Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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DNA polymerase epsilon was purified to near homogeneity from human placenta. The enzyme has one subunit (170 kDa, sedimentation coefficient 8.2S), intrinsic 3'-5'-exonuclease activity, it is independent on PCNA and high processivity on poly(dA)-oligo(dT) template-primer without PCNA. It was shown, that the enzyme incorporates 3'-amino-2',3'-dideoxythymidine 5'-triphosphate in DNA, after that synthesis is stopped. Simultaneously DNA polymerase alpha was purified.
Mol Biol (Mosk)
PMID:[A method of isolation and properties of DNA-dependent DNA-polymerase epsilon from human placenta]. 147 Jan 82

A GH cDNA was specifically amplified from cDNAs constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses employing the DNA polymerase chain reaction. Sequencing analysis revealed that the cDNA clone thus obtained was 654 bp in length, and included an open reading frame encoding the entire sequence of mature GH, with its signal peptide. Slight discrepancies were noted between the deduced amino acid sequence and that determined by direct protein sequencing of purified bullfrog GH or that deduced from the nucleotide sequence reported previously. The length of the bullfrog GH mRNA was estimated to be about 1.2 kb by Northern blot analysis. Homologies of nucleotide and amino acid sequences between GH and prolactin of bullfrog origin were 48% and 26% respectively. Using the cDNA as a probe, the content of GH mRNA in the pituitary of larval and adult bullfrogs was measured. GH mRNA levels were relatively low at the preclimax stage, and rose markedly during climax. In juvenile frogs, GH mRNA levels in the pituitary were extremely high and declined towards adulthood. This finding suggests that the increase in plasma and pituitary GH levels reported previously accompanies the increase in GH synthesis.
J Mol Endocrinol 1992 Dec
PMID:Cloning of a bullfrog growth hormone cDNA: expression of growth hormone mRNA in larval and adult bullfrog pituitaries. 147 15

We measured the relative steady-state levels of the mRNA transcribed from the Saccharomyces cerevisiae REV3 gene in cells at different stages of the mitotic and meiotic cycles, and after UV irradiation. This gene is thought to encode a DNA polymerase concerned only with a specific recovery function, the replication on mutagen-damaged templates that produces damaged-induced mutations. In keeping with this proposed function, the REV3 gene showed no evidence of the periodic transcription at the G1/S boundary of the mitotic and meiotic cycle that occurs with genes encoding replication enzymes. However, levels of REV3 mRNA were much increased in late meiotic cells, like those of transcripts of some other DNA repair-related genes. Steady-state levels of REV3 transcript were increased only slightly in response to UV irradiation.
Mol Gen Genet 1992 Dec
PMID:The REV3 gene of Saccharomyces cerevisiae is transcriptionally regulated more like a repair gene than one encoding a DNA polymerase. 149 46

Substrate properties of dNTP analogues in the DNA synthesis reaction catalyzed by Thermus aquaticus DNA polymerase were studied. It was shown that most of dNTP analogues which were known as terminators of DNA synthesis of E. coli DNA polymerase I were able to terminate DNA synthesis catalyzed by Thermus aquaticus DNA polymerase. An interesting feature of Thermus aquaticus DNA polymerase was the ability to utilize 3'-azido-2',3'-dideoxythymidine triphosphate as terminating substrate. Relative efficiency of tested dNTP analogues incorporation into the DNA growing chain was estimated.
Mol Biol (Mosk)
PMID:[Analogs of nucleotides, modified by a sugar residue and pyrimidine base, in a DNA synthesis reaction, catalyzed by Thermus aquaticus DNA polymerase]. 150 69

