Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.
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PMID:Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes. 329 56

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.
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PMID:A closed tube format for amplification and detection of DNA based on energy transfer. 917 Nov 7

Fragmentation is a major factor limiting mass range and resolution in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protonation of the nucleobase leads to base loss and backbone cleavage by a mechanism similar to the depurination reactions employed in the chemical degradation method of DNA sequencing. In a previous study [Tang,W., Zhu,L. and Smith,L.M. (1997) Anal. Chem ., 69, 302-312], the stabilizing effect of substituting the 24 hydrogen with an electronegative group such as hydroxyl or fluorine was investigated. These 24 substitutions stabilized the N-glycosidic linkage, blocking base loss and subsequent backbone cleavage. For such chemical modifications to be of practical significance, it would be useful to be able to employ the corresponding 24-modified nucleoside triphosphates in the polymerase-directed synthesis of DNA. This would provide an avenue to the preparation of 24-modified PCR fragments and dideoxy sequencing ladders stabilized for MALDI analysis. In this paper methods are described for the polymerase-directed synthesis of 24-fluoro modified DNA, using commercially available 24-fluoronucleoside triphosphates. The ability of a number of DNA and RNA polymerases to incorporate the 24-fluoro analogs was tested. Four thermostable DNA polymerases [Pfu (exo-), Vent (exo-), Deep Vent (exo-) and UlTma] were found that were able to incorporate 24-fluoronucleotides with reasonable efficiency. In order to perform Sanger sequencing reactions, the enzymes' ability to incorporate dideoxy terminators in conjunction with the 24-fluoronucleotides was evaluated. UlTma DNA polymerase was found to be the best of the enzymes tested for this purpose. MALDI analysis of enzymatically produced 24-fluoro modified DNA using the matrix 2,5-dihydroxy benzoic acid showed no base loss or backbone fragmentation, in contrast to the extensive fragmentation evident with unmodified DNA of the same sequence.
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PMID:2'-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry. 935 69

Novel anti-inflammatory compounds, terpeno-benzoic acids, were found from the plant, Myrsine seguinii. The strongest of these anti-inflammatory agents, 3-geranyl-4-hydroxy-5-(3'-methyl-2'-butenyl) benzoic acid (compound 1), showed an inhibitory effect against enzymes involved in replication, such as calf DNA polymerase alpha (pol. alpha), rat DNA polymerase beta (pol. beta) and one of the beta family polymerases, calf thymus terminal deoxynucleotidyl transferase (TdT). The minimum inhibitory concentration (MIC) and IC50 values were 82 and 22 microM for pol. alpha, 86 and 11 microM for pol. beta, 140 and 46 microM for TdT, respectively. However, compound 1 did not influence the activities of plant DNA polymerases, human immunodeficiency virus type-1 reverse transcriptase, any of the prokaryotic DNA polymerases or DNA and RNA metabolic enzymes tested. Dose-dependent relationships were observed between the anti-inflammatory activities and the DNA polymerase-inhibitory activities of the four derivatives. The carboxylic acid moiety in the benzoic acid of the compounds appeared to be related to the inhibitory effects. The mode of action of the terpeno-benzoic acids against the polymerases and their relationships to the anti-inflammatory activity are discussed.
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PMID:Novel anti-inflammatory compounds from Myrsine seguinii, terpeno-benzoic acids, are inhibitors of mammalian DNA polymerases. 1080 30