Strand displacement DNA synthesis assay. (A) Assay design. DNA polymerase incorporates dNTP thereby extending the primer strand and displacing the downstream reporter strand labeled with a 3′-fluorophore donor (F), leading to an increased fluorescence signal. F and Q denote 6-TAMRA and Black Hole Quencher-2 (BHQ-2), respectively. Chemical structures of 6-TAMRA and BHQ-2 used in the present study are also provided. (B) and (C) Assay demonstration. (B) DNA polymerase β concentration effect on reporter strand-displacement assay signal was tested using substrate 0–17 (Table 1). Time courses were run at room temperature using 50 nM of 0–17 and 100 μM of dTTP without (open square) or with pol β at different concentrations: 10, 20, 40 and 80 nM (filled squares, triangles, rhombs and circles, respectively). (C) dTTP concentration effect on the signal evolution was tested using 20 nM of pol β and 50 nM of 0–17 at different concentrations of dTTP : 0 (open squares), 25, 50, 100, 200 and 400 μM (filled triangles, inverted triangles, rhombs, circles and squares, respectively). (D) and (E) Template strand titration. Time course studies were performed at 50 nM substrate 0–10 with (filled squares) or without (open squares) 10 nM pol β (D) or 20 nM pol ι (E), respectively. When the fluorescence signal reached near saturation, 100 nM of template-strand labeled with BHQ-2 was added to the reaction mixture (arrow) and the fluorescence monitoring was resumed. Signal quenching upon addition of excess template strand is indicative of re-annealing of the intact displaced reporter strand.

Effect of DNA substrate structure on reaction profile. Time course studies with (A) pol β, (B) pol η and (C) pol ι were carried out at room temperature using 10 nM enzyme and 50 nM of substrates 0–17 (open squares), 2–15 (open triangles), 6–17 (open rhombs), 2–10 (filled squares), 0–12 (filled triangles) and 0–10 (filled rhombs).

Utilization of a modified dNTP substrate. Pol β (A) and pol ι (B) reactions were conducted using 10 nM enzyme and 50 nM substrate 0–10 in the presence of 100 μM dTTP as control (squares) and 8-oxodGTP (circles). Filled and open symbols represent the presence or absence of enzyme, respectively.

Characterization of tripartite DNA substrates. (A) Maximum dynamic range, defined as the fluorescence signal ratio between free reporter strand and tripartite substrate (Table 1) measured at 50 nM. (B) Substrate stability. Substrate 0–10 was subjected to repeat freeze-thaw cycles and tested in the assay at days 0 (squares), 1 (triangles), 4 (rhombs), 7 (circles) and 10 (inverted triangles). Time courses were carried out using 50 nM substrate with (filled symbols) and without (empty symbols) 10 nM pol β.

Effect of divalent ions on the activity of pols ι and η. Reactions (20 min) were carried out in presence of increasing concentrations of either Mg²⁺ or Mn²⁺ ions using 10 nM enzyme [pol ι (A) and pol η (B)] and 50 nM substrate 0–12. Empty and solid bars indicate Mn²⁺ or Mg²⁺ ion concentrations, respectively.

Assay miniaturization to 1536-well format. Time course assays for pol β (A), pol η (B), pol ι (C) and pol κ (D) were conducted in 4-μl reaction volumes with 10 nM of enzyme and 50 nM substrate 0–10. Filled and open symbols represent the presence or absence of enzyme, respectively. Averages and standard deviations shown are from 64 wells per reaction condition.