Fig. 3.

TPP1-POT1 remains stably associated with primers during competition assay. (a) Flow chart of TPP1 equilibration experiment. Primer 1 was bound to TEL-patch mutant TPP1-POT1 (EE mut) and in a separate tube primer 2 was bound by wild-type TPP-POT1. The two primers were mixed and allowed to pre-equilibrate for different amounts of time to allow TPP1 (or TPP1-POT1) to exchange between the primers. Primers were then extended for 10 min.

(b) TPP1 exchange equilibration experiment. Primer 1 was bound to TEL-patch mutant TPP1-POT1 (EE mut) and primer 2 was bound by wild-type TPP-POT1, as noted on left side of gel. No T-P, control experiment consisting of a competition between primer 1 and primer 2 with no TPP1-POT1 present. TPP1 Pre-mix, control experiment in which primers 1 and 2 were first mixed and then added to a mixture of wild-type TPP1-POT1 and E169A;E171A TPP1-POT1. Primer pre-equilibration time above gel; note that products visualized on gel were extended by telomerase for 10 min in addition to the time noted above the gel. Asterisk above 2 min pre-equilibration time corresponds to the final time point (12 min) in the initial velocity competitions shown in Figure 2 (i.e. 2 min of TPP1 pre-equilibration + 10 min telomerase extension = 12 min of TPP1 exchange). Products corresponding to +2 and +4 additions indicated on the right side of the gel. Precipitation and loading control (LC) shown below competition panel.

(c) Plot of the product ratio versus TPP1 pre-equilibration time. Product ratios were calculated by summing the +2, +3, +4 products for each primer and then dividing the total counts of primer 1 by the total counts of primer 2. The ratio of the TPP1-premix control is shown as a horizontal line at the top of the plot, and the pre-equilibration time (min) at which TPP1 has undergone 50% exchange is denoted with a dashed line, taking the starting product ratio as 15% and the pre-mix control ratio of 91% as the point at which equilibrium is reached.