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)

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.
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PMID:Purification and properties of DNA polymerase-beta from guinea pig liver. 70 39

We have investigated the biochemical basis of the mevalonate dependence of DNA replication. Stimulating quiescent rat hepatoma cells to proliferate in the presence of compactin, an inhibitor of mevalonate synthesis, prevented DNA replication in as many as 80% of these cells. The percentage of cells that failed to replicate DNA increased with the increased duration of quiescence. Aphidicolin-sensitive DNA polymerase and ornithine decarboxylase activities were selectively decreased in compactin-treated cells, whereas RNA and protein synthesis, the level of dihydrofolate reductase and aphidicolin-resistant DNA polymerase activity were unaffected. Adding putrescine, the product of ornithine decarboxylase and the precursor of other polyamines, did not restore DNA replication. Our results demonstrate that the decreased activities of at least two DNA-replication enzymes are among the proximal causes of the failure of mevalonate-deprived cells to synthesize DNA. More importantly, our data indicate that a mevalonate-dependent factor(s) is progressively depleted during quiescence, and that inability to resynthesize this factor(s) may be the ultimate cause of the failure of resting cells to replicate DNA when stimulated to proliferate in the absence of mevalonate.
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PMID:The effect of mevalonic acid deprivation on enzymes of DNA replication in cells emerging from quiescence. 128 82

The proliferation of neoplastic and nonneoplastic hepatocytes is caused by various humoral growth factors with autocrine and paracrine mechanisms, and the proliferative activity of both hepatocytes and nonhepatocytic cells contributes to neoplastic growth. The authors attempted to detect various kinds of proliferating cells immunohistochemically in small hepatocellular carcinoma (HCC) using a monoclonal antibody against DNA polymerase alpha. Most of the HCC cells that stained for this enzyme were small, had basophilic cytoplasm with poorly developed organelles, and aggregated to form clusters distributed randomly within cancer nests. Nonhepatocytic cells also were stained, including some endothelial cells, Kupffer's cells, macrophages, and lymphocytes. Fat-storing cells were not stained. The number of stained sinusoidal (capillary) cells decreased in this order: Kupffer's cells and macrophages, endothelial cells, and fat-storing cells. Nonhepatocytic cells, including lymphocytes, proliferated more actively in areas with actively growing HCC cells than in those with quiescent cancer cells. The relationship between stained HCC cells and stained sinusoidal cells was clearly defined; the correlation coefficient was 0.97. These findings suggest the possibility of a relationship between the proliferative activity of neoplastic hepatocytes and that of sinusoidal cells, including lymphocytes.
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PMID:An analysis of proliferating cells in biopsy specimens from patients with small hepatocellular carcinoma. 131 88

We have previously described a mutant hepatitis B virus (HBV) with a fused X-C open reading frame (ORF) resulting from a single nucleotide insertion in the X-C overlapping region. A stably transformed cell line producing HBV particles, HepG2-K8, was established by transfecting the human hepatoma cell line HepG2 with a plasmid carrying four tandem repeats of the mutant HBV genome. The virus particles secreted into the culture medium were characterized by density gradient centrifugation and electron microscopy. The particles, similar to Dane particles by morphology and density, contained the mature HBV genome and endogenous DNA polymerase activity. Six HBV-specific transcripts of 4.0, 3.5, 2.2, 2.1, 1.2 and 0.9 kb were detected in HepG2-K8 cells by Northern blot analysis. cDNA cloning and sequence analysis of X mRNA showed that an elongated X ORF encoding 193 amino acids was created by a frameshift mutation in the 3'-terminal region of the wild-type X ORF and that the formation of an in-frame termination codon (TAA) resulted from polyadenylation. This elongated X gene product exerted transcriptional trans-activation.
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PMID:Replication of a mutant hepatitis B virus with a fused X-C reading frame in hepatoma cells. 132 98

