T4 polynucleotide kinase: macromolecular crowding increases the efficiency of reaction at DNA termini |
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| Authors: | B Harrison S B Zimmerman |
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| Institution: | 1. BioEcoUva, Research Institute on Bioeconomy, High Pressure Process Group, Department of Chemical Engineering and Environmental Technology, Universidad de Valladolid, Spain;2. Planta Piloto de Ingeniería Química (PLAPIQUI), Chemical Engineering Department, Universidad Nacional del Sur (UNS) - CONICET, Camino La Carrindanga Km7, 8000B, Bahía Blanca, Argentina;3. BioEcoUva, Research Institute on Bioeconomy, Research Group TERMOCAL, Thermodynamics and Calibration, Universidad de Valladolid, Escuela de Ingenierías Industriales, Paseo del Cauce 59, E-47011, Valladolid, Spain;4. Thermodynamics Research Unit, School of Engineering, University of KwaZulu-Natal, Howard College Campus, King George V Avenue, Durban 4041, South Africa;1. Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;2. CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan;3. Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan;1. II. Institut für Theoretische Physik, Universität Stuttgart, Stuttgart, Germany;2. Laboratory of Statistical Biophysics, Institute of Physics, School of Basic Science and Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland |
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| Abstract: | The amount of reaction catalyzed by T4 polynucleotide kinase on a variety of its substrates is greatly increased in the presence of polyethylene glycol 8000 (PEG 8000). Both the forward and reverse reactions as well as the exchange reaction can be stimulated. The stimulation is a general effect on T4 polynucleotide kinase reactions involving high molecular weight DNA substrates. The use of PEG 8000 is particularly advantageous for labeling or removing terminal 5'-phosphate groups which are only slowly or incompletely labeled or removed under ordinary conditions, such as those at recessed termini or at "nicks" in duplex DNA, although the reaction on blunt-ended or protruding termini is also increased. It is further advantageous for labeling very low concentrations of substrates. |
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