A homogeneous, high-throughput fluorescence resonance energy transfer-based DNA polymerase assay.
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.
Shapiro
Adam
A
Department of Biochemistry, Infection Discovery, AstraZeneca R&D Boston, Waltham, MA 02451, USA. adam.shapiro@astrazeneca.com
Rivin
Olga
O
Gao
Ning
N
Hajec
Laurel
L
D001483:Base Sequence; D002623:Chemistry Techniques, Analytical; D002851:Chromatography, High Pressure Liquid; D004258:DNA Polymerase III / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000706:statistics & numerical data; D006193:Haemophilus influenzae / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008954:Models, Biological; D011994:Recombinant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III