FIG. 3.
Direct telomerase assay with POT1 preloaded primers. (A) Schematic of the experimental design to preload substrate primers with POT1 (see the text). (B) Direct telomerase activity assay. Biotinylated primer tel3.0 bound to streptavidin beads was incubated with POT1 dialysis buffer (lanes 1, 2, 6, and 7), a molar excess of POT1 (lanes 3 to 5), or an equivalent volume of vector control lysate (lanes 8 to 10). Following washes to remove unbound proteins, extension reactions were initiated by the addition of affinity-purified, baculovirus-expressed recombinant telomerase and POT1 dialysis buffer (lanes 3 and 8), 0.032 pmol (3.2 nM) of POT1 (lanes 1 and 4), 0.13 pmol (13 nM) of POT1 (lanes 2 and 5), vector lysate diluted 1:100 (lanes 6 and 9), or vector lysate diluted 1:25 (lanes 7 and 10). (C) The schematic of the EMSA control is indicated in the upper panel. Biotinylated primer tel3.0 bound to streptavidin beads was incubated with POT1. To test if the wash steps also removed some transiently dissociated POT1, trace amounts of 5′-labeled tel3.0 were added following the wash steps, and competition by preloaded biotinylated primer tel3.0 was assessed by EMSA (lower panel). Biotinylated primer tel3.0 bound to streptavidin beads was incubated with POT1 dialysis buffer (lanes 1 to 4) or a molar excess of POT1 (lanes 5 to 10). Protein-DNA complexes were washed (lanes 2 to 7) or not washed (lanes 1 and 8 to 10), and trace 5′-labeled tel3.0 was added. Dialysis buffer (lane 1) or POT1 at 3.2 nM (lanes 2, 5, and 8), 6.4 nM (lanes 3, 6, and 9), or 13 nM (lanes 4, 7, and 10) was added, and reactions (rxn) were incubated for 30 min at 37°C. Reaction products were separated on a 6% polyacrylamide TGE gel.