Hepatitis B virus (HBV) contains a particle-associated DNA polymerase/reverse transcriptase activity encoded by the P (pol) open reading frame. Due to its low abundance, the corresponding protein has so far escaped direct detection and structural analysis. As a first step to overcome these difficulties, a series of recombinant vaccinia viruses was constructed and used for the synthesis in human hepatoma cells of both the authentic full length protein and of its functional domains. Pulse chase experiments demonstrated that the P-proteins had very short half lives in striking contrast to the viral core protein expressed in parallel with the same system. No evidence was obtained for a specific proteolytic processing of the P-protein as occurring with retroviral pol gene products. Overexpression of P-protein by recombinant vaccinia viruses was then employed to develop a highly sensitive detection method based on the in vitro phosphorylation of newly introduced target sites for protein kinase A. The usefulness of this method was demonstrated in the analysis of encapsidated P-gene products that were transiently expressed from an appropriately modified HBV genome. The results obtained indicate that the P-protein acts unprocessed, at least during the initial steps of nucleocapsid assembly and reverse transcription, and that a fraction of the P-protein molecules is linked as such to the viral DNA. Direct detection of the hepadnaviral P-protein by in vitro phosphorylation should greatly facilitate future analyses on P-protein structure and function.
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PMID:Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites. 137 44

DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies. These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases. Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta. Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP. The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies. Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M. Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320. At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.
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PMID:Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain. 138 Aug 29

In retroviruses, the pol gene is expressed in the form of a gag-pol fusion protein by the mechanism of ribosomal frameshifting. In studies of the possible mechanism of hepadnaviral pol protein synthesis, recent results have ruled out core-pol fusion protein synthesis by ribosomal frameshifting. In this study, an in vitro transcription and translation coupling system was used to demonstrate that the HBV core and pol proteins could be synthesized independently using the pregenome RNA template. The result has led us to design experiments to distinguish between the involvement of a termination-reinitiation, internal initiation, or leaky scanning mechanism in the pol protein synthesis. In vitro experiments were then carried out to measure the amount of pol proteins being synthesized from (i) the preC mRNA, which contained an extra AUG and seven more nucleotides at the 5'-end in comparison with the pregenome RNA; (ii) the pregenome RNA in the presence of various amounts of antisense RNA annealing to the 5'-end of the pregenome RNA; and (iii) the pregenome RNA with an additional hairpin structure located upstream of the C gene. Results indicated that the synthesis of both core and pol proteins was concomitantly reduced in these three conditions, which suggested that leaky scanning is the most probable mechanism for pol protein synthesis in vitro. To further verify the mechanism in vivo, experiments were performed to assay the activity of DNA polymerase in virions, which were obtained from hepatoma cells transfected by plasmids containing either a wild-type sequence (5'-GGCATGG-3') or an optimal initiation context (5'-ACCATGG-3') of the C gene. Transfection results showed that the plasmid-containing mutations of the C gene significantly decreased the DNA polymerase activity in virions. This observation supports our hypothesis that the leaky scanning model is involved in the synthesis of pol protein.
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PMID:Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA. 156 78

In vivo examination of the occupancy of DNA elements that can regulate transcription is critical to reveal which proteins actually take part in establishing and maintaining gene expression. We describe a new genomic sequencing method involving the rapid purification of relevant DNA segments from the bulk of the genomic DNA using a biotinylated riboprobe. The purified sequences are revealed by a single primer extension using Taq DNA polymerase. We used this technique to study the promoter and the enhancer of mouse transthyretin (TTR), a gene highly expressed in the liver. Footprints showed high liver-specific occupancy of some, but not all, of the DNA sites that had been identified as important for expression by transfection studies in hepatoma cells. In addition, several previously undetected sites were observed that bound proteins specifically in liver. These results suggest that not all demonstrable binding sites are involved in ongoing transcription and that in vivo studies may reveal additional and probably more relevant sites.
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PMID:Rapid in vivo footprinting technique identifies proteins bound to the TTR gene in the mouse liver. 198 8

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.
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PMID:Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis. 200 53

Hepatitis B virus (HBV) variants with a nonsense mutation in the distal pre-C region have been detected in patients positive for anti-HBe, and the complete nucleotide sequence of one cloned pre-C variant has been determined. Transfection of this HBV variant clone into the human hepatoma cell line HepG2 resulted in the appearance of major HBV transcripts, replicative forms of viral DNA evidenced by both molecular hybridization and endogenous DNA polymerase assay, as well as the expression and secretion of HBsAg and HBcAg particles. Western blotting revealed only the 21-kDa HBcAg but not the 17-kDa HBeAg. These results demonstrate the replication capacity of the HBV variant with a nonfunctional pre-C region despite its inability to express HBeAg.
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PMID:In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region. 201 46


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