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 replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.
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PMID:Purification of DNA polymerase delta as an essential simian virus 40 DNA replication factor. 167 Oct 44

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.
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PMID:Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. 167 Oct 46

Saccharomyces cerevisiae replication factor C (RF-C) was purified 25,000-fold from a protease-deficient strain of yeast. RF-C is a complex of 6 subunits of 130, 86, 41, 40, 37, and 27 kDa. None of the subunits are related through proteolysis or differential phosphorylation. The assay for RF-C used as a substrate single-stranded DNA binding protein-coated singly primed single-stranded mp 18 DNA. This DNA was poorly replicated by yeast DNA polymerase delta with or without its cofactor proliferating cell nuclear antigen (PCNA). In the presence of RF-C, however, replication of the template proceeded efficiently when both ATP and PCNA were present as well. Formation of this replication-proficient complex of DNA polymerase delta required an input of one to two molecules of PCNA per replicated DNA molecule. DNA polymerase epsilon also formed an ATP-dependent complex with PCNA and RF-C. RF-C has a DNA-dependent ATPase activity, equally active on single-stranded and primed single-stranded mp18 DNA. Addition of PCNA stimulated the ATPase of RF-C on primed but not on unprimed DNA, indicating that the increase in ATPase was due to PCNA-enhanced binding of RF-C to the primer terminus. Calf thymus PCNA also stimulated the ATPase activity of yeast RF-C and participated in holoenzyme formation with DNA polymerase delta. These results attest to the structural and functional homology between yeast and mammalian cells for these components of the replication machinery.
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PMID:Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity. 168 21

Lag times in DNA synthesis by DNA polymerase delta holoenzyme were due to ATP-mediated formation of an initiation complex on the primed DNA by the polymerase with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). Lag time analysis showed that high affinity binding of RF-C to the primer terminus required PCNA and that this complex was recognized by the polymerase. The formation of stable complexes was investigated through their isolation by Bio-Gel A-5m filtration. A stable complex of RF-C and PCNA on primed single-stranded mp18 DNA was isolated when these factors were preincubated with the DNA and with ATP, or, less efficiently with ATP gamma S. These and additional experiments suggest that ATP binding promotes the formation of a labile complex of RF-C with PCNA at the primer terminus, whereas its hydrolysis is required to form a stable complex. Subsequently, DNA polymerase delta binds to either complex in a replication competent fashion without further energy requirement. DNA polymerase epsilon did not associate stably with RF-C and PCNA onto the DNA, but its transient participation with these cofactors into a holoenzyme-like initiation complex was inferred from its kinetic properties and replication product analysis. The kinetics of the elongation phase at 30 degrees, 110 nucleotides/s by DNA polymerase delta holoenzyme and 50 nucleotides/s by DNA polymerase epsilon holoenzyme, are in agreement with in vivo rates of replication fork movement in yeast. A model for the eukaryotic replication fork involving both DNA polymerase delta and epsilon is proposed.
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PMID:Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. 168 22

Human fibroblasts repair DNA damaged by bleomycin through both short-patch and long-patch pathways, mediated by an aphidicolin-resistant (beta) and aphidicolin-sensitive (delta) DNA polymerase respectively (DiGiuseppe, J.A. and Dresler, S.L. (1989) Biochemistry, 28, 9515-9520). Despite certain similarities, aphidicolin-sensitive repair synthesis induced by bleomycin can be distinguished genetically and biochemically from that elicited by UV radiation. Permeable xeroderma pigmentosum fibroblasts of complementation groups A and G, completely deficient in UV-induced repair, display aphidicolin-sensitive repair synthesis dependent upon dose of bleomycin. Furthermore, the ribonucleotide dependence of long-patch repair induced by bleomycin differs from that of UV repair with respect to substrate specificity and apparent Km for ATP. This novel ATPase activity mediates a step prior to polymerization. By contrast, short-patch repair synthesis does not require ATP. These data suggest that, in addition to short-patch repair, human cells possess two distinct long-patch excision repair pathways. We propose that these pathways represent strand-break, base and nucleotide excision repair respectively.
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PMID:Aphidicolin-sensitive DNA repair synthesis in human fibroblasts damaged with bleomycin is distinct from UV-induced repair. 169 20

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.
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PMID:Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity. 169 61

