FlashPlate scintillation proximity assays for characterization and screening of DNA polymerase, primase, and helicase activities.
DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gel-based techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [(3)H]-dNTPs onto the growing primer 3' end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [(3)H]-rNTPs or by subsequent polymerase extension with [(3)H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [(33)P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.
Earnshaw
D L
DL
Molecular Interactions and New Assay Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Essex, UK.
Pope
A J
AJ
D004265:DNA Helicases / Q000032:analysis*; D019915:DNA Primase / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D018259:Herpesvirus 1, Human / Q000201:enzymology; D009838:Oligodeoxyribonucleotides; D011869:Radioligand Assay / Q000379:methods*; D012588:Scintillation Counting; D013539:Simian virus 40 / Q000201:enzymology; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases