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Herbert Van Amerongen – 1st expert on this subject based on the ideXlab platform
Structural Heterogeneity in DNA: Temperature Dependence of 2‐Aminopurine Fluorescence in DinucleotidesChemPhysChem, 2005Co-Authors: O J G Somsen, Linda B. Keukens, M. Niels De Keijzer, Arie Van Hoek, Herbert Van AmerongenAbstract:
The fluorescent base analogue 2-Aminopurine is a sensitive probe for local dynamics of DNA. Its fluorescence is quenched by interaction with the neighboring bases, but the underlying mechanisms are still under investigation. We studied 2-Aminopurine fluorescence in dinucleotides with each of the natural bases. Consistently, two of the four fluorescence-decay components depend strongly on temperature. Our results indicate that these components are due to the excited-state dynamics of a single conformational state. We propose a variation of the gating model in which transient unstacking occurs in the excited state.
ultrafast transient absorption and steady state fluorescence measurements on 2 Aminopurine substituted dinucleotides and 2 Aminopurine substituted dna duplexesPhysical Chemistry Chemical Physics, 2004Co-Authors: O F A Larsen, Ivo H M Van Stokkum, Frank L De Weerd, Mikas Vengris, Charuvila T Aravindakumar, Rienk Van Grondelle, Nicholas E Geacintov, Herbert Van AmerongenAbstract:
Ultrafast transient-absorption and steady-state fluorescence measurements have been performed on dinucleotides comprising the fluorescent adenine analogue 2-Aminopurine and guanine, adenine, cytosine, thymine or hypoxanthine, respectively. Two oligodeoxyribonucleotide duplexes that were site-selectively substituted with a single 2-Aminopurine moiety were also studied. A strong quenching of the steady-state fluorescence was observed in all samples. The transient-absorption spectra were remarkably similar to those of the isolated 2-Aminopurine (Larsen et al.; O. F. A. Larsen, I. H. M. van Stokkum, M.-L. Groot, J. T. M. Kennis, R. van Grondelle and H. van Amerongen, Chem. Phys. Lett., 2003, 371, 157–163), exhibiting both a fluorescent and a non-fluorescent excited state. There was no evidence for significant amounts of charge-separated states in the transient-absorption spectra. The probability that an excitation of 2AP leads to stable charge transfer products was estimated to be very low (∼0.1%). In the systems we studied, the observed fluorescence quenching can largely be explained by a shift of the equilibrium between the two excited states in 2AP, in which the non-fluorescent state is favored.
David T F Dryden – 2nd expert on this subject based on the ideXlab platform
2 Aminopurine flipped into the active site of the adenine specific dna methyltransferase m taqi crystal structures and time resolved fluorescenceJournal of the American Chemical Society, 2007Co-Authors: Thomas Lenz, David T F Dryden, Robert K Neely, Anita C Jones, Eleanor Y M Bonnist, Goran Pljevaljcic, Axel J Scheidig, Elmar G WeinholdAbstract:
We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-Aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short (∼50 ps) lifetime component associated with fully base-stacked 2-Aminopurine in DNA, and 2-Aminopurine is subject to considerable quenching by π-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-Aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the propo…
evidence of tautomerism in 2 Aminopurine from fluorescence lifetime measurementsJournal of Physical Chemistry B, 2004Co-Authors: Robert K Neely, David T F Dryden, Steven W Magennis, Anita C JonesAbstract:
The fluorescence decay characteristics of 2-Aminopurine (2AP) and 2-Aminopurine riboside (2APr) have been investigated as a function of excitation and emission wavelength in aqueous and ethanolic solutions. Global analysis of the decay data shows that 2AP exists as two emitting species, whereas 2APr exists as a single species. This is attributed to 9H/7H tautomerism of 2AP. The proportion of 7H tautomer is estimated to be 20% in ethanol and 40% in water.
unusual 2 Aminopurine fluorescence from a complex of dna and the ecoki methyltransferaseNucleic Acids Research, 2004Co-Authors: Tsueuju Su, Bernard A Connolly, C Darlington, R Mallin, David T F DrydenAbstract:
The methyltransferase, M.EcoKI, recognizes the DNA sequence 5¢-AACNNNNNNGTGC-3¢ and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-Aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-Aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-Aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosylmethionine to the M.EcoKI:DNA complex, the 2Aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-Aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-Aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-Aminopurine.
Thomas W Conway – 3rd expert on this subject based on the ideXlab platform
2 Aminopurine inhibits the double stranded rna dependent protein kinase both in vitro and in vivoJournal of interferon research, 1993Co-Authors: Yinghe Hu, Thomas W ConwayAbstract:
ABSTRACT The autophosphorylation of interferon (IFN)-induced double-stranded RNA-dependent p68 protein kinase (PKR) and phosphorylation of the α-subunit of the translation initiation factor eIF-2 were inhibited by 10 mM 2-Aminopurine in vitro. High concentrations of ATP overcame the inhibition. Kinetic studies indicated that 2-Aminopurine is a competitive inhibitor with respect to ATP, suggesting that these two molecules bind the same site on the kinase. Treatment of HeLa cells with poly(I):poly(C) stimulated PKR autophosphorylation in vivo. The stimulated activity was inhibited by 10 mM 2-Aminopurine to approximately the same extent as the in vitro inhibition.