DNA polymerase activity

This enzyme is associated with the virions of RNA tumor viruses such as the Rous sarcoma virus (RSV). The enzyme has remarkable enzymatic activity in that it can catalyze several seemingly diverse steps in the synthesis of double-stranded DNA from the single-stranded RNA viral genome. The enzyme uses a tRNA for tryp-tophan as a primer to make a copy of DNA that is complementary to the viral RNA. The resulting RNA-DNA hybrid is converted to a double-stranded DNA molecule by ribon-uclease (RNase)H and DNA-dependent DNA polymerase activities that are intrinsic to reverse transcriptase. 

Linn, S Kairis, M. and Holliday, R. Decreased Fidelity of DNA Polymerase Activity Isolated from Aging Human Fibroblasts , Proc. Natl, Acad. Sci. (1976) 73, 2818. 

Nevirapine binds directly to reverse transcriptase (RT) and blocks the RNA-dependent and DNA-dependent DNA polymerase activities by causing a disruption of the enzyme s catalytic site. The activity of nevirapine does not compete with P.1118... 

DNA polymerase and also acts as an alternative substrate. The incorporation of one cidofovir molecule into the growing DNA chain slows replication sequential incorporation of two molecules halts DNA polymerase activity. 

Vidarabine s specific mechanism of action is not fully understood. Cellular enzymes convert this drug to a triphosphate that inhibits DNA polymerase activity. Vidarabine triphosphate competes with deoxyadeno-sine triphosphate (dATP) for access to DNA polymerase and also acts as a chain terminator. Although vidarabine is incorporated into host DNA to some extent, viral DNA polymerase is much more susceptible to inhibition by vidarabine. Vidarabine also inhibits ri-bonucleoside reductase and other enzymes. Resistance occurs as a result of DNA polymerase mutation. 

Where nucleic acids are concerned, the enhanced hydrophobicity of abiotic polyfluorinated aromatic bases (e.g., tetrafluorobenzene or tetrafluoroindole deoxyribose derivatives) was exploited as an alternative to natural hydrogen bonding to achieve selective and stable nucleic acid base pairing in duplex DNA [85], The DNA replication was examined using polyfluorinated-nucleotide analogs as substrates. A DNA polymerase active site was able to process the polyfluorinated base pairs more effectively than the analogous hydrocarbon pairs, demonstrating hydrophobic selectivity of polyfluorinated bases for other polyfluorinated bases [86]. 

J.S. Lai, E.T. Kool, Fluorous base-pairing effects in a DNA polymerase active site, Chem. Europ. J. 11 (2005) 2966-2971. 

Mechanism of Action A non-nucleoside reverse transcriptase inhibitor that binds directly to HlV-1 reverse transcriptase and blocks RNA- and DNA-dependent DNA polymerase activities. Therapeutic Effect Interrupts HIV replication, slowing the progression of HIV infection. 

The term purified distinguishes the enzyme from the product as it exists in nature. In an attempt to distinguish the claimed enzyme from enzymes which were known before the patent was filed, the enzyme is defined as having certain characteristics i.e. DNA polymerase activity, thermostability and a molecular weight within a specified range. 

Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) contains the multifunctional enzyme reverse transcriptase (RT). Retroviral RTs have a DNA polymerase activity that can use either an RNA or a DNA template and an RNase H activity. HIV-1 RT is essential for the conversion of single-stranded viral RNA into a linear double-stranded DNA that is subsequently integrated into the host cell chromosomes [1-4]. In this conversion process HIV-1 RT catalyzes the incorporation of approximately... 

A series of natural products, i.e., trihydroxyquinolone compounds isolated from Red Sea marine organisms, were reported to inhibit the DNA polymerase activity of HIV-1 RT [124,125]. This type of inhibitor appears to have a mechanism of inhibition that is different from either the NRTI inhibition mechanism or the NNRTI inhibition mechanism. The inhibition is reversible and noncompetitive with respect to both dNTP and template-primer [125]. This result indicates that there are other potential binding sites for inhibitors of HIV-1 RT. 

Fletcher RS, Arion D, Borkow G, Wainberg MA, Dmitrienko GI, Pamiak MA. Synergistic inhibition of HIV-1 reverse transcriptase DNA polymerase activity and virus replication in vitro by combinations of carboxanilide nonnucleoside compounds. Biochemistry 1995 34 10106-10112. 

Exonuclease activities, proofreading, and editing. DNA polymerase I not only catalyzes the growth of DNA chains at the 3 end of a primer strand but also, at about a 10-fold slower rate, the hydrolytic removal of nucleotides from the 3 end (31- 5 exonuclease activity). The same enzyme also catalyzes hydrolytic removal of nucleotides from the 5 end of DNA chains. This latter 5 - 3 exonuclease activity, the DNA polymerase activity, and the 3 -5 exonuclease activity all arise from separate active sites in the protein. DNA polymerases II and III do not catalyze... 

