase (dUTPase) activity with a molecular weight of approximately 48,000. The enzyme is maximally active at pH 8.5, being stimulated by Mg2+ and inhibited by EDTA. The enzyme is specific for dUTP among all the natural nucleotides tested, with an apparent Km for dUTP of 2 muM. Bacteriophage PBS2, whose DNA contains uracil instead of thymine, induces upon infection of B. subtilis a new 83,000-dalton protein which inhibits the host's dUTPase. The inhibitor acts immediately and reversibly in vitro to inhibit dUMP production from dUTP. The inhibitor's action is maximal in dUTPase assays performed at pH 6 to 7, and is minimal at pH 9.7. The inhibitor seems to form a higher molecular weight complex with the B. subtilis dUTPase. Increasing the pH of the medium for PBS2 infection from pH 7 to pH 8.85 caused a dramatic decrease in the synthesis of phage DNA and progeny phage. The newly synthesized DNA had an altered thymine/uracil ratio, being increased from less than 0.03 to greater than 1.0. We propose that infection at high pH prevents the PBS2-induced dUTPase inhibitor from blocking the B. subtilis dUTPase activity, thereby allowing the degradation of dUTP and the synthesis of dTTP (both of which are DNA polymerase substrates), so that thymine replaces some of the uracil normally found in PBS2 DNA.
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PMID:Bacillus subtilis deoxyuridinetriphosphatase and its bacteriophage PBS2-induced inhibitor. 81 Apr 87

We have recently demonstrated that mammalian uracil-DNA glycosylase activity is undetectable in adult neurons. On the basis of this finding we hypothesized that uracil, derived either from oxidative deamination of cytosine or misincorporation of dUMP in place of dTMP during DNA repair by the unique nuclear DNA polymerase present in adult neurons, DNA polymerase beta, might accumulate in neuronal DNA. Uracil residues could also arise in the herpes simplex 1 (HSV1) genome during latency in nerve cells. We therefore suggest a role for the virus encoded uracil-DNA glycosylase in HSV1 reactivation and in the first steps of DNA replication. We show here 1) that the viral DNA polymerase incorporates dUTP in place of dTTP with a comparable efficiency in vitro; 2) that virus specific DNA/protein interactions between the virus encoded origin binding protein and its target DNA sequence is altered by the presence of uracil residues in its central region TCGCA. Thus uracil, present in viral OriS or other key sequences could hamper the process leading to viral reactivation. Hence, HSV1 uracil-DNA glycosylase, dispensable in viral proliferation in tissue culture, could be essential in neurons for the "cleansing" of the viral genome of uracil residues before the start of replication.
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PMID:Uracil in OriS of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication? 133 82

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.
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PMID:Generation of single-nucleotide repair patches following excision of uracil residues from DNA. 154 15

We have shown that DNA polymerase beta, the only nuclear DNA polymerase present in adult neurons, cannot discriminate between dTTP and dUTP, having the same Km for both substrates. This fact suggests that during reparative DNA synthesis, in adult neurons, dUMP residues can be incorporated into DNA. Since uracil DNA-glycosylase functions to prevent the mutagenic effects of uracil in DNA coming as a product of deamination of cytosine residues or as a result of dUMP incorporation by DNA polymerase, we have studied the perinatal activity of uracil DNA-glycosylase and of 2 enzymes (nucleoside diphosphokinase and dUTPase) involved in dUTP metabolism. Our data indicate that during neuronal development there is a rapid decrease in uracil DNA-glycosylase which could impair the removal of uracil present in DNA in adult neurons. However, misincorporation of dUMP into DNA might be kept to a low frequency by the action of dUTPase present at all developmental stages.
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PMID:Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development. 169 67

