Abstract
A homogeneous, fluorescence resonance energy transfer (FRET)-based DNA polymerase assay that is suitable for high-throughput screening for inhibitors, and can also be used for steady-state kinetic investigations, is described. The activity, kinetic mechanism, and processivity of the isolated alpha subunit of DNA polymerase III, the product of the dnaE gene, from the gram-negative pathogen Haemophilus influenzae were investigated using the FRET assay.
MeSH terms
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Base Sequence
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Chemistry Techniques, Analytical
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Chromatography, High Pressure Liquid
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DNA Polymerase III / analysis*
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DNA Polymerase III / antagonists & inhibitors
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DNA Polymerase III / genetics
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DNA Polymerase III / metabolism
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DNA Primers / genetics
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DNA, Bacterial / genetics
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DNA, Bacterial / metabolism
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Electrophoresis, Capillary
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Escherichia coli / genetics
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Fluorescence Resonance Energy Transfer / methods*
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Fluorescence Resonance Energy Transfer / statistics & numerical data
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Haemophilus influenzae / enzymology
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Haemophilus influenzae / genetics
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Kinetics
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Models, Biological
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Recombinant Proteins / analysis
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
Substances
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DNA Primers
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DNA, Bacterial
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Recombinant Proteins
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DNA polymerase III, alpha subunit
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DNA Polymerase III