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)

Several thiosemicarbazone-metal complexes inhibit the RNA dependent DNA polymerase and the transforming ability of Rous sarcoma virus. Some complexes are equally as active as the free ligand whereas the activity of others is greatly enhanced. The 2-formyl pyridine thiosemicarbazone copper (II) complex is the most potent compound of this class that we tested. Some copper complexes of salicylaldehyde derivatives are very active also, particularly N-n-butyl, N-n-hexyl and N-benzylsalicylaldimine; no nickel complex of any salicylaldehyde compound is active. In addition, other metal ligands, such as dithizone, diacetyl bis (mercaptoethylimine), N-butyl thiocarbamate, 0,0' dimethyl dithiophosphate, potassium dithiooxalate, and cis-PtII(NH3)2Cl2 were tested with varying results.
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PMID:Inhibition of the RNA dependent DNA polymerase and the malignant transforming ability of Rous sarcoma virus by thiosemicarbazone-transition metal complexes. 7 67

The effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.
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PMID:On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase. 86 97

Foamy viruses form a separate group of retroviruses encoding a pol protein with at least four domains based on comparative sequence alignments. The polymerase and ribonuclease H domains of the human foamy virus (HFV) pol gene were expressed in Escherichia coli either individually or in combination. The histidine-tagged HFV fusion proteins were subsequently purified to near homogeneity by affinity Ni2+ chelate column chromatography. The polymerase and RNase H activities were characterized by performing conventional DNA polymerase and ribonuclease H assays and in situ gel assays. Six purified recombinant HFV proteins were enzymatically active either individually as DNA polymerase and ribonuclease H or as combined domains. The HFV enzymatic activities were characterized with respect to cation preferences and pH optima. Western blots with antibodies against the RNase H domain, in situ reverse transcriptase (RT), and RNase H gel assays showed that in HFV-infected cells pol proteins of 120 and 80 kDa were detectable. A novel activity band of 60 kDa was found in situ RT gel assays. Recombinant RNase H protein additionally purified by fast performance liquid chromatography was capable of removing the primer for minus-strand DNA synthesis when labeled tRNA(Lys1,2) model substrates were used. Specific cleavages occurred at the phosphodiester bonds one to three nucleotides 5' of the RNA-DNA junction. The results revealed biochemical properties of the HFV pol gene products that define functional domains of the HFV pol gene that are distinct but comparable to other retroviruses.
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PMID:Molecular biological characterization of the human foamy virus reverse transcriptase and ribonuclease H domains. 748 84

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.
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PMID:Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus. 754 60

The divalent nickel ion (Ni2+) is one of several metal ions that can substitute for Mg2+ in the activation of DNA polymerases in vitro, but usually with very low efficiency. We have purified and partially characterized a Ni(2+)-binding protein (p40) from HeLa cell extracts that can specifically enhance the polymerase activity of DNA polymerase alpha (pol alpha) and other DNA polymerases in response to Ni2+. This protein, with a molecular mass of 40 kDa, is a single stranded DNA binding protein that binds to a M13 DNA template-primer with an optimum stoichiometry of approximately 90 equiv of protein per equiv of DNA template and enhances the affinity of pol alpha for the primer-template. In the presence of Ni2+, p40 exhibits an increased affinity for DNA. The p40 increased by 3- to 6-fold the rates at which pol alpha and the Klenow fragment of Escherichia coli DNA polymerase I (KF) replicate different DNA templates in response to Ni2+. The low processivity of Ni(2+)-activated pol on primed M13 ssDNA was also enhanced by the presence of p40. The rates of Ni(2+)-dependent replication by inherently more processive enzymes, DNA polymerase delta and T4 DNA polymerase, were not significantly increased by p40 when M13 ssDNA was used as a template; however, p40 did increase the activity of T4 polymerase on an activated calf thymus DNA template. The protein did not stimulate Mg(2+)-activated DNA replication.
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PMID:A single stranded DNA binding protein isolated from HeLa cells facilitates Ni2+ activation of DNA polymerases in vitro. 799 74

The effects of the carcinogenic metal nickel on DNA polymerase alpha (pol alpha) activity and fidelity have been analyzed. In the absence of Mg2+, the presence of Ni2+ ions at concentrations below 0.25 mM gave rise to a dose-dependent activation of pol alpha as monitored by [3H]dTMP incorporation into an activated DNA template. The apparent Km for Ni(2+)-dependent pol alpha incorporation of dTTP was estimated to be 25 microM, which was about 10 times higher than the Km for Mg2+ (2.3 microM). Above 0.25 mM, Ni2+ caused a dose-dependent inhibition of pol alpha activity and the Ki was calculated to be 1.5 mM. Scatchard analyses showed that Ni2+ binds to affinity-purified pol alpha and associated proteins at two tight binding sites with a Kd of approximately 50 microM and at eight weak binding sites with a Kd of approximately 4 mM. In the presence of 2 mM Mg2+, the addition of Ni2+ to the reactions caused an inhibition of polymerase activity. The inhibition patterns tended to switch from competitive to mixed-type to noncompetitive as a function of Ni2+ concentration. Lastly, Ni2+ increased the incorporation of the modified nucleotide dideoxy-CMP in reactions using varying ratios of dideoxy-CTP/dCTP.
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PMID:The effect of divalent nickel (Ni2+) on in vitro DNA replication by DNA polymerase alpha. 816 78