A specific complex of proteins involved in bacteriophage T4 replication has been visualized by cryoelectron microscopy as distinctive structures in association with DNA. Formation of these structures, which we term "hash-marks" for their characteristic appearance in association with DNA, requires the presence of the T4 polymerase accessory proteins (the products of T4 genes 44, 45 and 62), ATP and appropriate DNA cofactors. ATP hydrolysis by the DNA-stimulated ATPase activity of the accessory proteins is required for visualization of the hash-mark structures. If ATP hydrolysis is stopped by chelation of Mg2+, by dilution with a non-hydrolyzable ATP analogue, or by exhaustion of the ATP supply, the DNA-associated structures disappear within seconds to minutes, indicating that they have a finite and relatively short lifetime. The labile nature of the structures makes their study by more conventional methods of electron microscopy, as well as by most other structural approaches, difficult if not impossible. Addition of T4 gene 32 protein increases the number of hash-mark structures, as well as increasing the rate of ATP hydrolysis. Using plasmid DNA in either a native (supercoiled) or enzymatically modified state, we have shown that nicked or gapped DNA is required as a cofactor for hash-mark formation. Stimulation of the ATPase activity of the accessory proteins has a similar cofactor requirement. These conditions for the formation and visualization of the structures parallel those required for the action of these complexes in promoting the enzymatic activity of the T4 DNA polymerase, as well as the transcription of late T4 genes. Substructure in the hash-marks has been examined by image analysis, which reveals a variation in the projected density of the subunits comprising the structures. The three-dimensional size of the hash-marks, modeled as a solid ellipsoid, is consistent with that of the gene 44/62 protein subcomplex. Density variations suggest an arrangement of subunits, either tetragonal or trigonal, viewed from a variety of angles about the DNA axis. The hash-mark structures often appear in clusters, even in DNA that has a single nick. We interpret this distribution as the result of one-dimensional translocation of the hash-marks along the DNA after their ATP-dependent initial association with, and injection into, the DNA at nicks or gaps.
J Mol Biol 1992 Mar 20
PMID:Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex. 153 38

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.
J Mol Biol 1992 Jun 20
PMID:Telomere end-replication problem and cell aging. 161 1

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.
Mol Cell Biol 1991 Sep
PMID:Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site. 165 64

(+/-)-(1 alpha,2 beta,3 alpha)-9-[2,3-Bis(hydroxymethyl)cyclobutyl] guanine [(+/-)-BHCG] is a nucleoside analog with potent in vitro activity against herpesviruses [Tetrahedron Lett. 30:6453-6456 (1989)]. The two enantiomers have been synthesized, and their biochemical characterization is reported here. [1S(1 alpha,2 beta,3 alpha)]-9-[2,3-Bis(hydroxymethyl)cyclobutyl]guanine [(S)-BHCG] was phosphorylated by herpes simplex virus type 1 (HSV-1) thymidine kinase (Vmax = 8 nmol/hr/micrograms of enzyme), whereas [1R(1 alpha,2 beta,3 alpha)]-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(R)-BHCG] was a poor substrate for the viral thymidine kinase under these conditions. The triphosphate of each enantiomer was enzymatically synthesized, and both enantiomers competitively inhibited HSV-1 DNA polymerase with respect to dGTP. However, the potency of (R)-BHCG-TP was 4 orders of magnitude greater than that of (S)-BHCG-TP. (R)-BHCG-TP inhibited HeLa DNA polymerase alpha, but the inhibition constant was 30-fold higher than that for the viral DNA polymerase. In comparison, (S)-BHCG-TP was a very poor inhibitor of DNA polymerase alpha. (R)-[3H]BHCG-TP could be incorporated into a synthetic DNA template by HSV-1 DNA polymerase at 80% the extent of dGTP under the assay conditions used and, therefore, could act as an alternative substrate. Incorporation of (R)-BHCG-TP was similar to that observed for acyclovir triphosphate and ganciclovir triphosphate, based on maximal velocities. In contrast, HSV-1 DNA polymerase did not incorporate (S)-BHCG-TP into DNA. Compared with dGTP, only limited extension (10%) of the DNA primer by HSV-1 DNA polymerase occurred after incorporation of (R)-BHCG-TP and, therefore, (R)-BHCG-TP acts as a nonobligate chain terminator.
Mol Pharmacol 1991 Oct
PMID:Inhibition of herpes simplex virus type 1 DNA polymerase by [1R(1 alpha,2 beta,3 alpha)]-9-[2,3-bis(hydroxymethyl)cyclobutyl] guanine. 165 94