Several N-(S)-(3-hydroxy-2-phosphonylmethoxypropyl) (HPMP) and N-(2-phosphonylmethoxyethyl) (PME) derivatives of purine bases (adenine, guanine, 2-aminoadenine, 3-deazaadenine) and cytosine inhibit the growth of various DNA viruses. PME-derivatives (PMEA, PMEG and PMEDAP) are also active against retroviruses. Both types of nucleotide analogues undergo phosphorylation by cellular nucleotide kinases to their mono- and diphosphates. The phosphorylation with crude extracts of L-1210 cells is potentiated by an ATP-regenerating system. HPMPA is phosphorylated faster than PMEA with or without the ATP-regenerating system. The HPMP and PME analogues inhibit several virus-encoded target enzymes and their cellular counterparts: (1) HSV-1 DNA polymerase is inhibited by the diphosphates of the PME series; the virus-encoded enzyme is more sensitive than HeLa DNA pol alpha and beta. PMEApp terminates the growing DNA chain; it specifically replaces dATP. HPMPApp also acts as an alternative substrate of dATP, but, in contrast with PMEApp, it permits limited chain growth. (2) Diphosphates of both series inhibit HSV-1 ribonucleotide reductase; the greatest inhibition of CDP reduction to dCDP is exhibited by HPMPApp and PMEApp. The enzyme isolated from a PMEA-resistant HSV-1 mutant proved less sensitive to PMEApp, hydroxyurea and HPMPApp. (3) Diphosphates of PME derivatives efficiently inhibit AMV(MAV) reverse transcriptase. (4) The purine HPMP and PME analogues and, even more so, their monophosphate derivatives inhibit purine nucleoside phosphorylase from L-1210 cells.
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PMID:Acyclic nucleotide analogues: synthesis, antiviral activity and inhibitory effects on some cellular and virus-encoded enzymes in vitro. 169 93

9-beta-D-Arabinofuranosyladenosine triphosphate (araATP) is a potent inhibitor of DNA primase. Primase readily incorporates araATP into primers, and primers containing araAMP are then elongated by DNA polymerase alpha (pol alpha) upon addition of dNTPs. AraATP did not inhibit utilization of primers under conditions where the ability of pol alpha to elongate primers was independent of the dATP concentration. The fraction of primers elongated by pol alpha was reduced by araATP only when elongation was dependent upon the dATP concentration. When the Ki for primase was measured in terms of the inhibition of the synthesis of primers that can be utilized by pol alpha, we obtained Ki = 2.7 microM (37 degrees C) and 2.0 microM (25 degrees C). Inhibition was competitive with ATP. Inhibition of pol alpha activity by araATP was measured under conditions where primase-catalyzed primer synthesis was required for the pol alpha activity. The decreased pol alpha activity was due to primase inhibition, and at constant dATP, araATP inhibition was competitive with ATP and gave Ki = 1.2 microM, similar to the Ki for primase alone. Increasing the dATP concentration had no effect on inhibition. In combination with previously reported in vivo data, we conclude that DNA primase is the primary in vivo target of the arabinofuranosyl nucleotides, not pol alpha.
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PMID:Inhibition of DNA primase by 9-beta-D-arabinofuranosyladenosine triphosphate. 170 21

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of retrovirus (i.e., human immunodeficiency virus) replication in vitro and in vivo. Uptake of PMEA by human MT-4 cells and subsequent conversion to the mono- and diphosphorylated metabolites (PMEAp and PMEApp) are dose-dependent and occur proportionally with the initial extracellular PMEA concentrations. Adenylate kinase is unable to phosphorylate PMEA. However, 5-phosphoribosyl-1-pyrophosphate synthetase directly converts PMEA to PMEApp with a Km of 1.47 mM and a Vmax that is 150-fold lower than the Vmax for AMP. ATPase, 5'-phosphodiesterase, and nucleoside diphosphate kinase are able to dephosphorylate PMEApp to PMEAp, albeit to a much lower extent than the dephosphorylation of ATP. PMEApp has a relatively long intracellular half-life (16-18 hr) and has a much higher affinity for the human immunodeficiency virus-specified reverse transcriptase than for the cellular DNA polymerase alpha (Ki/Km: 0.01 and 0.60, respectively). PMEApp is at least as potent an inhibitor of human immunodeficiency virus reverse transcriptase as 2',3'-dideoxyadenosine 5'-triphosphate. Being an alternative substrate to dATP, PMEApp acts as a potent DNA chain terminator, and this may explain its anti-retrovirus activity.
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PMID:Intracellular metabolism and mechanism of anti-retrovirus action of 9-(2-phosphonylmethoxyethyl)adenine, a potent anti-human immunodeficiency virus compound. 170 39

The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments.
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PMID:Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate. 170 19


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