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)

We recently found that inhibition of MYB protein synthesis in human peripheral blood mononuclear cells (PBMC) exposed to human c-myb (designated MYB) antisense oligodeoxynucleotides prevents entry into S phase and cell proliferation. To determine the mechanism(s) by which down-regulation of human c-myb protein (MYB) synthesis interferes with DNA synthesis, we analyzed mRNA levels of DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), transcripts of two genes required for DNA synthesis, in normal and leukemic T lymphocytes exposed to MYB antisense oligodeoxynucleotides. Expression of DNA polymerase alpha was inhibited both in normal T lymphocytes progressing from G0 to S phase and in exponentially growing CCRF-CEM leukemic cells, whereas expression of PCNA was inhibited only in mitogen-stimulated PBMC and remained essentially unaffected in the leukemia T-cell line. The functional link between expression of MYB and DNA polymerase alpha mRNAs was further demonstrated by analyzing DNA polymerase alpha mRNA levels in a temperature-sensitive (ts) fibroblast cell line (TK-ts13; TK is thymidine kinase) constitutively expressing human MYB mRNA driven by the simian virus 40 (SV40) promoter. In the MYB-expressing TK-ts13 cells, DNA polymerase alpha mRNA levels were unaffected following shift to the nonpermissive temperature of 39.6 degrees C, whereas in the parental line, DNA polymerase alpha mRNA levels were readily down-regulated. These findings indicate that the expression of MYB is related to that of DNA polymerase alpha in cells expressing MYB at high levels and suggest that there is a functional link between c-myb and DNA polymerase alpha mRNA expression during cell cycle progression of normal T lymphocytes.
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PMID:Inhibition of T-cell proliferation by a MYB antisense oligomer is accompanied by selective down-regulation of DNA polymerase alpha expression. 169 13

The DNA polymerase-alpha of Plasmodium falciparum was characterized according to aphidicolin sensitivity and immunological reactivity with monoclonal anti-sera against human DNA polymerase-alpha. Two major (105 and 72 kDa) and two minor (180 and 130 kDa) catalytic subunits of P. falciparum DNA polymerase-alpha were detected on activity gels. Activity gels did not indicate the presence of a DNA polymerase-beta in P. falciparum. Metabolically labeled polypeptides at 180, 105, 72, and 52 kDa were immunoprecipitated from Plasmodium nuclear extracts with the anti-KB cell DNA polymerase-alpha monoclonal antibody and, by size, correspond to the major subunits of mammalian DNA polymerase-alpha. The monoclonal antibody also neutralized Plasmodium DNA polymerase activity. Plasmodium DNA polymerase was synthesized predominantly at an early schizont stage at which time the parasite began to synthesize its DNA and multiply. No evidence for phosphorylation of the major catalytic subunit was obtained. Plasmodium growth, DNA synthesis, and DNA polymerase activity were inhibited significantly in parallel by aphidicolin. These results suggest that P. falciparum has a typical eukaryotic DNA polymerase-alpha and that regulation of its activity appears to be at the transcriptional level.
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PMID:Cell cycle-dependent biosynthesis of Plasmodium falciparum DNA polymerase-alpha. 190 42

The Drosophila melanogaster gene and cDNA which span the entire open reading frame for DNA polymerase alpha, were cloned, and their nucleotide sequences were determined. The gene consists of 6 exons separated by 5 short introns. The major transcription initiation site was localized 85 bp upstream from the initiation codon. The nucleotide sequence of the open reading frame revealed a polypeptide of 1,505 amino acid residues with a molecular weight of 170,796. The amino acid sequence of the polypeptide was 37% homologous with that of the catalytic subunit of human DNA polymerase alpha. This sequence contains six regions, the orders and amino acid sequences of which are highly conserved among a number of other viral and eukaryotic DNA polymerases. We found 7 amino acid residues in the region between the 639th and 758th positions, identical to those essential for the active site of Escherichia coli DNA polymerase I-associated 3'----5' exonuclease. Thus, the exonuclease activity may be associated with Drosophila DNA polymerase alpha. Levels of the DNA polymerase alpha mRNA were high in unfertilized eggs and early embryos, relatively high in adult female flies and second-instar larva, and low in bodies at other stages of development. This feature of the expression is similar to that of the proliferating cell nuclear antigen (an auxiliary protein of DNA polymerase delta) and seems to coincide with the proportions of proliferating cells in various developmental stages. As the half life of the mRNA for DNA polymerase alpha in cultured Drosophila Kc cells was 15 min, expression of the DNA polymerase alpha gene is probably strictly regulated at the step of transcription.
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PMID:Structure and expression during development of Drosophila melanogaster gene for DNA polymerase alpha. 192 67

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.
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PMID:The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins. 194 34

Partial hepatectomy (PH) of rats (Wistar strain) resulted in acceleration of DNA synthesis in liver which reached a maximum at 36 h after PH. Whole-body radiation exposure (10 Gy) of the rats at 12 h after PH completely arrested this stimulation in DNA synthesis. The elevation of DNA synthetic rate in response to PH and complete obliteration of this stimulation by whole-body radiation exposure were found to be the reflection of levels of DNA polymerase-alpha in nuclei and nuclear matrices isolated from the rat livers. Studies based on assays of DNA polymerase in nuclei and nuclear matrices, with and without exogenous DNA template (activated calf thymus DNA), revealed that whole-body irradiation blocked induction of DNA polymerase-alpha and, in turn, assembling of DNA polymerizing apparatus. Irradiation of nuclei (suspended in buffer) in vitro at doses as high as 500 Gy did not have any inhibitory effect on DNA polymerase-alpha activity.
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PMID:DNA synthesis in nuclei and nuclear matrices of regenerating rat liver: effect of whole-body gamma irradiation. 196 15

