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)

We have previously shown the presence of sphingomyelin and sphingomyelinase in cell nuclei, suggesting that they may play a role in the intranuclear production of sphingosine, a potent bioactive molecule modulating diverse cellular functions. In the present study, the direct effects of sphingosine (C18:1) on the activity of DNA replication/repair polymerases were studied in vitro. Sphingosine had no effect on DNA polymerases alpha and beta and slightly inhibited DNA polymerases gamma, delta, and epsilon. In contrast, sphingosine strongly inhibited the activity of primase in a dose-dependent manner. On the other hand, dihydrosphingosine (C18:0), glycolipids, sphingomyelin, and ceramide had no effect on primase activity. Sphingosine equally inhibited the activity of primase complexed with DNA polymerase alpha, as well as its free form, with a Ki value of 4 microM. A gel-retardation analysis showed that the binding of primase with 32P-labeled template DNA was suppressed by sphingosine. Inhibition by sphingosine was competitive with the DNA template, but not with the substrate NTPs. After product analysis, a dose-dependent decrease in the amount of RNA primer products, consisting mainly of 10- and 11-mers, was observed in the presence of sphingosine, indicating that it inhibits the synthesis of RNA primers by primase. Sphingosine, however, had no effect on T7 RNA polymerase.
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PMID:Sphingosine inhibits the synthesis of RNA primers by primase in vitro. 751 54

A variety of isosteres of the DNA polymerase inhibitor aphidicolin were synthesized as potential antiherpes agents. Modeling studies indicated that the bicyclooctane C, D rings of aphidicolin could be replaced by an aromatic moiety while maintaining the spatial arrangement of the hydroxyl group equivalent to the essential C18 hydroxyl group of aphidicolin. Of the racemic isosteres synthesized only 13, the compound with the greatest structural similarity to aphidicolin, showed any significant antiviral activity in primary assays. An enantioselective synthesis of the compound was carried out and the 4aS isomer 36 was shown to account for the observed antiviral activity noted against herpes simplex virus 1 and human cytomegalovirus.
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PMID:Isosteres of the DNA polymerase inhibitor aphidicolin as potential antiviral agents against human herpes viruses. 824 19

We found previously that long-chain fatty acids could inhibit eukaryotic DNA polymerase activities in vitro [1,2]. The purpose of the present study was to investigate the mode of this inhibition in greater detail. Among the C18 to C24 fatty acids examined, the strongest inhibitor was a C24 fatty acid, nervonic acid (NA), and the weakest was a C18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase beta (pol. beta), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase alpha (pol. alpha) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. beta could be stopped with a non-ionic detergent, but the binding to pol. alpha or HIV-RT could not. The inhibition mode of LA showed the same characteristics, except that the minimum inhibitory dose of the longer chain was much lower. We also tested the effects of NA and LA using pol. beta and its proteolytic fragments, as described by Kumar et al. [3,4]. Both of the fatty acids were found to bind to the 8 kDa DNA-binding domain fragment, and to suppress binding to the template-primer DNA. We found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity. The possible modes of inhibition by these long-chain fatty acids are discussed, based on the present findings.
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PMID:The inhibitory action of fatty acids on DNA polymerase beta. 936 79

The glycolipid galactosyldiacylglycerol (GDG), containing C16:0 and C18:1 fatty acids, was isolated from the sea alga Petalonia bingbamiae as a potent inhibitor of the activities of mammalian DNA polymerase alpha (pol. alpha). GDG, however, had no effect on pol. alpha from a fish or a higher plant. The inhibition of pol. alpha by GDG was dose-dependent with an IC50 value of 54 microM. The compound did not influence the activities of other replicative DNA polymerases such as mammalian pol. delta, or repair-related enzymes such as mammalian pol. beta. GDG also did not influence the activities of prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, DNA polymerases from the higher plant, cauliflower, or DNA metabolic enzymes such as calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type 1 reverse transcriptase and deoxyribonuclease 1. Kinetic analysis of the compound showed that pol. alpha was non-competitively inhibited with respect to both the DNA template and the nucleotide substrate. In this study, we demonstrated the structure-function relationship in the selective inhibition of pol. alpha by the glycolipid group.
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PMID:Galactosyldiacylglycerol, a mammalian DNA polymerase alpha-specific inhibitor from a sea alga, Petalonia bingbamiae. 1155 81

