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)

Primer recognition proteins (PRP) enable DNA polymerase alpha to utilize efficiently DNA substrates with low primer to template ratios. We have previously identified the protein-tyrosine kinase substrate annexin II, and the glycolytic enzyme 3-phosphoglycerate kinase as components of PRP. As a step towards elucidation of the role of PRP in the process of DNA replication, we have investigated the subcellular distribution and specific association of these proteins with the nuclear matrix in HeLa cells. Nuclear extracts prepared from HeLa cells in S phase contain the enzymatic activity of 3-phosphoglycerate kinase (PGK) and phospholipase A2 inhibitory activity of annexin II. Monomer annexin II is approximately equally distributed between the nuclear and cytoplasmic fractions, while a majority of PGK is in the cytoplasm. Immunoblot analyses reveal the presence of these two proteins in nuclei, specifically associated with the nuclear matrix. This is further confirmed by observation of the presence of annexin II and PGK in isolated nuclear matrices by immunoelectron microscopy. The phospholipase A2 inhibitory activity of annexin II colocalizes with the nuclear matrix-bound annexin II. A related protein, annexin I, is not detectable in the nuclear extracts and nuclear matrix. A slower-migrating (perhaps modified) form of annexin II is found to be associated with the nuclear matrix. Attempts to dissociate PGK and annexin II from the nuclear matrix with octyl-beta-glucoside, high salt or metal ion chelators were unsuccessful, suggesting that the interaction is very strong.
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PMID:The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells. 153 25

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.
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PMID:Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates. 284 66

The dnaZX gene of Escherichia coli directs the synthesis of two proteins, DnaZ and DnaX. These products are confirmed as the gamma and tau subunits of DNA polymerase III because antibody to a synthetic peptide present in both the DnaZ and DnaX proteins reacts also with the gamma and tau subunits of holoenzyme. To characterize biochemically the tau subunit, for which there has been no activity assay, the dnaZX gene was fused to the beta-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (tau) were replaced by beta-galactosidase lacking only 7 N-terminal amino acids. The 185-kDa fusion protein, which retained beta-galactosidase activity, was overproduced to the level of about 5% of the soluble cellular protein by placing the gene fusion under control of the tac promoter and Shine-Dalgarno sequence. The fusion protein was isolated in one step by affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. The purified fusion protein also had ATPase (and dATPase) activity that was dependent on single-stranded DNA. This activity copurified with the beta-galactosidase activity not only through the affinity column but also through a subsequent gel filtration. We conclude that the DnaX protein function involves binding to single-stranded DNA and hydrolysis of ATP or dATP, in addition to binding to other DNA polymerase III holoenzyme components, increasing the processivity of the core enzyme, and serving as a substrate for the production of the gamma subunit.
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PMID:Escherichia coli DnaX product, the tau subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity. 303 60

Peanut agglutinin (PNA), a D-galactose specific lectin, agglutinated 10-18% of lymphocytes isolated from tonsils of 3- to 6-yr-old children. PNA+ cells were found to be mainly B lymphocytes showing a 9.6 times higher specific activity of DNA polymerase alpha compared to the PNA- cells. The specific activity of deoxycytidine kinase as well as the incorporation of [5-3H]deoxycytidine were also much higher in PNA+ cell fraction than in PNA- fraction (7.7-fold and 6-fold, respectively). On the other hand, thymidine kinase activity and [5-3H]deoxythymidine incorporation were only 3.6 and 3.9 times higher, respectively. The data presented here show a high degree of DNA synthesis and preferential utilisation of [5-3H]deoxycytidine for DNA synthesis in this undifferentiated B lymphocyte fraction.
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PMID:Preferential utilisation of deoxycytidine by undifferentiated (peanut positive) tonsillar lymphocytes. 349 70

