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)

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.
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PMID:Effect of molecular crowding on DNA polymerase activity. 1689 71

The ability to monitor DNA polymerase activity with single-nucleotide resolution has been the cornerstone of a number of advanced single-molecule DNA sequencing concepts. Toward this goal, we report the first observation of the base-by-base DNA polymerase activity with single-base resolution at the single-molecule level. We describe the design and characterization of a supramolecular nanopore device capable of detecting up to nine consecutive DNA polymerase-catalyzed single-nucleotide primer extensions with high sensitivity and spatial resolution (<or=2.4 A). The device is assembled in a suspended lipid membrane by threading and mechanically capturing a single strand of DNA-PEG copolymer inside an alpha-hemolysin protein pore. Single-nucleotide primer extensions result in successive displacements of the template DNA strand within the protein pore, which can be monitored by the corresponding stepped changes in the ion current flowing through the pore under an applied transmembrane potential. The system described thus represents a promising advance toward nanopore-mediated single-molecule DNA sequencing concept and, in addition, might be applicable to studying a number of other biopolymer-protein interactions and dynamics.
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PMID:A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. 1816 54

A method for studying steady-state kinetics of nucleotide analogues incorporation into DNA strand by non-gel CE (NGCE) with LIF was developed. Nucleoside analogue is a kind of antiviral agent used to inhibit viral replication in infected cells, especially HIV. Steady-state parameter K(m) for nucleotide analogues is determined to imply the relationship between nucleoside analogues and the enzyme in the DNA chain elongation and predict the antiviral efficacy in vivo. Samples were prepared by single nucleotide incorporation assays catalyzed by Taq DNA polymerase at 58 degrees C and HIV reverse transcriptase (RT) at 37 degrees C, and then were separated using NGCE under optimized conditions: 25 mmol/L Tris-boric-EDTA buffer (pH 8.0) with 7 mmol/L urea in the presence of 20% w/v PEG 35000 at 30 degrees C and -20 kV. K(m(dTTP)), K(m(d4TTP)) and K(m(AZTTP)) were measured by NGCE for the first time and their values for Taq DNA polymerase were 0.29+/-0.04, 32.1+/-3.3 and 74.5+/-6.6 micromol/L, respectively. For HIV RT, the values were 0.15+/-0.05, 0.31+/-0.03 and 0.17+/-0.03 micromol/L, respectively. The trend of data for HIV RT measured by NGCE was consistent with that measured by PAGE. The reported method by NGCE for the K(m) determination was powerful, sensitive and fast, and required less amounts of reagents compared with PAGE. It be employed as a reliable alternative method and further applied in other relative studies of nucleoside analogue substrates and DNA polymerases or RTs.
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PMID:Study on steady-state kinetics of nucleotide analogues incorporation by non-gel CE. 2011 62

Polymerase chain reaction (PCR), a versatile DNA amplification method, is a fundamental technology in modern life sciences and molecular diagnostics. After multiple rounds of PCR, however, nonspecific DNA fragments are often produced and the amplification efficiency and fidelity decrease. Here, we demonstrated that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve the PCR specificity and efficiency. PEG-nGO allows the specificity to be maintained even after multiple rounds of PCR, allowing reliable amplification at low annealing temperatures. PEG-nGO decreases the nonspecific annealing of single-stranded DNA (ssDNA), such as primer dimerization and false priming, by adsorbing excess primers. Moreover, PEG-nGO interrupts the reannealing of denatured template DNA by preferentially binding to ssDNA. Thus, PEG-nGO enhances the PCR specificity by preferentially binding to ssDNA without inhibiting DNA polymerase, which is analogous to the role of ssDNA binding proteins.
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PMID:Facilitation of Polymerase Chain Reaction with Poly(ethylene glycol)-Engrafted Graphene Oxide Analogous to a Single-Stranded-DNA Binding Protein. 2796 Apr 6

Since the origin of life on Earth, the role of carrying genetic information has been presumably transferred from RNA to DNA. At present, cellular environments are extremely dense, packed with cosolutes and macromolecules. Hence, the preference between RNA-dependent RNA and DNA polymerization may be affected by molecular crowding. In this study, we investigated both RNA-dependent RNA and DNA polymerizations by tC9Y polymerase ribozyme, T7 RNA polymerase (T7 RNAP), and Klenow fragment DNA polymerase (KF) under different molecular crowding conditions. Poly(ethylene glycol) (PEG) of various molecular weights was used as a crowding agent and found to promote both RNA and DNA ribozyme-catalyzed polymerizations. In contrast, PEG with an average molecular weight of 200 (PEG200) reduced the level of RNA polymerization by proteinaceous T7 RNAP but simultaneously promoted DNA polymerization, without affecting the activity of KF. Thus, proteinaceous RNA polymerase might potentially display bisubstrate specificity, which can be switched in response to changes in the dielectric constant and excluded volume in crowded environments. Our findings validate the bisubstrate activity of RNA polymerase from an evolutionary perspective for the development of non-natural materials.
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PMID:Bisubstrate Function of RNA Polymerases Triggered by Molecular Crowding Conditions. 3071 21