Figure 3

DNA 3′ end protection when Rap1 is bound immediately adjacent to the ds-ss junction. (a) Schematic figure indicating the interaction of Rap1 with telomeric substrate D15S11. Black bars indicate the 14 nt annealing guide sequence, bold text indicates Rap1 MBS, “*” indicates ³²P radioactive label. Numbers above the sequence denotes the distance in nt relative the ds-ss junction. (b) Sequencing gel showing 3′DEPA products of D15S11 incubated with 0.015 U/μl ExoT for 25 s, 1 min, 2 min, 4 min and 7 min, following pre-incubation with BSA or Rap1 as indicated.“–” indicates no enzyme added, “LC” loading control. The sequence of the 3′ overhang is written beside the gel and numbered from the ds-ss junction (T0) to the 3′ end (C11). The G1 and T2 bases protected by Rap1 are in bold. (c) Lane profiles showing the intensities of each band in the lanes, corresponding to the indicated time points for each graph, after pre-incubation with Rap1 (red line) or the BSA control (blue line). The sequence corresponding to each peak is written as in (b). (d) The proportion of overhangs at lengths of ≥1 nt and 11 nt quantified at each reaction time for Rap1 containing reactions (red line) and BSA control (blue line). Error bars indicate SEM for n ≥ 5 experiments. (e) Schematic figure showing the 1 nt 3′ overhang that Rap1 protects from degradation by ExoT when bound adjacent to the ds-ss junction. Uncropped gel is shown in Supplementary Fig. S3.