Figure 2.

Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. (a) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. (b) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). (c) Graph showing the quantification of the gel shown in (b). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2.