It was found that 1 acted as an antimitotic agent, not binding to tubulin, but by disorganizing the microtubule network in some fashion. In addition, it is a DNA minor groove guanine-specific alkylating agent [1]. The Et s showed potent inhibition of DNA and RNA synthesis and of RNA polymerase activity, but its inhibition of DNA polymerase activity is much less marked [75]. The potent activity of Et s was attributed, at least in part, to the unit C since the related saframycin A lacks this unit and has lower efficacy than Et 729 in comparable tumor models [74, 75]. More recent structural information on Et 743-DNA adduct was obtained by NMR spectroscopy [78]. An enantioselective total synthesis of Et 743 has been achieved by Corey et al. [79]. 

Nevirapine is a member of the dipyridodiazepinone class of chemicals and is a nonnucleoside reverse transcriptase inhibitor that induces a conformational change in HIV-1 reverse transcriptase. Although the conformational change is at a distance from its active site, it disrupts its catalytic activity. It blocks both RNA-dependent and DNA-dependent DNA polymerase activity but does not affect the activity of the template or nucleoside triphosphate. Nevirapine does not inhibit HIV-2 reverse transcriptase or human DNA polymerases a, (3 or y. The resistance to the drug results from site-directed mutagenesis at codons 103 or 181, and also at 100, 106, 108, 188 and 190 of viral reverse transcriptase. The development of resistance to one nonnucleoside reverse transcriptase implies that HIV will also be resistant to the rest of the drugs in this class. 

Delavirdine is a synthetic nonnucleoside reverse transcriptase inhibitor which after directly binding to HIV-1 reverse transcriptase blocks RNA-dependent and DNA-dependent DNA polymerase activities. It disrupts the catalytic activity of the enzyme after causing a conformational change in HIV-1 reverse transcriptase and does not... 

A single-stranded DNA library was also screened against the HIV-1 RT. IQ values for the selected DNA sequences were in the nanomolar range and they inhibited the DNA polymerase activity of this enzyme with a K, as low as 1 nM (Schneider et al., 1995). The best DNA aptamer folds as a hairpin with an internal loop and competes with the RNA pseudo-knot for RT binding. These two ligands share very little structural similarity. 

Mechanism of Action. Cidofovir works like acyclovir and ganciclovir these drugs inhibit viral DNA replication by inhibiting DNA polymerase activity and by halting elongation of viral DNA chains.42... 

Ellipticine inhibits DNA, RNA and protein synthesis. The inhibition is not reversible by removal of the alkaloid. It has no appreciable effects on thymidine and uridine kinases or on RNA polymerase, but it markedly inhibits DNA polymerase activity [240, 241]. The actual mechanism of action of ellipticine and related compounds has not yet been elucidated. Ellipticine and derivatives have been found to interact with bacterial membranes [242]. Many investigators have categorized these compounds as DNA-interacting or intercalating agents [230, 235, 237,243-246]. It has recently been postulated that mammalian DNA topoisomerase II may be a common target of these antitumour compounds [247],... 

Bacterial family C polymerases are the major chromosomal replicative enzyme (Kornberg and Baker, 1992). Like other replicative polymerases, the holoenzyme interacts with other proteins and forms a large multisubunit complex consisting of at least 10 subunits (Kornberg and Baker, 1992). The a-subunit contains the DNA polymerase activity that is tightly associated with the e-subunit, which contains a 3 -5 exonuclease activity (Kelman and O Donnell, 1995). 

The first DNA polymerase activity was identified in 1956 in E. coli (Kornberg et al, 1956 Lehman et al., 1958). The enzyme was subsequently named DNA polymerase I (pol I). E. coli pol I is a 109-kDa enzyme that supports a multidomain architecture containing a polymerase activity, a 5 -3 exonuclease activity, and a 3 -5 exonuclease activity. The C-terminal portion of E. coli pol I, called the Klenow fragment, which lacks the 5 -3 ... 

Hsieh, J. C., Zinnen, S., and Modrich, P. (1993). Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. / Biol. Chem. 268, 24607-24613. 

Tabor, S., Huber, H. E., and Richardson, C. C. (1987). Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7./. Biol. Chem. 262, 16212-16223. 

Perhaps the best understood example of induction involves induction of the aromatic hydrocarbon receptor (AhR) by compounds such as TCDD and 3-methylcholanthrene. The use of suitable inhibitors of RNA and DNA polymerase activity has shown that inhibitors of RNA synthesis such as actinomycin D and mercapto(pyridethyl)benzimida-zole block aryl hydrocarbon hydroxylase induction, whereas hydroxyurea, at levels that completely block the incorporation of thymidine into DNA, has no effect. Thus it appears that the inductive effect is at the level of transcription and that DNA synthesis is not required. 

Several approaches have been reported for the screening of polymerase activity, for example radioisotope assays such as scintillation proximity assays [56, 57] or fluorescence-based assays [58-61], Most of these assays, however, suffer from use of tedious procedures, the use of radioisotopes, or use of expensive reagents for fluorescence signal generation. A convenient means of online monitoring of DNA polymerase activity has recently been presented by Andreas Marx and Daniel Summerer [62]. Their technique involves a DNA template that forms a stable hairpin structure labeled at two positions ... 

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