We examined the effect of specific inhibitors of DNA polymerases alpha and delta, and beta, on cisplatin (DDP) cytotoxicity in DDP-sensitive and -resistant human 2008 ovarian carcinoma cells. Under conditions of continuous exposure to drug combinations, neither aphidicolin glycinate (AG) nor dideoxythymidine enhanced the cytotoxicity of DDP in either cell line as determined by clonogenic survival assays. However, when clonogenic survival was determined following short-term drug exposure, AG exhibited strong synergism with DDP in the DDP-resistant, but not the DDP-sensitive cells, as indicated by median effect analysis of the data. DNA polymerase alpha mRNA levels were the same in both cell lines under basal conditions. DDP-sensitive cells, but not DDP-resistant cells, were able to increase their expression of DNA polymerase alpha in response to DDP exposure. Levels of mRNA for DNA polymerase beta and for the human DNA repair gene ERCC-1 were not elevated in resistant cells, either under basal conditions or 18 hr after a 1 hr exposure to IC20 concentrations of DDP. In another human ovarian carcinoma cell line, A2780, AG and DDP were synergistic in both DDP-sensitive and -resistant variants in short-term exposure. We conclude that DNA polymerases alpha and/or delta play a role in the DDP sensitivity of human ovarian carcinoma cells.
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PMID:The effect of DNA polymerase inhibitors on the cytotoxicity of cisplatin in human ovarian carcinoma cells. 211 28

HeLa cells treated with 10 J/m2 of ultraviolet (UV) radiation were examined for inhibition of DNA excision by inhibitors of DNA polymerase-alpha and -beta. DNA repair synthesis and excision were inhibited by aphidicolin, a specific inhibitor of DNA polymerase-alpha. Decreased release of radioactive nucleotides from UV-damaged DNA by the inhibitor indicates that the action of DNA polymerase-alpha activity is closely associated with DNA excision. Dideoxythymidine also inhibited DNA repair synthesis but failed to block the excision, suggesting that polymerization by DNA polymerase-beta might precede removal of damaged DNA.
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PMID:Inhibition of DNA excision by DNA polymerase-alpha inhibitor in UV-damaged HeLa cells. 211 83

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.
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PMID:The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I. 219 44

A synthetic procedure has been developed by which stable abasic sites are introduced into oligodeoxynucleotides at any desired position in the sequence. A modified tetrahydrofuran moiety, isosteric with 2'-deoxyribofuranose, serves as a structural analog of the natural apurinic/apyrimidinic site. We have also prepared oligodeoxynucleotides that lack cyclic structure at the abasic site but retain the carbon atoms of the phosphodiester backbone. These synthetic oligodeoxynucleotides are cleaved on the 5' side of the abasic site by endonuclease IV and by exonuclease III; they serve also as templates for avian myeloblastosis virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow fragment), and calf thymus DNA polymerase-alpha. Extension of primed templates by these DNA polymerases is blocked initially at the position immediately 3' to the abasic site; nucleoside monophosphates are subsequently incorporated opposite the lesion. The nucleotide most frequently incorporated opposite all abasic sites, regardless of structure, is dAMP. Significant "readthrough" at the abasic site was observed in experiments using avian myeloblastosis virus reverse transcriptase and DNA polymerase-alpha and, to a much lesser degree, with DNA polymerase I. We conclude that a modified tetrahydrofuran group can serve as a stable structural analog of 2'-deoxyribose in the apurinic/apyrimidinic site. These modified oligodeoxynucleotides should prove useful for studies of chemical mutagenesis.
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PMID:Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases. 244 Aug 61

Aphidicolin is a specific inhibitor of DNA polymerase-alpha and -delta from eukaryotic cells. Because of the specificity of this inhibitor, it is potentially a useful probe for the detailed studies of the function of these polymerases. DNA polymerase-alpha mutants isolated on the basis of resistance to aphidicolin have been described. We have isolated four variants that exhibit hypersensitivities to aphidicolin (Aphhs) from Chinese hamster V79/743X fibroblasts. These variants are designated aphhs-1, aphhs-2, aphhs-3 and aphhs-4. We reported here results of studies involving immunochemical characterization. The Aphhs phenotype in all mutants was stable for at least 30 days in the absence of selection pressure. The dCTP pools in the 743X and Aphhs cell lines were not significantly different. The level of total DNA polymerase activity in the crude extract from aphhs-2 cells was 30% of that observed in the parental 743X clone. We developed a method to quantitate DNA polymerase-alpha antigen at single cells in situ using monoclonal antibody SJK 132-20 and fluorescence pseudocolor image. We found that the antigen of DNA polymerase-alpha in aphhs-2 was 30-50% of that in the parental 743X cells. The underproduction of the antigen of DNA polymerase-alpha provides a basis for the observed Aphhs phenotype. Possible mechanisms for the underproduction of DNA polymerase-alpha in aphhs-2 clone are presented.
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PMID:Aphidicolin hypersensitive mutant of Chinese hamster V79 fibroblasts that underproduces DNA polymerase-alpha antigen. 250 94


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