FIG. 2.
Binding of POT1a and -b to 3′ and 5′ telomeric sites. (A and B) Gel-shift reactions with the probes indicated below the gels and increasing amounts of POT1a and POT1b. (A) Binding of POT1a and -b to the probes a, a3, and a3′. The red letters indicate nucleotide changes that interfere with POT1 binding to the 5′ site. The minimal binding sites are highlighted by the red boxes. Protein amounts in each binding reaction are indicated above the lanes in the left panel, and the same amounts were used in other panels. (B) POT1a and -b bind to the telomeric site at a 5′ end with diminished affinity. The mutations in primer a5 block POT1a and POT1b from binding to the 3′ end of the probe. Probes with no POT1 recognition site are not bound by POT1a or -b. The gel shifts shown are representatives of three independent experiments. (C) Summary of the Kd values (nM) of POT1a and POT1b in gel-shift experiments with the indicated probes. Values derived from three or more experiments are given as averages with standard deviations. Where probes were tested only twice, both values are given. All experiments were done with POT1a and -b in parallel, and Kd values were derived from quantitative analysis of the phosphorimager data as shown in panels A and B.