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)

Recently developed techniques based on 'in situ end-labelling' (ISEL) of DNA strand breaks may help to identify apoptotic cells in tissue sections. We have applied ISEL techniques at the electron microscopic (EM) level, in order to verify if ultrastructural features of apoptosis are indeed associated with evidence of DNA fragmentation, and whether cells committed to, but which have not yet entered the stage of cell death are also labelled. Terminal transferase and DNA polymerase assays were applied to thin sections of Araldite and LR Gold-embedded medulloblastomas and embryonic mouse dorsal root ganglia. Digoxigenin-labelled nucleotides were used; incorporation was demonstrated by immunogold staining. Apoptotic cells in various stages of the death process were easily labelled in both tissues. In addition, DNA fragmentation was demonstrated in cells with initial chromatin condensation, but otherwise indistinguishable from adjacent unstained cells. Our results show that EM-ISEL techniques effectively demonstrate the occurrence of DNA strand breaks in apoptotic and possibly 'pre-apoptotic' cells in neural tissues. Since the labelling is easily obtained on tissue that is routinely processed for electron microscopy, this technique may allow retrospective studies on archival material.
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PMID:Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labelling techniques. 754 32

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Pol beta and TdT to investigate the region of Pol beta required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154-212 of TdT was replaced by the corresponding region encoding aa 1-60 of POL beta. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of POL beta represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Pol beta protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Pol beta are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Pol beta possesses ssDNA-binding activity [Kumar et al., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar et al., Biochemistry 29 (1990b) 7156-7159; Prasad et al., J. Biol. Chem. 268 (1993) 22746-22755].
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PMID:Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase beta. 759 Feb 83

TrwC is required for conjugal DNA transfer of the broad host range plasmid R388. The purified protein shows in vitro DNA helicase activity. Here we report that it also has in vitro oriT-endonuclease activity. TrwC specifically nicks oriT-containing supercoiled plasmid DNA in the presence of Mg2+, and the nicked DNA can be visualized after treatment with SDS. Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence. Both the 5' and the 3' ends of the nick were mapped. The 5' end was not accesible to phosphorylation by T4 polynucleotyde kinase, suggesting a covalent association with TrwC. Analysis of a collection of deletions in oriT indicated that the nucleotide sequences immediately surrounding the nic site are important, but not the only essential feature, for the nicking reaction. Comparison of the R388 nic site with previously published nic DNA sequences suggests that IncF, IncN and IncW plasmids form a family of related nic sites. During the course of this work we have also demonstrated a terminal transferase activity of Sequenase Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems.
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PMID:Nicking activity of TrwC directed against the origin of transfer of the IncW plasmid R388. 785 4

Two new techniques were used to quantify cell death (i.e. DNA fragmentation) in situ: (1) 3' overhangs of the fragmented DNA were end labelled with biotin-7-dATP and TdT (peroxidase/DAB). (2) In situ nick translation (ISNT) was performed with DNA polymerase 1 and biotin-7-dATP, to label single strand segments of DNA (peroxidase/DAB). Both methods were tested to be negative in ischemic and tumor necrosis, and negative for mitotic figures. In 26 centroblastic Non Hodgkin lymphomas (CB) (monomorphous subtype [n = 9], polymorphous subtype [n = 7], secondary [n = 10]), 14 chronic lymphocytic leukemias and two immunocytomas these methods were employed to quantify the rate of cell death. ISNT proved to be more sensitive than end labelling. By ISNT, CB had a mean cell death rate of 250/10HPF (monomorphous type: 429/10HPF, polymorphous type: 222/10HPF, secondary: 111/10HPF). CLL showed a significantly lower rate (28/10HPF). These data suggest, that the low rate of cell turnover in CLL is indicated by a low rate of cell proliferation and a low rate of programmed cell death. In CB the high proliferation rate was accompanied by a high level of cell death. In CB/monomorphous a high turnover state with a very high proliferation and cell death rate was found, whereas CB/polymorphous represents an expansive state as indicated by a lower rate of cell death. CB/secondary showed almost no programmed cell death and therefore was interpreted as a high expansive state neoplasia.
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PMID:[Specific in situ labeling of apoptosis shows different rates of programmed cell death in non-Hodgkin lymphomas]. 788 32

