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)

Previously, we found that a ganciclovir (GCV)-resistant clinical human cytomegalovirus (HCMV) isolate had an amino acid substitution at codon 501 (Leu --> Phe) in the delta-region C of the DNA polymerase gene. DNA polymerases have now been (partially) purified from both the GCV-resistant and sensitive parental strains and the activity of DNA polymerase and 3'-5' exonuclease compared. With respect to DNA polymerase activity, the Michaelis constant (Km) and maximum velocity (Vmax) of the GCV-resistant strain for the DNA template were lower than those of the GCV-sensitive strain. With respect to 3'-5' exonuclease activity, the Km and Vmax of the GCV-resistant strain for the DNA substrate in the presence of ammonium sulfate were lower than those of the GCV-sensitive strain, while being similar in the absence of ammonium sulfate. Although the polymerase activity of the two strains showed almost the same sensitivity for the different polymerase inhibitors, the 3'-5' exonuclease activity of the GCV-resistant strain was more resistant to these inhibitors than that of the GCV-sensitive strain.
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PMID:Comparison of human cytomegalovirus DNA polymerase activity for ganciclovir-resistant and -sensitive clinical strains. 1080 20

The whole genome sequences of Helicobacter pylori strain 26695 have been reported. Whole cell proteins of H. pylori strain 26695 cells were obtained and analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips. The most abundant proteins were shown in the region of pI 4.0-9.5 with molecular masses from 10 to 100 kDa. Soluble proteins were precipitated by the use of 0-80% saturated solutions of ammonium sulfate. Soluble proteins precipitated by the 0-40% saturations of ammonium sulfate produced similar spot profiles and their abundant protein spots had acidic pI regions. However, a number of soluble proteins precipitated by more than 60% saturation of ammonium sulfate were placed in the alkaline pI regions, compared to those precipitated by 40% saturation. In addition, we have performed an extensive proteome analysis of the strain utilizing peptide MALDI-TOF-MS. Among the 345 protein spots processed, 175 proteins were identified. The identified spots represented 137 genes. One-hundred and fifteen proteins were newly identified in this study, including DNA polymerase III beta-subunit. These results might provide guidance for the enrichment of H. pylori proteins and contribute to construct a master protein map of H. pylori.
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PMID:Identifying the major proteome components of Helicobacter pylori strain 26695. 1198 66

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).
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PMID:PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi. 1244 50

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
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PMID:Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli. 1275 14

Over the past years several methods using mass spectrometry for high-throughput genotyping of single nucleotide polymorphisms (SNPs) have been developed. Most of these procedures require stringent purification. Only the GOOD assay does not need any sample purification. Here, several new implementations of this assay are presented. The molecular biological procedure of the GOOD assays is based on the principle that the analysis of DNA by matrix-assisted laser desorption/ionization (MALDI) is strongly dependent on the charge state. A 100-fold increase in sensitivity can be achieved if the analyzed DNA product is conditioned by a chemical procedure termed 'charge-tagging'. The GOOD assay starts with a PCR; allele-specific DNA molecules are generated by extension of modified primers. These contain up to three phosphorothioates and optionally a quaternary ammonium charged group with ddNTPs or alpha-S-ddNTPs. Then the unmodified part of the primers is digested by phosphodiesterase II and the negative charges of the phosphorothioates are neutralized by an alkylation reaction resulting in charge-tagged DNA products. Through the use of a novel DNA polymerase for the primer extension, which preferably incorporates ddNTPs over dNTPs, an enzymatic degradation of residual dNTPs from the PCR is not required. Additionally, the unique property of charge-tag technology is demonstrated to detect specifically on the same sample allele-specific DNA products carrying a positive charge-tag in the positive ion mode while products carrying a negative charge-tag are analyzed in the negative ion mode. We also generated zwitterionic allele-specific products that were detectable with high sensitivity in positive ion mode. The findings of this study raise interesting questions about the ionization process of nucleic acids in MALDI. The new variations of the GOOD assay were applied to genotype SNPs of a candidate gene for cardiovascular disease.
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PMID:Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry. 1281 49

Two crystal forms of DNA polymerase from Thermus aquaticus have been grown at room temperature. Rhombohedral crystals (form I) grown from ammonium sulfate solution diffracted poorly to 10 A only and thus are not suitable for X-ray structure determination. Trigonal crystals (form II) grown from polyethylene glycol solution are more suitable for structure determination since their diffraction pattern extends to 2.5 A at cryogenic temperature upon exposure to synchrotron X-rays. They belong to space group P3(1)21 (or its enantiomorph P3(2)21) and their unit-cell dimensions are a = 106.7 and c = 169.7 A, for flash-frozen crystals. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.0 A(3) Da(-l) and a solvent content of 58% by volume. X-ray data have been collected to 2.7 A Bragg spacing from native crystals.
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PMID:Crystallization and preliminary X-ray crystallographic analysis of DNA polymerase from Thermus aquaticus. 1529 82

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.
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PMID:Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III. 1545 36

