Figure 3. DNA–protein complexes generated by Cdc13 and CST.

(A) The apparent K_d of Cdc13 and CST for the CgTELX1 probe (TGTGGGGTCTGGGTGC) was estimated in gel shift assays using different concentrations of the protein or protein complex. Data (average ± standard deviation) are from three independent experiments. (B) The stability of the CST-DNA complex was estimated in a competition experiment. CST (10 nM) was allowed to form complexes with labeled CgTELX1 (7.5 nM) for 20 min. One-hundred fold excess unlabeled CgTELX1 was then added, and the mixture applied to a native gel at various time points following the addition of the competitor. The non-uniform mobility of the free probe and the complex was due to the fact that samples were applied at different times. The fractions of labeled complexes remaining at various time points are determined and used to calculate t_½. The experiments were repeated three times and the estimated t_½ ranges from 5 to 13 min. (C) Increasing concentrations of CST and Cdc13 (15 nM, 30 nM and 60 nM) were incubated with P³²-labeled CgTELX1 oligo (7.5 nM) and the resulting complexes analyzed by electrophoresis through a native gel. Complexes of decreasing mobility are designated by I, II, etc. (D) Same as part C except that P³²-labeled CgTELX3 oligo ([TGTGGGGTCTGGGTGC]₃) was used as the probe.