Some sulfoquinovosylacylglycerols (SQAG) have been shown to be potent DNA polymerase inhibitors, and to have strong antitumor activity in vivo. In this study, we investigated the mode of action of SQAG with regard to the interaction with the tumor cell surface. Of the SQAG used, the monoacyl forms (SQMG) with C18-, C18:1- or C16-fatty acids (SQMG-alphaC18, -alphaC18:1 or -alphaC16) effectively inhibited cell proliferation of a human adenocarcinoma cell line, DLD-1, but SQMG-alphaC14 and the diacyl forms (SQDG) did not. Analysis of the interaction of SQMG-alphaC18 and -alphaC18:1 on three oligosaccharides of cell surface, sLe(A), Le(X), and SM3, by flow cytometry demonstrated that the most effective interaction was observed on sLe(A). DLD-1 cells bound to SQMG-alphaC18:1-coated plates, and this binding was inhibited by monoclonal antibody against sLe(A) or SM3. However, these cells did not bind to SQMG-alphaC14-coated plates. Moreover the cytotoxic effects of SQMG-alphaC18, -alphaC18:1 on DLD-1 cells was inhibited by monoclonal antibodies against sLe(A) or SM3. Our results suggested that the interaction of SQMGs and sLe(A) plays an important role in suppression of the DLD-1 cell proliferation.
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PMID:Specific interaction between an oligosaccharide on the tumor cell surface and the novel antitumor agents, sulfoquinovosylacylglycerols. 1168 93

Acyclovir (ACV) is an antiviral drug, which selectively inhibits replication of members of the herpes group of DNA viruses with low cell toxicity. Valaciclovir (VACV), a prodrug of ACV is usually preferred in the oral treatment of viral infections, mainly herpes simplex virus (HSV). Also other analogues such as ganciclovir and penciclovir are discussed here. The former acts against cytomegalovirus (CMV) in general and the latter against CMV retinitis. The action mechanism of these antiviral drugs is presented briefly here, mainly via phosphorylation and inhibition of the viral DNA polymerase. The therapeutic use and the pharmacokinetics are also outlined. The measurement of the concentration of acyclovir and related compounds in biological samples poses a particularly significant challenge because these drugs tend to be structurally similar to endogenous substances. The analysis requires the use of highly selective analytical techniques and chromatography methods are a first choice to determine drug content in pharmaceuticals and to measure them in body fluids. Chromatography can be considered the procedure of choice for the bio-analysis of this class of antiviral compounds, as this methodology is characterised by good specificity and accuracy and it is particularly useful when metabolites need to be monitored. Among chromatographic techniques, the reversed-phase (RP) HPLC is widely used for the analysis. C18 Silica columns from 7.5 to 30 cm in length are used, the separation is carried out mainly at room temperature and less than 10 min is sufficient for the analysis at 1.0-1.5 ml/min of flow-rate. The separation methods require an isocratic system, and various authors have proposed a variety of mobile phases. The detection requires absorbance or fluorescence measurements carried out at 250-254 nm and at lambdaex=260-285 nm, lambdaem=375-380 nm, respectively. The detection limit is about 0.3-10 ng/ml but the most important aspect is related to the sample treatment, mainly when body fluids are under examination. The plasma samples obtained from human blood are pre-treated with an acid or acetonitrile deproteinization and the supernatant after centrifugation is successively extracted before RP-HPLC injection. Capillary Electrophoresis methods are also discussed. This new analytical approach might be the expected evolution, in fact the analyses are improved with regard to time and performance, in particular coated capillary as well as addition of stabilisers have been employed. The time of analysis is shortened arriving at less than half a minute. Furthermore by using an electrochemical detection, and having a calibration linearity in the range of 0.2-20.0 ng/ml, the detection limit is 0.15 microg/ml. The measurements of acyclovir and penciclovir have been presented but in the future other related drugs will probably be available using CE methods.
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PMID:Separation methods for acyclovir and related antiviral compounds. 1181 33

