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)

Three forms of DNA polymerase alpha [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] were partially purified from the combined nuclear extract and postmicrosomal supernatant solution of synchronized HeLa cells. These enzymes, designated DNA polymerases alpha 1, alpha 2, and alpha 3, on the basis of their order of elution from DEAE-Bio-Gel, differ in their abilities to utilize single-strand DNA templates. DNA polymerase alpha 2 has equal catalytic activities with activated and single-strand DNAs as template-primers. DNA polymerase alpha 1 has only partial catalytic activity with single-strand DNA templates, and DNA polymerase alpha 3 is essentially inactive with this template. Successive steps of hydrophobic affinity chromatography and phosphocellulose chromatography of DNA polymerase alpha 2 resolved the polymerase alpha activity and two protein factors (C1 and C2) that are required for its catalytic activity with a DNA template-primer that contains extended single-strand regions. In the absence of the factors, DNA polymerase alpha activity is measurable with activated but not single-strand DNA templates. In the presence of the C1 and C2 factors DNA polymerase alpha activity with single-strand DNA templates is restored to about 75% of the catalytic activity of DNA polymerase alpha 2 with this template.
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PMID:Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates. 694 21

Homogeneously purified DNA polymerase alpha subunit-free primase was used to analyze primer RNA synthesis. On a chemically synthesized 36 mer DNA template, a part of upstream region of human c-myc gene, the primer synthesis started from a doublet of deoxythymidine (TT) in the deoxypyrimidine-rich sequence. The primase in DNA polymerase alpha-primase complex synthesized 21-mer reaction product, while DNA polymerase alpha-free primase gave the similar products, 21- and 22-mer, indicating that the site recognition was carried out by primase itself and DNA polymerase alpha subunit has an auxiliary role on it. Product analysis using DNA fragments carrying base substitutions further revealed that the existence of deoxypyrimidine residues around the starting sites was important for priming frequencies. Competition analysis showed that the priming was strongly competed by poly(dC), and to a much lesser extent by poly(dA). Gel-shift analysis showed that the primase could bind to the DNA template, and this complex formation was also competed by poly(dC), but not by poly(dA). These results indicate that primase subunit interacts with the starting site by binding directly with deoxypyrimidine residues.
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PMID:Deoxypyrimidine cluster mediates the priming by calf thymus DNA primase subunit. 768 46

Calcium and its receptor protein calmodulin function in the regulation of proliferation of mammalian cells. A 68 kDa calmodulin-specific binding protein was shown previously to be associated with growth factor-dependent progression of a variety of mammalian cells from G1 to S phase and to stimulate DNA synthesis in permeabilized hematopoietic progenitor cells. In this report we show that the 68 kDa calmodulin-specific binding protein in HeLa cells is tightly associated with the DNA polymerase alpha-primase component of the 21S complex of enzymes for DNA synthesis. The 68 kDa calmodulin-binding protein and the DNA polymerase alpha-primase complex cofractionate during Q-Sepharose chromatography to isolate the 21S enzyme complex, native and denatured DNA-cellulose to dissociate the 21S complex, and DEAE-Bio-Gel chromatography to isolate the multiprotein DNA polymerase alpha-primase complex. The 68 kDa calmodulin-specific binding protein and DNA polymerase alpha also bind and coelute during affinity chromatography on calmodulin-agarose. They also coprecipitate with C10-agarose-linked monoclonal antibody SJK 132-20 to human DNA polymerase alpha. The tight association of the 68 kDa calmodulin-binding protein to the DNA polymerase alpha-primase complex supports a function for this protein in the regulation of DNA synthesis in vivo.
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PMID:The 68 kDa calmodulin-binding protein is tightly associated with the multiprotein DNA polymerase alpha-primase complex in HeLa cells. 769 50

Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha 180-kDa catalytic subunit gene contain a common 8 base pair (bp) promoter element, 5'-TATCGATA (DRE, Drosophila DNA replication-related element). We have generated various base substitutions and internal deletions in and around DRE (nucleotide positions -93 to -100 with respect to the transcription initiation site) of the PCNA gene in vitro and subsequently examined their effects on the binding to DREF (DRE-binding factor) and PCNA gene promote activity in cultured Drosophila Kc cells as well as in living flies. Gel mobility shift assays using nuclear extracts of Kc cells with and without competitor DNA fragments carrying the mutations indicated that the 10-bp sequence from positions -91 to -100 is essential for complex formation with DREF. Transient expression assays of chloramphenicol acetyl-transferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying the mutations revealed that the 8-bp sequence from -93 to -100 is essential for activation of the promoter in Kc cells. Examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying the mutations, introduced into flies by germ-line transformation, revealed that the 8-bp sequence is also important for DRE function during development. However, we obtained two exceptional mutations in the 8-bp sequence that did not or only marginally affected the PCNA gene promoter activity in transgenic flies. Both of these mutations effectively reduced the promoter activity in CAT transient expression assay in Kc cells and the binding to DREF in vitro. Therefore, the 8-bp sequence requirement for DRE function appears to be less stringent in living flies than in the cultured cell or in vitro cases.
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PMID:A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNa replication-related genes. 779 83

Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filter binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase will dissociate from the template after each cycle of catalysis.
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PMID:Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template. 803 92

We identified cis elements in the 5'-flanking region of rat Na,K-ATPase alpha 2 subunit gene (Atp1a2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5'-deletion mutation analysis, the region between nucleotide positions -175 and -108 was identified as a positive regulatory region. In the region, the distal E box (nucleotides -144 to -139) acts as a negative regulatory element, and the Sp1 consensus sequence (nucleotides -123 to -118) and the GGGAGG sequence (nucleotides -114 to -109) act as positive regulatory elements. Gel-retardation analysis revealed that binding factors are an E-box-binding protein and Sp1. DNase I footprinting and methylation-interference analyses revealed that Sp1 binds to the region from nucleotides -122 to -101 and the E-box-binding protein to the region from nucleotides -144 to -136. T4 DNA polymerase footprinting revealed that there are three Sp1-binding sites in the region and that Sp1 binds to one of the three sites in a mutually exclusive manner. The mechanism by which Sp1 activates the Atp1a2 promoter is discussed.
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PMID:Anomalous interaction of Sp1 and specific binding of an E-box-binding protein with the regulatory elements of the Na,K-ATPase alpha 2 subunit gene promoter. 824 64

It has been confirmed that water-soluble eumelanins often extracted together with DNAs from natural black hairs act as an inhibitor of Taq DNA polymerase in the polymerase chain reaction (PCR). In the present investigation, an attempt to amplify the non-coding 333-bp region of mitochondrial DNA (mt333DNA) produced the following results: 1) Water-soluble preparations made from chemically synthesized melanin (Sigma products), as well as natural black eumelanins, inhibited the PCR amplification of mt333DNA at concentrations of more than 2 micrograms/ml. 2) Quantitative measurement of Taq DNA polymerase-catalyzed DNA synthesis in terms of the amount of [alpha-32P] dCMP incorporated into activated calf thymus DNA showed that both of the water-soluble melanins had the same inhibition activity as represented by the sigmoidal curve derived from a quadratic equation of melanin concentration. This observation suggested that Taq DNA polymerase combined with two molecules of melanin to form an inactivated complex. 3) Melanins did not appear to affect either the thermostability of Taq DNA polymerase at 94 degrees C, or the step of primer-annealing to template DNAs. On the other hand, we established a simple and useful method for removal of water-soluble eumelanins contaminating DNA preparations from hairs. The method was based on the adsorption of melanins to Bio-Gel. When a Bio-Gel P-60 minicolumn was equilibrated with 10 mM sodium acetate buffer, pH 4.2, water-soluble melanins were completely adsorpted to it whereas DNAs passed through, although the melanins showed incomplete adsorption to the gel when it was equilibrated with TE (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Water-soluble eumelanin as a PCR-inhibitor and a simple method for its removal]. 837 74

Gel filtration chromatography was used to isolate both preprimosomal and primosomal complexes formed on single-stranded DNA-binding protein-coated phiX174 DNA by the combination of PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG. The presence and relative amounts of primosomal proteins in these complexes were determined by Western blotting. Protein-DNA complexes isolated (i) after assembly in the presence of 10 microM ATP, (ii) after preprimosome movement in the presence of 1 mM ATP, (iii) after priming in the presence of the four ribonucleoside triphosphates, or (iv) after complementary strand DNA replication in the presence of the DNA polymerase III holoenzyme all had the same protein composition; preprimosomes contained PriA, PriB, PriC, DnaT, and DnaB, whereas primosomes included DnaG. The stable association of DnaG with the protein-DNA complex could be attributed partially to its ability to remain bound to the primers synthesized. In the absence of PriC, the efficiencies of priming and replication were reduced by one-third and one-half, respectively, even though PriC was not required for the formation of stable protein-DNA complexes on a 304-nucleotide-long single strand of DNA containing a primosome assembly site (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15642-15648). We hypothesize that maintenance of the primosome on the replicated DNA may provide a mechanism to allow primosomes to participate in the resolution of recombination intermediates and intermediates formed during double strand break repair by permitting the re-establishment of a replication fork.
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PMID:The ordered assembly of the phiX174-type primosome. II. Preservation of primosome composition from assembly through replication. 866 5

Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16-72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.
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PMID:Early detection of DNA strand breaks in the brain after transient focal ischemia: implications for the role of DNA damage in apoptosis and neuronal cell death. 920 15

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5, 6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage lambda DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.
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PMID:Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system. 1006 45


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