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)

A comparison was made between two methods of isolating DNA polymerases from MRC-5 fibroblasts. The first method produces DNA polymerase-alpha with a lower molecular weight and other properties that are not normally found for this enzyme. It was concluded that this method produces proteolytically degraded DNA polymerase-alpha. A second method was developed which produces DNA polymerase-alpha with all the normal properties of this enzyme. The specific activity of DNA polymerase was reduced in senescent MRC-5 fibroblasts approximately 2--4-fold. DNA polymerase-alpha accounts for 95% of polymerase activity in young cells and its specific activity during the fibroblast lifespan correlates with the declining cellular growth rate. DNA polymerase-beta is present at 0.3-3% of total cellular activity and its specific activity does not correlate with cellular growth rate. DNA polymerase-gamma accounts for 5% of the polymerase activity in young cells and 20% in old cells. However, the specific activity of the polymerase-gamma is constant throughout the lifespan of MRC-5. The 5 S DNA polymerase-alpha has an increased in vitro error frequency (average 3.6) compared to the 7 S polymerase-alpha. In addition the proportion of 5 S polymerase-alpha rises from 7% in young cells to 29% in senescent cells in an apparently exponential fashion.
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PMID:Properties of DNA polymerases from young and ageing human fibroblasts. 730 Apr 56

The direct-acting carcinogens acetoxyacetylaminofluorene, methylnitrosourea, and N-methyl-N'-nitro-N-nitrosoguanidine were tested for their ability to inhibit rat liver DNA polymerase-alpha, -beta, and -gamma activity in vitro. DNA polymerase-alpha was the most sensitive, polymerase-beta was the most resistant, and polymerase-gamma exhibited an intermediate response. When the reactions were reassayed in the presence and absence of dithiothreitol, a thiol reducing agent, it was shown that the inhibition by carcinogens was generally reversible with increasing dithiothreitol, except that polymerase-beta recovered only 80-90% of control values. These and binding data suggest that DNA polymerase-beta, the putative repair enzyme, is highly resistant to carcinogen damage. This resistance may contribute to the retention of normal function and fidelity of the repair enzyme during carcinogen exposure in vivo and to a normal cellular repair.
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PMID:Differential inhibition of rat liver DNA polymerases in vitro by direct-acting carcinogens and the protective effect of a thiol reducing agent. 730 70

Analysis of extracts of the bloodstream forms of Trypanosoma brucei showed that both DNA polymerase-alpha and DNA polymerase-beta activities were present. The detection of DNA polymerase-beta in T. brucei demonstrates the presence of this enzyme in unicellular organisms. DNA polymerase-beta is present also in Leishmania mexicana. The DNA polymerases in T. brucei are immunologically distinct from the host enzymes. The structural differences between the parasite and the host enzymes could be exploited for the development of agents to combat parasitic diseases.
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PMID:DNA polymerases in parasitic protozoans differ from host enzymes. 736 75

In order to determine the effect of maternal diabetes on the somatic growth of the rat fetus and to elucidate mechanisms underlying the control of fetal growth, concentrations of DNA and proteins and DNA polymerase-alpha activities in neonates were examined. The maternal status was classified as normal (no urinary glucose excretion), mildly diabetic (0.01-0.99 g/day urinary glucose), and severely diabetic (1.00 g/day or more urinary glucose). The total DNA contents in mg/neonate were 26.8 +/- 2.2 (mean +/- SEM), 31.3 +/- 2.5, and 29.4 +/- 2.7 for neonates from normal, mildly diabetic and severely diabetic mothers, respectively. The DNA polymerase activities in (cpm/g neonate) X 10(-3) for the same groups of neonates were 432 +/- 58, 1,008 +/- 74, and 888 +/- 118, respectively. These results indicate that the neonatal macrosomia disappears as the severity of maternal diabetes increases. Furthermore, DNA polymerase is one of possible biochemical sites through which macrosomia is manifested in diabetic pregnancies.
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PMID:Neonatal macrosomia in maternal diabetes. 742 59

Chinese hamster DDN cells have been exposed to low doses of X-irradiation (< 15 Gy). These doses have been shown to induce significant levels of repair replication. Levels of activity of DNA polymerase were subsequently measured in cells exposed in G1 phase of mitosis to 7.5 Gy of X-rays. Following exposure, the activity of this enzyme was observed to rise to a peak within 15 min post-irradiation, then decline and then rise again over a long period. The initial rise at least was found to be sensitive to inhibition by N-ethylmaleimide, a known inhibitor of DNA polymerase-alpha and DNA polymerase-gamma at the concentration used. It is therefore suggested that repair of X-ray-induced damage in these DON cells was at least in part dependent upon induced DNA polymerase activity and this may involve DNA polymerase-alpha or DNA polymerase-gamma, or both.
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PMID:X-ray induced activity of N-ethylmaleimide-sensitive DNA polymerase in Chinese hamster cells. 744 87

The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus.
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PMID:Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis. 751 Mar 88

In virtually all eukaryotes, mitosis starts after the completion of DNA synthesis. This orderly process is ensured by the checkpoint mechanism that blocks the onset of mitosis while DNA is being synthesized or is damaged. In the fission yeast Schizosaccharomyces pombe, this mechanism involves some rad+ and hus+ genes. However, it is not known how the checkpoint system monitors these events. Recently a multicopy suppressor of a temperature-sensitive DNA polymerase-alpha mutant was isolated. This gene, named cds1+ (checking DNA synthesis), encodes a typical protein kinase. Here we report that this protein kinase is a key component of the DNA replication-monitoring S/G2 checkpoint system. Our data suggest that its primary role is to monitor DNA synthesis by interacting with DNA polymerase alpha and send a signal to block the onset of mitosis while DNA synthesis is in progress.
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PMID:A kinase from fission yeast responsible for blocking mitosis in S phase. 772 27

Cell cycle is regulated by the activation of complexes of cyclins and cyclin-dependent protein kinases at specific points. Quiescent cells lack both cyclins and cyclin-dependent kinases but their expression is induced after proliferative activation. Cyclin A/cdk2 complexes are involved in the onset of DNA replication whereas cyclin B/cdc2 trigger mitosis. We report here that Ca2+ and calmodulin regulate the expression of cdk2, cdc2, cyclin B and the proliferating cell nuclear antigen (a co-factor of DNA polymerase-delta) in human T lymphocytes. Likewise, the expression of cdk4, cyclin A and DNA polymerase-alpha is dependent of the synergistic effect of both the Ca2+/calmodulin and the protein kinase C pathways. Thus, calmodulin controls DNA synthesis by regulating the levels of cdk2 and proliferating cell nuclear antigen and mitosis entry by modulating the expression of cyclin B and cdc2.
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PMID:Calmodulin regulates the expression of cdks, cyclins and replicative enzymes during proliferative activation of human T lymphocytes. 790 33

Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.
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PMID:Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. 791 Mar 75

Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA polymerase alpha catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had polymerase activity ranging from 4 to 60% compared with the wild type polymerase alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their Km values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the Km for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg(2+)-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA polymerase alpha are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in kcat, an increase in Km for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the alpha-like DNA polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the polymerase alpha catalytic site.
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PMID:Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis. 822 63


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