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)

DNA polymerase (deoxynucleosidetriphosphate: DNA nucleotidyltransferase, EC 2.7.7.7 or DNA nucleotidyltransferase) activity, isolated from late and early passage cells of the diploid human fibroblast line, MRC-5, was compared. The level of activity dropped with increasing passage. In addition, when the fidelity of polymerization was monitored with four synthetic templates under a variety of conditions, it was observed that the enzyme from late passage cells was more error-prone. The possible relation of these observations to "senescence" of the fibroblasts is discussed.
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PMID:Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts. 106 93

The metabolism and mode of action of penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were studied and compared with those of acyclovir. In uninfected MRC-5 cells, low concentrations of the triphosphates of penciclovir and acyclovir were occasionally just detectable, the limit of detection being about 1 pmol/10(6) cells. In contrast, in cells infected with either herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV), penciclovir was phosphorylated quickly to give high concentrations of the triphosphate ester. Following the removal of penciclovir from the culture medium, penciclovir-triphosphate remained trapped within the cells for a long time (half-lives, 20 and 7 h in HSV-2- and VZV-infected cells, respectively). In HSV-2-infected cells, acyclovir was phosphorylated to a lesser extent and the half-life of the triphosphate ester was only 1 h. We were unable to detect any phosphates of acyclovir in VZV-infected cells. (S)-Penciclovir-triphosphate inhibited HSV-1 and HSV-2 DNA polymerase competitively with dGTP, the Ki values being 8.5 and 5.8 microM, respectively, whereas for acyclovir-triphosphate, the Ki value was 0.07 microM for the two enzymes. Both compounds had relatively low levels of activity against the cellular DNA polymerase alpha, with Ki values of 175 and 3.8 microM, respectively. (S)-Penciclovir-triphosphate did inhibit DNA synthesis by HSV-2 DNA polymerase with a defined template-primer, although it was not an obligate chain terminator like acyclovir-triphosphate. These results provide a biochemical rationale for the highly selective and effective inhibition of HSV-2 and VZV DNA synthesis by penciclovir and for the greater activity of penciclovir than that of acyclovir when HSV-2-infected cells were treated for a short time.
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PMID:Mode of antiviral action of penciclovir in MRC-5 cells infected with herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. 133 46

The activities of the purine acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against two human and five animal strains of cytomegalovirus were compared with those of acyclovir. DHPG was significantly more active than acyclovir against all but one (mouse cytomegalovirus) of the strains tested, with 50% effective doses ranging from 5 to 13 microM, as determined by plaque reduction assays in human embryonic lung (MRC-5) and human embryonic tonsil cells. Both DHPG and acyclovir inhibited virus replication at concentrations considerably lower than those necessary to inhibit cell proliferation. In mode-of-action studies, the triphosphates of DHPG and acyclovir inhibited human cytomegalovirus DNA polymerase. DHPG phosphorylation to the active triphosphate was enhanced in infected cells; however, this enzymatic activity was unrelated to thymidine kinase. In animal studies, DHPG was slightly more effective than acyclovir in reducing mouse cytomegalovirus-induced mortality.
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PMID:Activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine compared with that of acyclovir against human, monkey, and rodent cytomegaloviruses. 301 Aug 40

The activity of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) against several herpesviruses was compared with that of acyclovir (ACV). In plaque reduction tests with clinical isolates of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus, mean 50% inhibitory concentrations (IC50S) (n = number tested) for BRL 39123 were 0.4 (n = 17), 1.5 (n = 13), and 3.1 (n = 5) micrograms/ml, respectively. Corresponding IC50S for ACV were 0.2, 0.6, and 3.8 micrograms/ml. Cytomegalovirus was relatively resistant to BRL 39123 (IC50, 51 micrograms/ml), but equid herpesvirus 1, bovid herpesvirus 2, and felid herpesvirus 1 were susceptible (IC50S, 1.6, 1.2, and 0.9 micrograms/ml, respectively). BRL 39123 was inactive against an HSV-1 strain which does not express thymidine kinase activity, but a DNA polymerase mutant selected for resistance to ACV was sensitive to BRL 39123 (IC50, 1.5 micrograms/ml). In contrast to the results from plaque reduction tests, BRL 39123 was more active than ACV against HSV-1 and of equal activity against HSV-2 in virus yield reduction assays in MRC-5 cells. After treatment of HSV-infected cultures for short periods, BRL 39123 was considerably more effective than ACV at reducing virus replication, and furthermore, after removal of extracellular BRL 39123, virus replication remained depressed for long periods, whereas such persistent activity was not observed with ACV. Neither compound significantly affected MRC-5 cell replication at 100 micrograms/ml, but at 300 micrograms/ml BRL 39123 was more inhibitory than ACV.
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PMID:Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxy-methylbut-1-yl)guanine (BRL 39123) in cell culture. 363 45