An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2). Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E. coli in a time- and concentration-dependent manner. The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types. In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent [3H]dTMP incorporation into activated calf thymus DNA. However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment. In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract.
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PMID:The stimulatory effect of nickel chloride on DNA replication in human HeLa cells and Escherichia coli. 820 62

Nickel is a genotoxic carcinogen. However, the mechanisms of nickel-induced genotoxicity are not well understood. We have investigated the effects of Ni2+ ions on DNA polymerase activity and the fidelity of DNA replication in vitro. The effect of Ni2+ on different DNA polymerases is quite variable. The amount of enzyme inhibition and degree of alteration in replication fidelity induced by Ni2+ are dependent both on the polymerase and its associated 3'-5' exonuclease activity. Some polymerases, such as E. coli DNA polymerase I, AMV reverse transcriptase and human DNA polymerase alpha, can utilize Ni2+ as a weak substitute for Mg2+ during DNA replication. Other polymerases are very sensitive to inhibition by Ni2+ and the IC50 can vary by an order of magnitude. T4 polymerase is relatively insensitive to inhibition by Ni2+, although the sensitivity is enhanced in the absence of added Mg2+, and Ni preferentially inhibits the 3'-5' exonuclease function of T7 DNA polymerase. The fidelity and processivity of DNA polymerases may be either increased or decreased by Ni ions in a polymerase dependent manner. The inhibition DNA polymerase activity and altered replication fidelity may contribute significantly to Ni-induced mutagenesis and genotoxicity in vivo.
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PMID:Effects of nickel ions on polymerase activity and fidelity during DNA replication in vitro. 840 77

Heterocyclic thiosemicarbazones, thioureas and 2-substituted pyridine N-oxides as well as representative nickel, cobalt and copper complexes were shown to be potent antineoplastic/cytotoxic agents. The cytotoxicity was demonstrated against single cell leukemia as well as cell lines derived from solid tissue (colon adenocarcinoma, HeLa, KB, skin, bronchogenic lung, bone osteosarcoma and glioma). In L1210 cells, DNA synthesis and subsequently RNA synthesis were particularly inhibited by the agents. IMP dehydrogenase activity and thus purine de novo synthesis was reduced significantly by the agents. Dihydrofolate reductase, ribonucleoside reductase, nucleoside kinase and DNA polymerase alpha activities were inhibited by the agents. d(NTP) pool levels were reduced by most of the agents. DNA strand scission was present with all of the derivatives; however, there was no evidence of intercalation, cross linking or alkylation/binding to bases of DNA. This new group of compounds may offer novel exploratory derivatives for future investigations in the treatment of cancer.
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PMID:The cytotoxicity of heterocyclic thiosemicarbazones and their metal complexes on human and murine tissue culture cells. 849 Feb 2

The tau subunit dimerizes DNA polymerase III via interaction with the alpha subunit, allowing DNA polymerase III holoenzyme to synthesize both leading and lagging strands simultaneously at the DNA replication fork. Here, we report a general method to map the limits of domains required for heterologous protein-protein interactions using surface plasmon resonance. The method employs fusion of a short biotinylation sequence at either the NH2 or COOH terminus of the protein to be immobilized on streptavidin-derivatized biosensor chips. Inclusion of a hexahistidine sequence permits rapid purification and separation of the fusion protein from the endogenous Escherichia coli biotin carboxyl carrier protein. Ten deletions of the alpha subunit were constructed and purified by Ni2+-nitrilotriacetic acid chromatography and, when required, monomeric avidin chromatography. Each alpha deletion protein was captured by streptavidin immobilized on a Pharmacia Biosensor BIAcore chip, and the tau binding activity of each alpha deletion was analyzed using surface plasmon resonance. The tau subunit bound very tightly to a full-length amino-terminal fusion of the biotinylation sequence with alpha (KD approximately 70 pm). Four additional NH2-terminal alpha deletion proteins (60, 240, 360, and 542 residues deleted) retained strong binding activity to the tau subunit (KD = 0.19-0.39 nM), whereas deletion of 705 residues or more from the NH2 terminus of the alpha subunit abolished tau binding activity. Full-length alpha that contained a carboxyl-terminal fusion with the biotinylation sequence bound tau strongly (KD = 0.37 nM). However, deletion of 48 amino acids from the COOH terminus totally eliminated tau binding. These results indicate that the COOH-terminal half of the alpha subunit is involved in tau interaction.
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PMID:Biotin tagging deletion analysis of domain limits involved in protein-macromolecular interactions. Mapping the tau binding domain of the DNA polymerase III alpha subunit. 870 19


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