It is well-known that there are multiple forms of DNA polymerase alpha. In order to determine which form(s) is (are) tightly bound, the activities were dissociated from DNA-poor nuclear matrices, with octyl beta-D-glucoside. Sucrose gradient sedimentation analysis revealed three bands with s values of 7.5, 10.5, and 13. The 7.5S form was free of DNA primase and represented only 10% of the total DNA polymerase alpha bound to the nuclear matrix. The 13S and the 10.5S forms each contained DNA primase activity. The 10.5S form comprised 85% of the DNA polymerase alpha activity and 95% of the DNA primase activity, dissociated from the nuclear matrix. Neither temperature of nuclease digestion nor various salt treatments of nuclei had significant effects on the proportions of DNA polymerase alpha and DNA primase activities bound to, or subsequently dissociated from, nuclear matrices. In a comparison of primase activity bound to the nuclear matrix, dissociated from the nuclear matrix, and in the soluble fraction, it was found that the bound activity had a lower ATP dependence, had less KCl inhibition, and was less sensitive to heat, compared to the dissociated and soluble activities. No differences in Mg2+ or pH dependence were noted. The amounts of DNA polymerase alpha and DNA primase activities bound to the nuclear matrix varied over the cell cycle of synchronized cells. Over the S phase, there were two peaks of matrix-bound DNA primase and two peaks of subsequently dissociated DNA polymerase alpha-DNA primase complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of the DNA polymerase alpha-DNA primase complex to the nuclear matrix in HeLa cells. 367 71

We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule.
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PMID:Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. 434 68

Multiple forms of DNA polymerase alpha activity (alpha 1, alpha 2, and alpha 3) from human neuroblastoma IMR-32 cells untreated or treated with tunicamycin (3 microgram/ml) were separated by DEAE-cellulose column chromatography. Loss of 40--60% of DNA polymerase alpha 2 activity was observed in tunicamycin-treated cells. Ricin 1B, a subunit of intact ricin (Mr, 64,000), was found to be a specific inhibitor of DNA polymerase alpha 2 isolated from control IMR-32 cells. However, DNA polymerase alpha 2 isolated from tunicamycin-treated cells was insensitive to ricin 1B. Heat treatment studies at 50 degrees C showed two completely different inactivation profiles for the DNA polymerase alpha 2 enzymes isolated from the tunicamycin-treated and untreated cells. A probable involvement of a beta-linked galactose-containing carbohydrate chain in the catalytic subunit of DNA polymerase alpha 2 is suggested.
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PMID:Probable involvement of a glycoconjugate in IMR-32 DNA synthesis: decrease of DNA polymerase alpha 2 activity after tunicamycin treatment. 695 Nov 91

Swertifrancheside [1], a new flavonone-xanthone glucoside isolated from Swertia franchetiana, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2], a triterpene isolated from the roots of Maprounea africana, and protolichesterinic acid [3], an aliphatic alpha-methylene-gamma-lactone isolated from the lichen Cetraria islandica, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT), with 50% inhibitory doses (IC50 values) of 43, 3.7, and 24 microM, respectively. They were not cytotoxic with cultured mammalian cells. The kinetic mechanisms by which compounds 1-3 inhibited HIV-1 RT were studied as was their potential to inhibit other nucleic acid polymerases. Swertifrancheside [1] bound to DNA and was shown to be a competitive inhibitor with respect to template-primer, but a mixed-type competitive inhibitor with respect to TTP. On the other hand, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] were mixed-type competitive inhibitors with respect to template-primer and noncompetitive inhibitors with respect to TTP. Therefore, the mechanism of action of 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] as HIV-1 RT inhibitors involves nonspecific binding to the enzyme at nonsubstrate binding sites, whereas swertifrancheside [1] inhibits enzyme activity by binding to the template-primer.
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PMID:Mechanistic evaluation of new plant-derived compounds that inhibit HIV-1 reverse transcriptase. 756 95

A DNA polymerase beta (pol. beta) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris. These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-beta-D-glucoside, prunasin. The compound inhibited the activity of rat pol. beta at 150 microM, but did not influence the activities of calf DNA polymerase alpha and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined. The compound dose-dependently inhibited pol. beta activity, the IC(50) value being 98 microM with poly dA/oligo dT(12-18) and dTTP as the DNA template and substrate, respectively. Inhibition of pol. beta by the compound was competitive with the substrate, dTTP. The inhibition was enhanced in the presence of fatty acid, and the IC(50) value decreased to approximately 40 microM. In the presence of C(10)-decanoic acid, the K(i) value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site.
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PMID:The cyanogenic glucoside, prunasin (D-mandelonitrile-beta-D-glucoside), is a novel inhibitor of DNA polymerase beta. 1042 40

The highly purified DNA Pol-alpha from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488-1492]. Binding of ConA and RCA to human recombinant DNA polymerase-alpha showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231-6237]. The catalytic polypeptide, DNA polymerase-alpha of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-alpha from embryonic chicken brain (ECB) contains an alpha-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-alpha reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567-573] by the treatment with methyl-alpha-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an alpha-galactose binding, 56kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-alpha as determined by immunostaining with the polymerase-alpha-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-alpha and a complete separation of polymerase complex and primase.
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PMID:DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex. 1076 11


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