Human terminal deoxynucleotidyl transferase (TdT) was overexpressed in a baculovirus system. The pure recombinant enzyme was identical in size, activity, kinetic constants, and metal effects to native enzyme. Three amino acids, within either the putative nucleotide binding domain and part of a DNA polymerase consensus sequence, YGDTDSLF, or a TdT consensus sequence, GGFRRGK, were altered by site-directed mutagenesis. The four mutant forms of terminal transferase were also overexpressed in the baculovirus expression system and purified from Trichoplusia ni larvae by a monoclonal antibody affinity column and compared with wild-type enzyme with respect to thermostabilities, secondary structure, metal effects, and kinetic parameters. Three of the four mutants retained 3-16% of wild-type activity under varying metal conditions, and one of the mutants, D343E, consistently exhibited less than 0.2% of wild-type TdT activity with dATP and no activity with dGTP. All mutants had alterations in the Km for dATP. Variations in Km for dGTP were not as consistent. The Km for the other substrate, DNA initiator (dA)50) in the presence of dATP remained essentially the same as that of wild-type TdT for all mutants except D343E. The enzyme activity of all mutants was stimulated by Zn2+ at low concentrations, and this effect was diminished and reversed at higher concentrations of ZnSO4. All mutants still retained significant amounts of the secondary structure as measured by circular dichroism. These results indicated that the aspartic acid residue at position 343 is located at or near the active site and is critical for the nucleotide binding and catalytic activity.
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PMID:Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase. 816 85

As acyclic oligonucleotides have been suggested as a primitive model of DNA or RNA in prebiotic times, we compared some biochemical properties of these analogues to that of natural ones. Firstly, an acyclic analogue of deoxyribonucleoside triphosphates was tested as a potential substrate of enzymes intervening in nucleic acids synthesis. GlyTTP, a dTTP analogue with a missing 2'-methylene group is not accepted as a substrate by either DNA polymerase or deoxynucleotidyl terminal transferase (TdT). Secondly, the modified dodecathymidylate (GlyT)12, the racemic acyclic sugar analogue of (dT)12, proved to be an efficient primer for DNA polymerase and TdT, though the associative properties of (GlyT)12 are very weak as shown by UV spectroscopy in phosphate buffer without magnesium chloride. But (GlyT)12 has the advantage to be 500-times more stable against hydrolysis by snake venom phosphodiesterase than the corresponding oligothymidylate.
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PMID:Some biochemical properties of an acyclic oligonucleotide analogue. A plausible ancestor of the DNA? 838 7

Terminal deoxynucleotidyltransferase (TdT) is a template-independent DNA polymerase that is expressed transiently during the earliest stages of B- and T-cell ontogeny. Previously, we characterized the promoter for the murine TdT gene and identified a novel DNA-binding protein, called LyF-1, that interacts with a DNA sequence element found to be critical for transcriptional activity in lymphoid cell lines. Here, we present a more detailed analysis of this 30-bp control element, called the TdT D' element, which is centered approximately 60 bp upstream of the transcription start site. We found that both the murine and human D' elements are recognized by multiple proteins, including LyF-1 and at least two Ets family proteins, Ets-1 and Fli-1. Additional protein-DNA interactions were identified through studies using unfractionated nuclear extracts, in which the D' element was apparently incorporated into a multiprotein complex, possibly containing an Ets protein as a core component. By analyzing a series of substitution mutations, two adjacent binding sites for LyF-1 were identified in the murine D' element, with the Ets protein binding site closely coinciding with the proximal, lower-affinity LyF-1 site. Transient transfection analysis with these mutations revealed that only a 10-bp region, containing precisely the Ets and proximal LyF-1 binding sites, was needed for D' activity. These results suggest an important role for an Ets family protein in the expression of the TdT gene. The role of LyF-1 is less clear; it might act in conjunction with the Ets protein bound at the D' element or it might be unnecessary for D' activity.
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PMID:Both LyF-1 and an Ets protein interact with a critical promoter element in the murine terminal transferase gene. 847 56

The aliphatic polyamines, putrescine, spermine and spermidine belong to a category of molecules implicated in DNA replication. Their synthesis is strongly activated during the G1 period and they have been implicated in the regulation of cell proliferation and differentiation. Terminal transferase is a DNA polymerase present in pre-T and pre-B cells and its expression can be modulated by phorbol ester treatment. In this study we have monitored the relationship of intracellular polyamine levels with terminal deoxynucleotidyl transferase down-regulation induced by 12-O-tetradecanoyl phorbol myristate 13-acetate treatment in the human pre-B KM-3 cell line. Phorbol myristate acetate can cause an increase, at 4 and 8 hours of differentiation, of intracellular levels of putrescine as well as a decrease in terminal deoxynucleotidyl transferase synthesis showing the probable involvement that polyamines have in the differentiation process.
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PMID:Polyamines and terminal deoxynucleotidyl transferase expression in KM 3 pre-B cell line during phorbol ester induced differentiation. 852 90

The SuperTth DNA polymerase from Thermus thermophilus exhibits template-independent terminal transferase (extendase) activity. This enzyme is proposed as a cheap alternative for both high performance PCR as well as quick T-vector cloning of amplicons, including reverse transcription and cDNA cloning.
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PMID:T-vector cloning and high performance PCR with SuperTth from Thermus thermophilus. 857 96


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