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)

We have overexpressed the vaccinia virus DNA polymerase using the hybrid vaccinia virus/T7 expression system. Accumulation of the DNA polymerase to levels as high as 10% of the total protein was observed following coinfection of BSC40 cells with the appropriate vaccinia recombinants. Although the DNA polymerase produced at 37 degrees C was largely insoluble, 25% of the recombinant protein could be recovered as soluble protein when infected cultures were maintained at 32 degrees C. Starting with cytoplasmic lysates of coinfected cells, a rapid and reproducible purification protocol that yielded apparently homogeneous preparations of the DNA polymerase after four chromatographic steps was established. Typically, 0.3 mg of purified DNA polymerase was obtained from 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vaccinia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7819, 1979), the purified recombinant enzyme displayed both polymerase and 3'-5' exonuclease activities but lacked detectable 5'-3' exonuclease activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent Km values of 0.9, 2.9, 4.0, and 2.7 microM for dGTP, dATP, TTP, and dCTP, respectively.
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PMID:Overexpression and purification of the vaccinia virus DNA polymerase. 795 Mar 89

Wheat germ DNA polymerase A, a gamma-like enzyme, recognized efficiently natural and synthetic RNA templates, resembling a retroviral reverse transcriptase (P. Laquel et al., Biochim Biophys Acta 1048 (1990): 139-148). Ammonium-21-tungsto-9-antimoniate (HPA-23), an antiviral drug, inhibited the DNA polymerase A activities, independently of the template primers used, i.e. activated DNA or polyriboadenylic acid oligodeoxythymidylate (poly(rA)-oligo(dT)). The inhibition observed in the poly(rA)-oligo(dT)-directed DNA polymerase A activity occurred in the presence of either Mg2+ or Mn2+ as divalent cation, and also with the 2'-fluoro analogue of poly(rA) as template. HPA-23 was a non-competitive inhibitor with respect to TTP, activated DNA, poly(rA)-oligo(dT), and poly(dAfl)-oligo(dT). A preincubation study showed a reversible HPA-23 binding to DNA polymerase A, in the presence of poly(rA)-oligo(dT) as the template primer.
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PMID:Inhibition of the wheat germ DNA polymerase A activity by the antiviral drug HPA-23. 826 Jun 25

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

DNA polymerase beta activity, its content and gene transcription levels in SMMC-LTNM hepatoma were investigated, using 3H-TTP incorporation, immunocytochemistry and cytoplasmic dot hybridization, respectively. The relations between the biological properties of the enzyme and DNA repair synthesis induced by gamma-ray irradiation were also studied. It was found that DNA polymerase beta activity, its content and the amount of its mRNA were much higher in hepatoma than those in normal hepatocytes (P < 0.01). Following whole-body irradiation of the nude mouse bearing SMMC-LTNM with 2 Gy of gamma ray, the polymerase beta activity in hepatoma increased temporarily and the gene transcription of the enzyme seemed to be more active. DNA polymerase beta participated in DNA repair synthesis and this effect was different between hepatoma and hepatocyte because of the biological differences of DNA polymerase beta. The results presented here indicated that DNA polymerase beta could affect radiation damage and radiotherapy of cancer.
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PMID:[The radiobiologic characteristics of DNA polymerase beta in hepatomas]. 873 5

The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.
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PMID:DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site. 879 44

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying DNA that is based upon the combined action of a DNA polymerase and restriction enzyme. Previously, a form of SDA was developed which utilizes the exonuclease deficient Klenow fragment of E. coli polymerase I (exo Klenow) and the restriction enzyme HincII to achieve 10(8)-fold amplification in 2 h at 37 degrees C (Walker, G.T., 1993, PCR Methods and Applications 3; 1-6). A new thermophilic form of SDA is reported here which uses a restriction endonuclease from Bacillus stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient polymerase from Bacillus caldotenax (exo Bca). SDA was used to amplify DNA from Mycobacterium tuberculosis. An amplification factor of 10(10)-fold was achieved after 15 min of SDA at 60 degrees C. The new thermophilic system is much more specific than the previous mesophilic system as evidenced by a dramatic decrease in background amplification products. Thermophilic SDA was also optimized with dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA glycosylase.
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PMID:Detection of M. tuberculosis DNA using thermophilic strand displacement amplification. 886 73

The multiple mutations associated with high-level AZT resistance (D67N, K70R, T215F, K219Q) arise in two separate subdomains of the viral reverse transcriptase (RT), suggesting that these mutations may contribute differently to overall resistance. We compared wild-type RT with the D67N/K70R/T215F/K219Q, D67N/K70R, and T215F/K219Q mutant enzymes. The D67N/K70R/T215F/K219Q mutant showed increased DNA polymerase processivity; this resulted from decreased template/primer dissociation from RT, and was due to the T215F/K219Q mutations. The D67N/K70R/T215F/K219Q mutant was less sensitive to AZTTP (IC50 approximately 300 nM) than wt RT (IC50 approximately 100 nM) in the presence of 0.5 mM pyrophosphate. This change in pyrophosphate-mediated sensitivity of the mutant enzyme was selective for AZTTP, since similar Km values for TTP and inhibition by ddCTP and ddGTP were noted with wt and mutant RT in the absence or in the presence of pyrophosphate. The D67N/K70R/T215F/K219Q mutant showed an increased rate of pyrophosphorolysis (the reverse reaction of DNA synthesis) of chain-terminated DNA; this enhanced pyrophosphorolysis was due to the D67N/K70R mutations. However, the processivity of pyrophosphorolysis was similar for the wild-type and mutant enzymes. We propose that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyrophosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination. The increased processivity of viral DNA synthesis may be important to enable facile HIV replication in the presence of AZT, by compensating for the increased reverse reaction rate.
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PMID:Phenotypic mechanism of HIV-1 resistance to 3'-azido-3'-deoxythymidine (AZT): increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase. 984 96

To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A.
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PMID:Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases. 1021 27

The 5'-triphosphate of 4-thiothymidine (4S-TTP) is an excellent substrate for the Klenow fragment of Escherichia coli DNA polymerase 1 and HIV-1 reverse transcriptase with values of k(cat)/Km within a factor of approximately 3 of those for TTP. A large UV change (deltaepsilon= -9770 M(-1)cm(-1) at 340 nm) associated with incorporation of 4S-TMP into nucleic acid duplexes makes possible a rapid, continuous spectrophotometric assay of the reaction progress.
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PMID:Incorporation of 4-thiothymidine into DNA by the Klenow fragment and HIV-1 reverse transcriptase. 1085 57

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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