FIG. 2.

RID1 mutants display specific defects in repeat addition processivity. Pulse-chase time course experiments were performed to determine whether the product profiles of RID1 mutants were attributable to defects in repeat addition processivity or elongation kinetics. Wild-type (WT) hTERT (A) and RID1 variants (B to E) were synthesized in RRL in the presence of wild-type hTR and in the absence (B and C) or presence (D and E) of DNA encoding the minimal RID1 region required for complementation of processivity defects (amino acids 1 to 250). Since RID1 mutants were less active than wild-type hTERT (Fig. 1D), we performed RID1 pulse-chase experiments using three times more RRL-reconstituted telomerase and primer extension reagents (B to E). Experiments were performed with wild-type telomerase using standard assay conditions (A). Reconstituted telomerases were incubated with biotinylated (TTAGGG)₃ pulse primer for 5 min (5′) at 30°C in the presence of dATP, dTTP, and [α-³²P]dGTP (direct primer extension assay). A 150-fold excess of nonbiotinylated (TTAGGG)₃ chase primer was added, and the reactions continued for 15, 30, 60, or 120 min. Elongated biotinylated pulse primers were separated from the products of chase primer elongation using magnetic streptavidin beads. Two controls were used. In control 1 (lane 3), pulse and chase primers were added simultaneously to demonstrate the efficiency of chase conditions. In control 2 (lane 4), reactions were performed with chase primer alone to demonstrate that nonbiotinylated chase primer elongation products are not purified by streptavidin beads. A 3′ radiolabeled biotinylated TTAGGGT primer was added to stopped reaction mixtures prior to purification on streptavidin beads as a purification and loading control (LC). First repeat DNA synthesis and repeat addition processivity (R.A.P.) values for individual samples (expressed in arbitrary units) are indicated below the gels. All experiments were performed twice.