Polynucleotide Kinase (T4) (NEE101)
1. DNA oligo #2 was 5'-labeled with 33P by T4 polynucleotide kinase, following the manufacturer's protocol.
2. Equal molar quantities of DNA oligo #1 and the [33P]-labeled #2 oligo were annealed at 90C for 5 minutes and cooled to room temperature.
3. Approximately 1 ng of DNA duplex was applied to each well of the SMP103 FlashPlate in phosphate buffer containing 1 M NaCl (pH 7.4) and incubated for a minimum of 4 hours at room temperature or, alternately, overnight at 4C. Each well was washed twice with 200 μl of PBS at room temperature, and once with 200 μl of 50 mM Tris (pH 7.5), 50 mM NaCl at 37C. Each well contained approximately 10,000 to 12,000 CPM.
4. The coated plate was assayed with various amounts of SV40 Large T-Antigen (0 μg, 0.7 μg, 1.4 μg, 2.8 μg) in 50 μl of assay buffer, in the presence or absence of 1 mM ATP. The reaction was incubated at 37C and monitored in real time on a microplate scintillation counter, as indicated in Figures 2 and 3. Percentage of unwinding was calculated as follows:
% unwinding = [(CPM @ Time(0) - CPM @ Time(x))/CPM @ Time(0)] X 100 See Figure 1.
Results and Discussion
In the presence of ATP, double stranded DNA duplex can be unwound by T-Antigen (0.7 μg) beginning at the single/double strand junction, resulting in the release of the [33P]-labeled DNA strand (22 mer). As negative controls, the assay was also performed in the absence of ATP, and in the absence of T-Antigen, the results of which demonstrated that decreases in CPM were specific to T-Antigen helicase activity, and not a consequence of temperature-induced melting or