Fig. 4.

Mutations in the TEL-patch increase primer dissociation from extending telomerase.

(a) Cycling telomerase off-rate assays for free primer (left panel, No TPP1-POT1), primer bound by wild-type TPP1-POT1 (middle panel), or primer bound by E169A;E171A (right panel, EE Mut TPP1-POT1). “Pre-Ch” indicates pre-chase control samples in which 3′-phosphorylated primer was added to the telomerase prior to addition of the substrate primer. Time (min) of the chase denoted above gel; number of telomeric repeats indicated on left side of gel. Precipitation and loading control (LC) shown below each off-rate panel. The diffuse bands found near repeats 1, 3, and 7 are the result of contaminants present in the ³²P-α-dGTP. The intensity of these spurious bands was excluded from the calculation of the fraction of primer bound.

(b) The total counts (TC) incorporated in each lane at time n were expressed as fraction of the counts incorporated at time zero, TC(t = n)/TC(t = 0), and plotted versus time. The data were fit to a double exponential. Values of t_1/2^Apparent were estimated by determining the time at which half of the primer dissociated, represented by the dashed line. Fits for free primer (No TP, open square), wild-type TPP1-POT1 (WT TP, closed circle), and E169A/E171A mutant TPP1-POT1 (EE TP, closed triangle). Averaged data were plotted ± standard deviation (n = 2).