Several clinical, genetic and neuroimaging studies implicate mitochondrial dysfunction in the pathophysiology of bipolar disorder and schizophrenia. It has been reported that a mitochondrial DNA (mtDNA) deletion of 4,977 bp, known as the 'common deletion', is associated with both mental illnesses. A lack of normal age-related accumulation of this deletion in schizophrenia and increased occurrence of the common deletion in bipolar disorder have been reported. However, even in the affected bipolar samples, the levels of common deletion were relatively small, indicating that the common deletion did not play a pathophysiological role in respiratory function. We hypothesized that accumulation of multiple mtDNA deletions, rather than the common deletion alone, is involved in the pathophysiology of these two major mental disorders. To test this hypothesis, we assessed mtDNA deletion(s) by comparing the copy number of two regions in mtDNA -- ND1 and ND4 -- using real-time quantitative PCR in the frontal cortex of 84 subjects (30 control, 27 with bipolar disorder, and 27 with schizophrenia). We also assessed the relative amount of mtDNA vs. nuclear DNA and the expression level of DNA polymerase gamma (POLG), which is involved in replicating mtDNA. We observed no association between mtDNA deletions and the two major mental disorders in the frontal cortex, which did not support our hypothesis. We did, however, make the following observations, although they were not significant after Bonferroni correction: (1) the ratio of mtDNA to nuclear DNA was significantly higher in female patients with schizophrenia than in control females ( p =0.040) and (2) in bipolar disorder, the relative amount of mtDNA decreased with age ( p =0.016). furthermore, POLG expression was significantly up-regulated in bipolar disorder ( p =0.036). Our results suggest that abnormalities in the system maintaining replication of mtdna may underlie bipolar disorder and schizophrenia.
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PMID:Quantitative analysis of mitochondrial DNA deletions in the brains of patients with bipolar disorder and schizophrenia. 1620 81

GTP cyclohydrolase (GCH) III from Methanocaldococcus jannaschii, which catalyzes the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), has been shown to require Mg2+ for catalytic activity and is activated by monovalent cations such as K+ and ammonium [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084]. The reaction is formally identical to that catalyzed by a GCH II ortholog (SCO 6655) from Streptomyces coelicolor; however, SCO 6655, like other GCH II proteins, is a zinc-containing protein. The structure of GCH III complexed with GTP solved at 2 A resolution clearly shows that GCH III adopts a distinct fold that is closely related to the palm domains of phosphodiesterases, such as DNA polymerase I. GCH III is a tetramer of identical subunits; each monomer is composed of an N- and a C-terminal domain that adopt nearly superimposible structures, suggesting that the protein has arisen by gene duplication. Three metal ions were located in the active site, two of which occupy positions that are analogous to those occupied by divalent metal ions in the structures of a number of palm domain containing proteins, such as DNA polymerase I. Two conserved Asp residues that coordinate the metal ions, which are also found in palm domain containing proteins, are observed in GCH III. Site-directed variants (Asp-->Asn) of these residues in GCH III are less active than wild-type. The third metal ion, most likely a potassium ion, is involved in substrate recognition through coordination of O6 of GTP. The arrangement of the metal ions in the active site suggests that GCH III utilizes two metal ion catalysis. The structure of GCH III extends the repertoire of possible reactions with a palm fold to include cyclohydrolase chemistry.
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PMID:A new use for a familiar fold: the X-ray crystal structure of GTP-bound GTP cyclohydrolase III from Methanocaldococcus jannaschii reveals a two metal ion catalytic mechanism. 1805 7

Quinone methides (QMs) are involved in the metabolism of many drugs and carcinogens as reactive intermediates to form covalent nucleobase adducts in DNA that associate with high mutagenicity. Recently, a plethora of synthetic QM DNA alkylating agents have been developed to form various nucleobase adducts as potential antitumor agents. However, the mutagenic potential of these synthetic QM alkylating agents has not been fully investigated. In this report, N-methylquinolinium QM was developed as a synthetic model to study biological consequences of the formation of nucleobase adducts in a DNA target. N-Methylquinolinium QM was generated in situ via an elimination process from a bis-quaternary ammonium precursor that was synthesized from a quinoline derivative. Alkylation with N-methylquinolinium QM on a DNA target produced mostly a stable N(2)-dG adduct as revealed by gel electrophoresis and DNA digestion assays and confirmed by mass and NMR analyses. The formation of N(2)-dG adducts of a DNA target was found to cause extensive stops in the primer extension with high fidelity DNA polymerase T7 and even low fidelity error prone Dpo4. The direct biological impact of a prealkylated green fluorescence protein plasmid with N-methylquinolinium QM was demonstrated as significant suppression of protein expression in A549 cells. Overall, our results suggested that nucleobase-QM adducts could potentially block nucleobase mismatch/translesion in the error-prone process to reduce the mutagenic potential if designed carefully.
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PMID:DNA alkylation with N-methylquinolinium quinone methide to N2-dG adducts resulting in extensive stops in primer extension with DNA polymerases and subsequent suppression of GFP expression in A549 cells. 2130 16


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