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 further modification of Behrens ' method of nonaqueous cell fractionation, using glycols as media for homogenization and centrifugation, was presented. HeLa cells were frozen in melting Freon-12 ( CCl2F2 ), dried under vacuum at -30 degrees C, sonicated in hexylene glycol at -35 degrees C, and centrifuged through either propylene glycol or a mixture of the two glycols at -40 degrees C. The centrifugation yielded a nuclear pellet and a cytoplasmic supernatant. The supernatant was recentrifuged at -10 degrees C, yielding a cytoplasmic pellet. The success of the method depended on the temperature-dependent viscosities of the glycols and on the aggregation properties of cell structures in cold glycols. The allowed ranges of low temperatures were critical but not difficult to use; methods are given for sonication and for centrifugation. The two pellet fractions together contained 90% or more of cellular proteins and nucleic acids. Distribution of [3H]uridine-labeled nucleic acids showed that the first pellet (nuclei) contained over 95% of the nuclear markers, DNA, and ribosomal RNA precursors, plus about 10% of the cytoplasmic marker, 18 S ribosomal RNA. The cytoplasmic pellet contained less than 5% of the nuclear markers. Two enzyme activities were tested; DNA polymerase, a mostly soluble nuclear marker frequently eluted in aqueous fractionation, and lactate dehydrogenase, a cytoplasmic marker. The two enzymes each lost activity in propylene glycol but not in a mixture of 90% hexylene glycol and 10% propylene glycol, so the glycol mixture was used as a centrifugation medium when studying enzymes. The glycol mixture sometimes gave more cytoplasmic material, up to 20% of the 18 S ribosomal RNA, in the nuclear pellet. The fractionation showed, as expected, that DNA polymerase activity was 95% nuclear and lactate dehydrogenase activity was more than 68% cytoplasmic. The concentration of cytoplasmic material afforded by the glycol method allowed the detection of a small amount (approx. 5%) of DNA polymerase activity not associated with nuclei. The chief reason for use of the glycol method instead of other methods of cell fractionation is that easily solubilized cellular material can be recovered in concentrated pellet form in the appropriate nuclear or cytoplasmic fraction.
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PMID:Nonaqueous fractionation of HeLa cells in glycols. 674 32

L-glutamic acid, gamma-(p-hydroxyanilide), is a naturally occurring metabolic inhibitor found in mushrooms and shown to be active against B-16 melanoma in vivo. We have prepared and evaluated 2 analogs, the 3,4- and 2,5-dihydroxy derivatives, since these might represent more immediate precursors to the putative biologically active quinone. Both dihydroxy derivatives were more toxic than the parent phenol. The 2,5-dihydroxy derivative was significantly more cytotoxic with a 5-fold decrease in IC50 for both human and B-16 melanoma cells in vitro. In the presence of mushroom tyrosinase, both derivatives were potent inhibitors of isolated DNA polymerase with essentially complete inhibition occurring at concentrations of 10(-5) M. The 3,4-dihydroxy derivative exerted inhibitory effects primarily upon thymidine incorporation into melanoma cells in vitro while the 2,5-dihydroxy derivative also inhibited uridine and leucine incorporation. There was no significant antitumor activity observed in the B-16 system, a fact which might be attributed to the increased toxicity of the compounds.
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PMID:Antitumor effects of L-glutamic acid dihydroxyanilides against experimental melanoma. 676 71

Levodopa and dopamine are naturally occurring catecholamines with antitumor activity in several experimental tumor systems. Previous studies suggested that their cytotoxic effect was related in part to their inhibitory effect upon DNA polymerase. We have examined the effects of levodopa, dopamine, levodopa methyl ester, norepinephrine, and the analog 3,4-dihydroxybenzylamine upon human and murine melanoma cells. When exponentially growing cells were exposed to these drugs, a characteristic inhibition of thymidine incorporation was observed with much less inhibition of either uridine or leucine incorporation. In order to ascertain that inhibition was occurring at the level of DNA synthesis, we examined the effects of the drugs upon the incorporation of thymidine triphosphate by permeabilized melanoma cells. When melanoma cells were permeabilized by lysolecithin, thereby permitting the direct incorporation of labeled thymidine triphosphate, a similar inhibition of incorporation was observed. Dopamine at a concentration of 4.8 microM caused a 50% reduction in incorporation of label. These results suggested that inhibition did occur at the level of DNA synthesis. In the presence of the melanocyte-specific oxidase, tyrosinase, these derivatives are potent inhibitors of isolated DNA polymerase alpha with 50% inhibitory concentrations between 1 and 10 microM. The inhibition could be completely prevented by the presence of reducing agents such as dithiothreitol (1.0 mM). The quinols themselves were not inhibitors of DNA polymerase. Dopamine analogs represent an interesting class of antitumor agents with inhibitory activity for DNA polymerase.
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PMID:Levodopa and dopamine analogs as DNA polymerase inhibitors and antitumor agents in human melanoma. 676 47