To identify the sites in the Klenow fragment of Escherichia coli DNA polymerase I that interact with the ssDNA overhang of the template strand in the pre-polymerase ternary complex, we carried out UV-mediated photo-cross-linking of the enzyme-DNA-dNTP ternary complex. The template strand contained a nine-nucleotide overhang and was radiolabeled at the 5'-end. Since the enzyme-TP-dNTP ternary complex but not the E-TP binary complex is stable at high ionic strengths, the cross-linking was carried out in the presence of 0.5 M NaCl. The cross-linked E-TP-dNTP complex was purified and subjected to trypsin digestion. The radiolabeled TP cross-linked peptide was further purified by DEAE-Sepharose and C18 column chromatography and subjected to amino acid sequencing. The release of radiolabeled DNA during each sequencing cycle was also monitored. The sequencing results as well as the radioactivity release pattern show that F771, contained in a peptide spanning amino acids 759-775 of pol I, is the unequivocal site of the template cross-linking. A qualitative assessment of the cross-linking efficiency of the template overhang containing a TT sequence at different positions in the ternary complex further suggests that the major cross-linking site within the template overhang is at the second and/or third nucleotide. An examination of the F771A mutant enzyme showed that it was able to form the E-TP binary as well as E-TP-dNTP ternary complex; however, it could not cross-link to the template-primer in the ternary complex. Furthermore, the ternary complex with F771A was qualitatively defective and exhibited some salt sensitivity. These results suggest that F771 participates in the stabilization of the pre-polymerase ternary complex.
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PMID:Phe 771 of Escherichia coli DNA polymerase I (Klenow fragment) is the major site for the interaction with the template overhang and the stabilization of the pre-polymerase ternary complex. 1266 54

We have screened the inhibitors of mammalian DNA polymerases from natural products, and in the process found that either sulfoglycolipids or sulfoquinovosyl monoacylglycerol with a C18-saturated fatty acid (C18-SQMG), potently and selectively inhibited the activity of mammalian DNA polymerase (pol) and moderately the pol alpha. C18-SQMG was a cancer cell growth suppressor and a promissive anti-tumor agent. The purpose of this study was to elucidate the cell growth inhibition mechanism of C18-SQMG using HeLa cells. Analyses of the cell cycle and cyclin expression suggested that C18-SQMG arrested the cell cycle at intra-S phase, and the inhibition manner of DNA replication by C18-SQMG was similar to that by hydroxyurea. However, the DNA replication block by C18-SQMG did not induce degradation of Cdc25A protein, which was required for the replication block by hydroxyurea. C18-SQMG somewhat delayed mitosis because it induced phosphorylation of protein kinases, such as checkpoint kinases 1 and 2. These results suggest that C18-SQMG at first blocked DNA replication at the S phase by inhibiting replicative DNA polymerases, such as alpha, and then as the result of the inhibition, the other checkpoint signals associated with the pol might have responded.
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PMID:Mechanism of cell cycle arrest by sulfoquinovosyl monoacylglycerol with a C18-saturated fatty acid (C18-SQMG). 1501 53

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.
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PMID:Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis. 1565 80

Epolactaene (compound 1), a neuritogenic compound found in human neuroblastoma cells, was found to show anti-inflammatory activity in vivo in this study. DNA polymerases and DNA topoisomerase II (topo II) were some of the major molecular targets of compound 1. Since the agent seems to be a potential pharmaceutical medicine, we synthesized derivatives chemically and obtained seven compounds, 1 to 7 to screen clinically more efficient epolactaene derivatives. A comparison of its structural derivatives revealed that the long alkyl side chain seemed to have an important role in the inhibitory effect. Notably, C18-alkyl chain conjugated epolactaene (compound 5) was the strongest inhibitor of DNA polymerase alpha, beta, lambda (pol alpha, beta, lambda) and topo II, with IC50 values of 13, 135, 4.4 and 5 microM, respectively, and 500 microg of compound 5 caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 65.0%). Compound 5 did not influence the activities of plant or prokaryotic DNA polymerases, or of other DNA metabolic enzymes such as telomerase, RNA polymerase and deoxyribonuclease I. Based on these results, the relationship among the three-dimensional structure of epolactaene derivatives and the inhibition of polymerases and topo II, and anti-inflammation is discussed.
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PMID:Structural analysis of epolactaene derivatives as DNA polymerase inhibitors and anti-inflammatory compounds. 1580 99


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