The enzymes of DNA polymerization and DNA precursor synthesis are assembled in the replitase complex during the S phase of the cell cycle. Cross-inhibition is a phenomenon shown by enzymes of the replitase complex, in which inhibition of one enzyme of the complex leads to inhibition of a second, unrelated enzyme. This inhibition occurs only in vivo and only during S phase. The second enzyme shows no inhibition in vitro. In this study, using Chinese hamster embryo fibroblast cells, we have shown that direct allosteric interactions, i.e., structural interaction from a remote site within the replitase complex, is the cause of cross-inhibition of thymidylate synthase activity by the inhibitors of ribonucleotide reductase and DNA polymerase, because disruptions of the deoxynucleotide pools, which would be predicted for alternative explantations, do not occur. Cross-inhibition of DNA polymerase by hydroxyurea is demonstrated by the cessation of DNA synthesis when ribonucleotide reductase block is circumvented by the provision of all four deoxynucleosides. In addition to the cross-inhibition for thymidylate synthase and DNA polymerase, we have also presented evidence, on the basis of alterations of the in vivo conversion of deoxyuridine to dUMP, that cross-inhibition also occurs for the enzyme thymidine kinase. This conclusion is further supported by the lack of inhibition of the similar process in RNA synthesis, because enzymes of RNA synthesis are not included in the replitase complex. To facilitate the measurements, we have introduced a novel method of distinguishing between thymidine and deoxyuridine derivatives, making use of the fact that a tritium label placed in the 5'-position of deoxyuridine is removed on conversion to thymidine by methylation, whereas a tritium placed in the 6'-position is not.
Mol Pharmacol 1990 Jul
PMID:Allosteric interaction of components of the replitase complex is responsible for enzyme cross-inhibition. 169 15

Rubromycins, a class of quinone antibacterials, were discovered to selectively inhibit human immunodeficiency virus-1 (HIV-1) RNA-directed DNA polymerase (reverse transcriptase) (RT) activity more potently than cellular DNA polymerase alpha. beta- and gamma-rubromycin each inhibited equipotently HIV-1 RT and avian myeloblastosis virus RT, in a concentration-dependent manner, and were significantly weaker as inhibitors of calf thymus DNA polymerase alpha. These agents inhibited HIV-1 RT reversibly, were competitive with respect to template.primer, and were noncompetitive with respect to TTP. Dixon analyses yielded HIV RT Ki values of 0.27 +/- 0.014 and 0.13 +/- 0.012 microM for beta- and gamma-rubromycin, respectively. Similarly, using DNA polymerase alpha, the Ki values were 25.1 +/- 4.3 and 3.9 +/- 0.6 microM for beta- and gamma-rubromycin, respectively. Because these agents were toxic to noninfected human T lymphoid cells using concentrations at or above 6 microM, HIV-1 infectivity studies were carried out at 0.8-6 microM. At these concentrations, which are below the range expected to provide protection, no significant antiviral activity was observed. Although beta- and gamma-rubromycins did not possess sufficient HIV RT inhibitory potency or selectivity versus mammalian DNA polymerase to demonstrate antiviral activities, these studies support the hypothesis that specific molecules containing quinone functional groups can selectively inhibit viral polymerase activities over cellular polymerase activities. In addition, these studies suggest that rubromycins may be lead structures for the development of more potent and selective agents.
Mol Pharmacol 1990 Jul
PMID:Inhibition of human immunodeficiency virus-1 reverse transcriptase activity by rubromycins: competitive interaction at the template.primer site. 169 17


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