The enzymes of DNA polymerization and DNA precursor synthesis are assembled in the replitase complex during the S phase of the cell cycle. Cross-inhibition is a phenomenon shown by enzymes of the replitase complex, in which inhibition of one enzyme of the complex leads to inhibition of a second, unrelated enzyme. This inhibition occurs only in vivo and only during S phase. The second enzyme shows no inhibition in vitro. In this study, using Chinese hamster embryo fibroblast cells, we have shown that direct allosteric interactions, i.e., structural interaction from a remote site within the replitase complex, is the cause of cross-inhibition of thymidylate synthase activity by the inhibitors of ribonucleotide reductase and DNA polymerase, because disruptions of the deoxynucleotide pools, which would be predicted for alternative explantations, do not occur. Cross-inhibition of DNA polymerase by hydroxyurea is demonstrated by the cessation of DNA synthesis when ribonucleotide reductase block is circumvented by the provision of all four deoxynucleosides. In addition to the cross-inhibition for thymidylate synthase and DNA polymerase, we have also presented evidence, on the basis of alterations of the in vivo conversion of deoxyuridine to dUMP, that cross-inhibition also occurs for the enzyme thymidine kinase. This conclusion is further supported by the lack of inhibition of the similar process in RNA synthesis, because enzymes of RNA synthesis are not included in the replitase complex. To facilitate the measurements, we have introduced a novel method of distinguishing between thymidine and deoxyuridine derivatives, making use of the fact that a tritium label placed in the 5'-position of deoxyuridine is removed on conversion to thymidine by methylation, whereas a tritium placed in the 6'-position is not.
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PMID:Allosteric interaction of components of the replitase complex is responsible for enzyme cross-inhibition. 169 15

1. Pharmacodynamics and pharmacokinetics of antimetabolites. Antimetabolites are administered in the form of a base or its riboside, which is incorporated into the cell and converted to an active or inactive metabolite. The active metabolite remain in the cell inhibiting the enzymes to catalyze nucleotide synthesis for nucleotide triphosphate formation, but the inactive metabolites are rapidly excreted out of the cell. The inhibitory effect of antimetabolites on nucleotide formation is correlated with factors, such as maintenance of drug blood level, incorporation of the drug into the cell, activation and inactivation of the drug, affinity of the active form to the corresponding enzyme, and change in pool size of the intermediate metabolites in nucleotide synthesis. The salvage synthesis occurring at the higher level of the enzymes catalyzing nucleotide synthesis to counteract the inhibition by the drug is also correlated with the nucleotide formation. II. Pyrimidine antagonists 1. Cytosine arabinoside (ara-C) and its derivatives Ara-C is rapidly converted to ara-CTP and ara-U. The former remains in the cell and inhibits DNA polymerase, but the latter is excreted rapidly out of the cell. A small portion of ara-C is incorporated into DNA, which results in the degradation of DNA as demonstrated by reduced sedimentation of bulk DNA in alkaline sucrose gradient centrifugation and the ladder DNA fragmentation with a minimum fragment of approximately 180 base pairs and its conjugates in agarose gel electrophoresis. Behenoyl ara-C (BHAC) is highly lipophilic and highly distributed in the erythrocyte stroma and membrane fraction of leukocytes after iv infusion. The incorporated BHAC is released after the plasma BHAC level decreases, which suggests that erythrocytes can be a drug reservoir after iv infusion. Therefore, severe anemia should be treated before BHAC chemotherapy for longer maintenance of the plasma BHAC level. 2. 5-Fluorouracil (5-FU) and its derivatives Activation of 5-FU in the cells is metabolized by uracil metabolizing enzymes to FUMP and FdUMP. FUMP is further metabolized to FdUMP and is also incorporated to RNA. FdUMP produces a ternary complex with thymidylate synthetase and leucovorin; subsequently, conversion of dUMP to dTMP is strongly inhibited. Thus, FUMP and FdUMP inhibit RNA and DNA metabolism, respectively. Enzyme activity during 5-FU metabolism and consequently the degree of inhibition of DNA and RNA syntheses markedly differ with the tumor cell species. This should be taken into consideration when performing chemotherapy of malignancies.
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PMID:[Clinical pharmacology of anticancer agents (Part 4). Antimetabolites (1)]. 173 42


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