A comparison was made between two methods of isolating DNA polymerases from MRC-5 fibroblasts. The first method produces DNA polymerase-alpha with a lower molecular weight and other properties that are not normally found for this enzyme. It was concluded that this method produces proteolytically degraded DNA polymerase-alpha. A second method was developed which produces DNA polymerase-alpha with all the normal properties of this enzyme. The specific activity of DNA polymerase was reduced in senescent MRC-5 fibroblasts approximately 2--4-fold. DNA polymerase-alpha accounts for 95% of polymerase activity in young cells and its specific activity during the fibroblast lifespan correlates with the declining cellular growth rate. DNA polymerase-beta is present at 0.3-3% of total cellular activity and its specific activity does not correlate with cellular growth rate. DNA polymerase-gamma accounts for 5% of the polymerase activity in young cells and 20% in old cells. However, the specific activity of the polymerase-gamma is constant throughout the lifespan of MRC-5. The 5 S DNA polymerase-alpha has an increased in vitro error frequency (average 3.6) compared to the 7 S polymerase-alpha. In addition the proportion of 5 S polymerase-alpha rises from 7% in young cells to 29% in senescent cells in an apparently exponential fashion.
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PMID:Properties of DNA polymerases from young and ageing human fibroblasts. 730 Apr 56

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.
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PMID:Further characterization of the human cell multiprotein DNA replication complex. 853 May 40

In recent years, work from a large number of laboratories has greatly expanded our knowledge of the biochemical characteristics and the genetic structure of the DNA polymerases used during papovavirus DNA replication. The development of in vitro DNA replication systems for both SV40 and polyoma virus has been paramount in facilitating the development of the current models describing how DNA polymerase alpha and delta function to replicate the genomes of these two viruses. Our studies have demonstrated that the proteins recognized to be essential for both in vitro SV40 and polyoma viral origin-dependent DNA synthesis can be isolated from cells as an intact complex. We have shown that the human cell MRC closely resembles the murine cell MRC, in both its protein composition and its fractionation and chromatographic profile. In addition, our data regarding both the human and the murine MRC support the dipolymerase model proposed from in vitro DNA replication studies using reconstituted assay systems. In addition, analysis of the nucleotide sequence of the genes encoding DNA polymerase alpha and delta has revealed that the amino acids encoded by several regions of these two genes have been rigorously maintained across evolutionary lines. This information has permitted the identification of protein domains which mediate the complex series of protein-protein interactions that direct the DNA polymerases to the cell nucleus, specify complete or partial exonuclease active sites, and participate in the interaction of each DNA polymerase with the DNA template. Expression studies examining each of the genes encoding DNA polymerase alpha and delta clearly indicate that both DNA polymerases are cell cycle regulated and undergo a dramatic induction in their expression when quiescent cells are stimulated to enter the cell cycle. This is in contrast to the two- to three-fold upregulation in the level of expression of these two genes when cycling cells cross the G1/S boundary. In addition, both proteins are phosphorylated in a cell cycle-dependent manner, and phosphorylation appears to be mediated through the action of a cdc2-dependent protein kinase. Despite all of this new information, much remains to be learned about how papovavirus DNA replication is regulated and how these two DNA polymerases act in vivo to faithfully copy the viral genomes. Studies have yet to be performed which identify all of the cellular factors which potentially mediate papovavirus DNA replication. The reconstituted replication systems have yielded a minimum number of proteins which are required to replicate SV40 and polyoma viral genomes in vitro. However, further studies are needed to identify additional factors which may participate in each step of the initiation, elongation, and termination phases of viral genome replication. As an example, models describing the potential role of cellular helicases, which are components of the MRC isolated from murine and human cells, have yet to be described. It is also conceivable that there are a number of other proteins which serve to attach the MRC to the nuclear matrix, stimulate viral DNA replication, and potentially regulate various aspects of the activity of the MRC throughout viral DNA replication. We are currently working toward characterizing the biochemical composition of the MRC from both murine and human cells. Our goals are to identify all of the structural components of the MRC and to define the role of these components in regulating papovavirus and cellular DNA replication. We have also begun studies to visualize the spatial organization of these protein components within the MRC, examine the regulatory processes controlling the activity of the various components of the MRC, and then develop this information into a coherent picture of the higher order structure of the MRC within the cell nucleus. We believe that this information will enable us to develop an accurate view of the detailed processes mediating both pa
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PMID:Expression, purification, and characterization of DNA polymerases involved in papovavirus replication. 902 36

Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.
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PMID:Regulation of the expression or recruitment of components of the DNA synthesome by poly(ADP-ribose) polymerase. 964 17

The mode of action of (1'S,2'R)-9-([1', 2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl)guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2' site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.
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PMID:Mode of action of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl) cycloprop-1'-yl]methyl]guanine (A-5021) against herpes simplex virus type 1 and type 2 and varicella-zoster virus. 968 13

Cidofovir is the first nucleoside monophosphate analogue currently being used for the treatment of human cytomegalovirus (HCMV) retinitis in individuals with AIDS. Unfortunately, the period of therapy with the use of this compound may be limited due to the possible emergence of serious irreversible nephrotoxic effects. New drugs with improved toxicity profiles are needed. The goal of this study was to investigate the anticytomegaloviral properties and drug-induced toxicity of a novel phosphonate analogue, namely, (-)-2-(R)-dihydroxyphosphinoyl-5-(S)-(guanin-9'-yl-methyl) tetrahydrofuran (compound 1), in comparison with those of cidofovir. The inhibitory activities of both compounds on HCMV propagation in vitro were similar against the AD 169 and Towne strains, with 50% inhibitory concentrations ranging from 0.02 to 0.17 microgram/ml for cidofovir and < 0.05 to 0.09 microgram/ml for compound 1. A clinical HCMV isolate that was resistant to ganciclovir and that had a known mutation within the UL54 DNA polymerase gene and a cidofovir-resistant laboratory strain derived from strain AD 169 remained sensitive to compound 1, whereas their susceptibilities to ganciclovir and cidofovir were reduced by 33- and 10-fold, respectively. Both compound 1 and cidofovir exhibited equal potencies in an experimentally induced murine cytomegalovirus (MCMV) infection in mice, with a prevention or prolongation of mean day to death at dosages of 1.0, 3.2, and 10.0 mg/kg of body weight/day. In cytotoxicity experiments, compound 1 was found to be generally more toxic than cidofovir in cell lines Hs68, HFF, and 3T3-L1 (which are permissive for HCMV or MCMV replication) but less toxic than cidofovir in MRC-5 cells (which are permissive for HCMV replication). Drug-induced toxic side effects were noticed for both compounds in rats and guinea pigs in a 5-day repeated-dose study. In guinea pigs, a greater weight loss was noticed with cidofovir than with compound 1 at dosages of 3.0 and 10.0 mg/kg/day. An opposite effect was detected in rats, which were treated with the compounds at relatively high dosages (up to 100 mg/kg/day). Compound 1 and cidofovir were nephrotoxic in both rats and guinea pigs, with the epithelium lining the proximal convoluted tubules in the renal cortex being the primary target site. The incidence and the severity of the lesions were found to be dose dependent. The lesions observed were characterized by cytoplasm degeneration and nuclear modifications such as karyomegaly, the presence of pseudoinclusions, apoptosis, and degenerative changes. In the guinea pig model, a greater incidence and severity of lesions were observed for cidofovir than for compound 1 (P < 0.001) with a drug regimen of 10 mg/kg/day.
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PMID:Comparative study of the anti-human cytomegalovirus activities and toxicities of a tetrahydrofuran phosphonate analogue of guanosine and cidofovir. 1004 67


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