Table 1. Sequences and Imp4p-binding affinity of yeast telomeric DNA substrates.
Oligonucleotide TG15, TG20, TG25, TG30, TG35 or TG40 was 5′-end-labelled with [γ-32P]ATP and subsequently purified from a 10% sequencing gel after electrophoresis. To perform the assays, purified Imp4p was mixed with 5 nM 32P-labelled oligonucleotides in a total volume of 15 μl in buffer containing 50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl, 1 mM dithiothreitol and 1 μg of heat-denatured poly(dI-dC)·(dI-dC). The reaction mixtures were incubated at room temperature for 10 min and separated on a 6% non-denaturing polyacrylamide gel. The apparent binding constant of Imp4p to telomeric DNA was determined from an average of three experiments using EMSA and quantified using a PhosphorImager.
| Name | Sequence | Kd (app) (μM) |
|---|---|---|
| TG15 | 5′-TGTGTGGGTGTGGTG-3′ | 8.7±1.5 |
| TG20 | 5′-TGGTGTGTGTGGGTGTGGTG-3′ | 3.5±1.6 |
| TG25 | 5′-GGGTGTGGTGTGTGTGGGTGTGGTG-3′ | 1.3±0.6 |
| TG30 | 5′-TGTGTGGGTGTGGTGTGTGTGGGTGTGGTG-3′ | 1.2±0.1 |
| TG35 | 5′-TGGTGTGTGTGGGTGTGGTGTGTGTGGGTGTGGTG-3′ | 1.0±0.2 |
| TG40 | 5′-GGGTGTGGTGTGTGTGGGTGTGGTGTGTGTGGGTGTGGTG-3′ | 0.6±0.3 |