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)

Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs "D-NSLYP" and "K--NS(L/V)YG," regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.
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PMID:Phi 29 DNA polymerase active site. Mutants in conserved residues Tyr254 and Tyr390 are affected in dNTP binding. 152 62

Oligodeoxyribonucleotides containing N6-methoxyadenine (M) have been synthesized. The order of stability of duplexes consisting of synthesized oligodeoxyribonucleotides, 5'd(CCTGGTAXCAGGTCC)3'-5'd(GGACCTGNTACCAGG)3' (X = M, A, G. N = A, G, T, C), was M: A (Tm = 52 degrees C) greater than M: T (50 degrees C) greater than M: G (48 degrees C) greater than M: C (46 degrees C) observed by thermal denaturation in a buffer of 0.01 M Na cacodylate, and 0.1 M NaCl at pH 7.0. The Tms are within a range of 6 degrees of difference, which is smaller than those of Tms of the duplexes containing A:N pairs (11 degrees) and G:N pairs (11 degrees). DNA replication study on a template-primer system, 5'd(32p-CAGCTTTCGC)3' 3'd(GTCGAAAGCGMAGTCG)5', showed that TTP and dCTP were incorporated into DNA strands at a site opposite to M by Klenow DNA polymerase, but dATP and dGTP were not.
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PMID:The synthesis and properties of oligodeoxyribonucleotides containing N6-methoxyadenine. 154 73

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.
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PMID:Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine. 163 14

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

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.
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PMID:Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases. 170 19

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

The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.
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PMID:Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities. 170 71

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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