Figure 1.

Schematic illustration of the DNA End Protection Assay (DEPA) on the 3′ overhang. (a) DNA oligonucleotides are annealed to form model telomeres with a double-stranded part and a single-stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomeric part is varied to create different length overhangs and different 5′ end permutations. Exonuclease T (ExoT) removes nucleotides in the 3′ to 5′ direction of the single-stranded 3′ overhang (II) of the G-rich strand which is radioactively 5′ end labelled (*). To assay for 3′ end protection, Cdc13 is pre-bound to the telomere before adding ExoT to the reaction, which will inhibit the exonuclease (III). (b) Reactions are stopped at different incubation times, de-proteinized, a labelled oligonucleotide loading control (LC) is added, samples are ethanol precipitated and run on a denaturing 10% polyacrylamide sequencing gel. Initially, the uncleaved substrate (S) is visible at the top of the gel (I) but as the exonuclease reaction progresses, products of decreasing size (P) appear on the gel while the substrate diminishes (IIa). Lane I, no enzyme control showing the initial substrate (S) and the loading control (LC); lane IIa, possible digestion products at different incubation times; lane IIb, ds product after the substrate 3′ overhang has been fully digested; lane III, a reaction where the substrate was pre-incubated with either Cdc13 or Rap1, providing substrate protection (SP) which generates a product of a certain length due to enzyme stalling.