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

3'-Azido-2',3'-dideoxy-5-iodouridine (AzIdUrd) and 3'-azido-2',3'-dideoxy-5-bromouridine (AzBdUrd), previously shown to be potent and selective inhibitors of human immunodeficiency virus replication in vitro were minimally toxic to the uninfected human lymphoid cell line H9 (IC50 = 197 and 590 microM, respectively). Both compounds strongly inhibited the incorporation of [3H]thymidine but not [3H]deoxyadenosine into DNA, and we observed no significant inhibition of [3H]uridine incorporation into RNA or [3H]amino acid incorporation into protein. Exposure of H9 cells to AzIdUrd or AzBdUrd (100 microM, 24 hr) and pulse-labeling with [3H]thymidine resulted in approximately 80% reduction in levels of tritiated dTMP, dTDP, and dTTP relative to control. [125I]AzIdUrd was phosphorylated rapidly in H9 cells with the monophosphate accounting for over 90% of total soluble radioactivity. A relatively low but stable level of AzIdUTP was maintained over a 12-hr period. [125I]AzIdUrd was phosphorylated by a cell free extract of H9 cells at a rate approximately three times that of thymidine and its phosphorylation was inhibited by excess thymidine. AzIdUrd was found to be a competitive inhibitor of cytosolic thymidine kinase with a Ki of 2.63 microM and AzIdUMP a weak competitive inhibitor of thymidylate kinase with a Ki of 55.3 microM. Both AzIdUTP and AzBdUTP were potent competitive inhibitors of HIV-1 reverse transcriptase (Ki = 0.028 and 0.043 microM, respectively) and relatively poor inhibitors of H9 cell DNA polymerase alpha (Ki = 42.0 and 42.7 microM, respectively). Thus, the high therapeutic index of these compounds is due to the sensitivity of the viral reverse transcriptase, coupled with the relative insensitivity of the host cell DNA polymerase alpha.
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PMID:Metabolism and mode of selective inhibition of human immunodeficiency virus replication by 3'-azido-2',3'-dideoxy-5-iodouridine and 3'-azido-2',3'-dideoxy-5-bromouridine. 767 40

Northern blot analysis of the Epstein-Barr virus DNA polymerase mRNA identified two discrete sizes of virally encoded polymerase transcripts, 5.08 kb detected in strains P3HR1, Raji, W-91, and FF-41 and 3.7 kb detected solely in the prototype B95-8 strain. 3' S1-nuclease mapping and analysis of cDNA sequence generated by RNA-based PCR demonstrated that the 3.7-kb polymerase mRNA from B95-8 terminates 484 base pairs downstream of the open reading frame in a region of the genome remarkable for its lack of an apparent polyadenylylation signal. Moreover, between the cleavage point and the poly(A) tract of the cDNAs are a series of inserted nucleotides, mostly adenosine and uridine residues of unknown origin. A similar analysis of the 3' terminus of the 5.0-kb mRNA from the other cell lines revealed that polyadenylylation occurs 1.4 kb downstream of the B95-8 terminus. This region is deleted in B95-8, which accounts for the alternate upstream terminus used in B95-8. Like the 3.7-kb terminus, the 5.0-kb terminus lacks a canonical polyadenylylation signal, but contains a rarely used UAUAAA sequence 32 bp upstream of the poly(A) tail. These results indicate that the mRNA encoded by the Epstein-Barr virus DNA polymerase gene is polyadenylylated at two different termini without the use of canonical signals, raising the possibility of involvement of a virus-encoded factor in 3' processing of this message.
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PMID:Unconventional processing of the 3' termini of the Epstein-Barr virus DNA polymerase mRNA. 809 41

