Figure 4

DNA 3′ end protection when Rap1 is bound across the ds-ss junction. (a) Schematic showing the interaction of Rap1 with telomeric substrate D13S13. Black bars indicate the 14 nt annealing guide sequence, bold text indicates Rap1 MBS, “*” indicates ³²P radioactive label. Numbers denote the distance in nt relative the ds-ss junction. (b) Sequencing gel showing 3′DEPA products of D13S13 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 (G0) to the 3′ end (C13). The stalling point at T6 is shown in bold and the bar indicates the area of the overhang protected by Rap1. (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 ≥6 nt and 13 nt was quantified at each reaction time for BSA (blue line) and Rap1 (red line) containing reactions. Error bars indicate SEM for n ≥ 6 experiments. (e) Schematic figure showing the 6 nt 3′ overhang that Rap1 protects from degradation by ExoT when bound across the ds-ss junction. Uncropped gel is shown in Supplementary Fig. S3.