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)

African swine fever virus (ASFV) growth and plaque formation were inhibited by phosphonoacetic acid (PAA) concentrations of 200 micrograms/ml or more. One spontaneous mutant and two mutants isolated from mutagenized virus were resistant to PAA inhibition and showed practically normal viral DNA synthesis in the presence of PAA. DNA polymerase activity present in the cytoplasmic fraction from cells infected with the mutants required 10-fold higher concentrations of PAA for inhibition compared to equivalent inhibition of the wild-type enzyme. Like wild-type virus, the PAA-resistant mutants were resistant to inhibition by aphidicolin. Marker rescue analysis with mutant DNA fragments covering different regions of the ASFV DNA polymerase gene mapped the mutations within a fragment which was cloned and sequenced. A single nucleotide and amino acid change was assigned to each mutant. Two of the PAA-resistant mutations lie within the highly conserved region II common to alpha-like DNA polymerases, which has been implicated in pyrophosphate binding and probably also in dNTP binding. The other mutation was localized to within a region of moderate homology among viral DNA polymerases close to one of the motifs allegedly considered as constituting the 3'-5' exonuclease active site.
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PMID:Functional and molecular characterization of African swine fever virus mutants resistant to phosphonoacetic acid. 852 40

DNA polymerases alpha, delta and epsilon from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physiochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (Km values) and specific inhibitors (Ki values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases alpha and epsilon from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells, when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lower Ki values, indicating lower affinity to deoxyribonucleoside 5'-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K50 values). Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases delta and epsilon was measured using poly(dA.dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.
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PMID:Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells by inhibitors of cell proliferation. 857 84

By site-directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3'-5' exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3'-5' exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA-dependent DNA polymerases. Moreover, in contrast to the dual role in 3'-5' exonucleolysis and strand displacement previously observed for phi29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer-terminus at the 3'-5' exonuclease active site.
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PMID:Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. 860 89

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.
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PMID:Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity. 864 67

The functional significance of the conserved motif 'YxGG/A', located between the 3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization. Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the 'YxGG/A' motif. All different phenotypes could be directly related to defects in DNA binding at a particular active site. Thus, a high pol/exo ratio was related to a poor stability at the 3'-5' exonuclease active site. On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal or an increased accessibility to the exonuclease active site. These results allow us to propose that this motif, located in the connecting region between the N-terminal and C-terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross-talk between synthesis and degradation.
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PMID:A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases. 867 Aug 45

We investigated nuclease activities associated with the catalytic subunit of herpes simplex virus type 1 DNA polymerase. We confirm that a 3'-5' exonuclease copurifies with this enzyme. Previous reports suggested that a 5' DNase was intrinsic to the polymerase. Our preparation lacks such activity.
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PMID:Evidence that the nuclease activities associated with the herpes simplex type 1 DNA polymerase are due to the 3'-5' exonuclease. 867 14

We report the crystal structure of an NH2-terminal 388-residue fragment of T4 DNA polymerase (protein N388) refined at 2.2 A resolution. This fragment contains both the 3'-5' exonuclease active site and part of the autologous mRNA binding site (J. D. Karam, personal communication). The structure of a complex between the apoprotein N388 and a substrate, p(dT)3, has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7%. Two divalent metal ion cofactors, Zn(II) and Mn(II), have been located in crystals of protein N388 which had been soaked in solutions containing Zn(II), Mn(II), or both. The structure of the 3'-5' exonuclease domain of protein N388 closely resembles the corresponding region in the Klenow fragment despite minimal sequence identity. The side chains of four carboxylate residues that serve as ligands for the two metal ions required for catalysis are located in geometrically equivalent positions in both proteins with a rms deviation of 0.87 A. There are two main differences between the 3'-5' exonuclease active site regions of the two proteins: (I) the OH of Tyr-497 in the Klenow fragment interacts with the scissile phosphate in the active site whereas the OH of the equivalent tyrosine (Tyr-320) in protein N388 points away from the active center; (II) different residues form of the binding pocket for the 3'-terminal bases of the substrate. In the protein N388 complex the 3'-terminal base of p(dT)3 is rotated approximately 60 degrees relative to the position that the corresponding base occupies in the p(dT)3 complex with the Klenow fragment. Finally, a separate domain (residues 1-96) of protein N388 may be involved in mRNA binding that results in translational regulation of T4 DNA polymerase (Pavlov & Karam, 1994).
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PMID:Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions. 867 62

Family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol) is a monomer of about 100 kDa with two associated catalytic functions: 3'-5' exonuclease and DNA polymerase activities. The structure of this enzyme in the free and DNA-bound states was probed by limited proteolysis and fluorescence spectroscopy measurements. The results of partial trypsin proteolysis experiments on the recombinant Sso DNA pol pinpointed three major sites of protease sensitivity: near the N-terminus, within the center, and near the C-terminal end of the polypeptide chain. When partial trypsin digestion was carried out in the presence of either activated calf thymus DNA or primed M13mp18 single-stranded DNA, changes in cleavage pattern and in susceptibility to protease were detected. This phenomenon was dependent on the nucleic acid concentration and suggested the occurrence of DNA-induced conformational changes. These were also probed by steady-state fluorescence spectroscopy measurements using acrylamide as a quencher. Fine mapping of the DNA-specific cleavage sites allowed us to precisely locate the protein subdomains which were affected by these structural changes. Importantly, a specific proteolytic fragment of about 8 kDa was recovered after partial digestion of Sso DNA pol only in the presence of nucleic acid ligands. It was found to start at residues 392-394 and to span the protease-hypersensitive central region of the polypeptide chain. Its involvement in critical polymerase functions, such as substrate binding and/or enzyme processivity, was discussed. In addition, we found that controlled trypsin digestion of Sso DNA pol did not inactivate either polymerase or 3'-5' exonuclease activity concomitantly with the disappearance of full-sized enzyme. Activity gel analysis revealed that proteolytic products corresponding to the amino- and carboxyl-terminal halves of the enzyme retained 3'-5' exonuclease and DNA polymerase activity, respectively. These results are in line with the model of modular organization proposed for Sso DNA pol in a previous report.
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PMID:Domain organization and DNA-induced conformational changes of an archaeal family B DNA polymerase. 870 21

The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologous to Escherichia coli DNA polymerase I (Pol I) and likewise has domains responsible for DNA polymerase and 5' nuclease activities. The structures to the polymerase domains of Taq polymerase and of the Klenow fragment (KF) of Pol I are almost identical, whereas the structure of a vestigial editing 3'-5' exonuclease domain of Taq polymerase that lies between the other two domains is dramatically altered, resulting in the absence of this activity in the thermostable enzyme. The structures have been solved for editing complexes between KF and single-stranded DNA and for duplex DNA with a 3' overhanging single strand, but not for a complex containing duplex DNA at the polymerase active-site. Here we present the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft; the DNA neither bends nor goes through the large polymerase cleft, and the structural form of the bound DNA is between the B and A forms. A wide minor groove allows access to protein side chains that hydrogen-bond to the N3 of purines and the O2 of pyrimidines at the blunt-end terminus. Part of the DNA bound to the polymerase site shares a common binding site with DNA bound to the exonuclease site, but they are translated relative to each other by several angstroms along their helix axes.
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PMID:Structure of Taq polymerase with DNA at the polymerase active site. 871 47

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


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