The acute effect of RNA and DNA synthesis inhibitors on DNA topoisomerase (topo) I localization within cells was examined. Indirect immunofluorescence revealed that topo I was distributed throughout the nuclei but was concentrated in nucleoli of untreated K562 leukemia cells and A549 non-small cell lung cancer cells. Treatment with the DNA polymerase inhibitor aphidicolin did not alter this distribution. In contrast, 30-60 min after addition of the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) at concentrations that inhibited [3H]uridine incorporation into RNA by > or = 50%, topo I was visible throughout the nuclei without nucleolar accentuation. Western blotting and activity assays confirmed that the amount of topo I polypeptide and topo I activity were unaltered by the brief DRB treatment. Within 30 min of DRB removal, topo I relocalized to the nucleoli in the absence or presence of the protein synthesis inhibitor cycloheximide. Collectively, these results suggest a reversible translocation of topo I out of the nucleoli when RNA synthesis is inhibited. Treatment with the topo I poisons topotecan or camptothecin, agents that also inhibit RNA synthesis, likewise caused redistribution of topo I to nonnucleolar regions of the nucleus in a variety of cell types. In DC3F hamster lung fibroblasts, 2.5 microM topotecan or 1.25 microM camptothecin was sufficient to cause this topo I redistribution. In DC3F/C-10 cells that contain a mutant camptothecin-resistant topo I, topo I relocalization required 50-fold higher concentrations of topotecan or camptothecin but not DRB. These observations not only suggest that accumulation of topo I in the nucleolus is related to ongoing RNA synthesis but also raise the possibility of screening for some types of camptothecin resistance at the single-cell level using a rapid immunofluorescence-based assay.
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PMID:RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I. 860 19

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.
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PMID:Modeling of a possible conformational change associated with the catalytic mechanism in the hammerhead ribozyme. 882 31

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after cold injury-induced brain trauma (CIBT) in mice, in which apoptosis is assumed to participate. Immunohistochemistry showed the nuclear expression of XRCC1 in the entire region of the control brains. Fifteen minutes after CIBT, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, followed by a significant reduction of XRCC1 in the entire lesion 4 h after CIBT. A characteristic 70-kDa band was detected in the non-traumatic area, and was markedly decreased after CIBT as shown by Western blot analysis. DNA fragmentation was also observed after CIBT, and double staining with XRCC1 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between XRCC1 loss and DNA fragmentation 24 h after CIBT. These data indicate that early decrease of XRCC1 and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after CIBT.
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PMID:Reduction of the DNA base excision repair protein, XRCC1, may contribute to DNA fragmentation after cold injury-induced brain trauma in mice. 1086 64

Analogues of dUTP bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group linked via spacers of varying length (n = 2, 4, 7-13 atoms) to the 5-position of the uridine ring (NAB-n-dUTP) were synthesized and characterized. DNA polymerase beta efficiently incorporated these analogues into synthetic primer-template substrates in place of TTP, which allowed us to selectively introduce a photoreactive group at the 3' primer terminus. After completing photoreactive primer synthesis, the reaction mixtures were irradiated with monochromatic UV light (315 nm) in the presence of human replication protein A (RPA), a heterotrimer consisting of three subunits with molecular mass 70 kDa (p70), 32 kDa (p32), and 14 kDa (p14), and were separated by SDS-PAGE. The photoreactive primers cross-linked directly with p70 and p32, but cross-linking of p14 was not achieved even by varying the length of the spacer group. The data speak in favor of the protection of p14 by other RPA subunits from the interaction with 3'-end of the primer. Cross-linking of substrates to pol beta is inhibited when the analogue bears a short spacer (n = 2, 4, 7, and 8), but this is abrogated somewhat when longer spacers (n = 9-13) are examined. On the basis of these observations, we suggest that RPA and pol beta form a complex on primer-template substrates.
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PMID:Synthesis of base-substituted dUTP analogues carrying a photoreactive group and their application to study human replication protein A. 1089 64


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