qHTS Assay for Inhibitors of DNA Polymerase III Holoenzyme System
E. coli DNA polymerase III holoenzyme complex was assayed for DNA production by fluorescent detection of the double-stranded DNA product with PicoGreen dye. The holoenzyme complex was reconstituted from the following purified protein components: DNA Polymerase III, beta subunit processivity factor, SSB single-stranded DNA binding protein, and Primase. Single-stranded, circular M13Gori was used as more ..
BioActive Compounds: 169
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: 1 X01 MH077636-01
Assay Provider: Charles McHenry, University of Colorado
NCGC Assay Overview:
E. coli DNA polymerase III holoenzyme complex was assayed for DNA production by fluorescent detection of the double-stranded DNA product with PicoGreen dye. The holoenzyme complex was reconstituted from the following purified protein components: DNA Polymerase III, beta subunit processivity factor, SSB single-stranded DNA binding protein, and Primase. Single-stranded, circular M13Gori was used as a substrate with dNTPs and NTPs also supplied to the substrate reagent. Compounds from the NCGC collection were incubated with the holoenzyme mixture for 15 min at room temperature to allow binding to occur, and then the reaction was started by the addition of substrate. After 30 min incubation at room temperature, PicoGreen solution containing EDTA was added to stop the reaction and to visualize the double-stranded DNA produced. Reaction progress was measured in a ViewLux CCD imager in fluorescence intensity mode by using 480 nm excitation and 540 nm emission filter set.
NCGC Assay Protocol Summary:
Buffer. 50 mM Hepes-KOH, pH 7.5, 5 % Glycerol, 0.02% NP40, 30 mM potassium;
glutamate, 1.5 mM Magnesium Acetate, 16 uM TCEP.
Buffer in columns 3 and 4 as negative control (no enzyme).
Substrate solution: 5 uM each dATP, dGTP, dCTP and TTP, 50 uM each GTP, CTP, UTP and 200 uM ATP, 1.4 nM M13gori (final concentrations).
Enzyme: 154 nM SSB, 2 nM Primase, 1 nM Beta, 0.35 nM Pol III (final concentrations).
PicoGreen/EDTA: 10 mM Tris-HCl, 10 mM EDTA, 5 uM PicoGreen (final concentrations).
Control titration: ChemBridge 5805060 (from 10 mM, then 1:2, in duplicate) was pin-transferred to upper half of column 2.
Three uL of holoenzyme mixture were dispensed to 1536-well Greiner black solid bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 1 uL of substrate solution was added to start the reaction. After room temperature incubation for 35 minutes, 1 uL of PicoGreen solution was added to each well and the plates were read 480 nm excitation and 540 nm emission using ViewLux (Perkin-Elmer) High-throughput CCD imager and fluorescence protocol settings.
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, DNA Polymerase III, DNAPol3, DNAPoly3, DNA replication targets
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Class". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Data Table (Concise)