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. 2008 Nov 29;37(2):354–367. doi: 10.1093/nar/gkn830

Figure 1.

Figure 1.

The DBD of Cdc13p inhibits yeast telomerase. (a) Schematic of Cdc13p. The DBD resides near the C-terminus of the protein (amino acids 497–694). Purified Cdc13(DBD) (24.7 kDa with the His6 tag) is shown on a Coomassie-stained gel (right). E252, residue required for binding Est1p to recruit telomerase (32). Y522, amino acid important for high-affinity DNA-binding (27). (b) DBD inhibits telomerase in vitro. Cdc13(DBD) was incubated with the 15-nt primer TP1 and then telomerase was added along with deoxynucleotides A, C, T and [α-32P]-dGTP. M, reconstituted mini-telomerase. YWT, telomerase from extracts of yeast overexpressing ProA-Est2p and 1157-nt TLC1. The +1 and +7 telomerase extension products are indicated with filled and open triangles, respectively. Primer TP1 aligns with telomerase RNA as shown; arrow denotes template-directed DNA synthesis up to the boundary element (dotted line). Primer concentration was 0.3 µM. Reactions contained 0.6 µM Cdc13(DBD) (+) or its storage buffer (−). Gray lines show instances of GxGT, the essential 5′ binding portion of the Cdc13(DBD) binding consensus. (c) Cdc13(DBD) inhibits telomerase when present at a concentration similar to TP1 primer. Reaction conditions were essentially the same as when using reconstituted mini-telomerase in b. Water and Cdc13(DBD) storage buffer controls are shown in lanes 1 and 2, respectively. Lanes 3–9, increasing amounts of Cdc13(DBD) were incubated with DNA prior to adding telomerase. Lane 10, boiled (denatured) Cdc13(DBD). Lane 11, telomerase incubated for 10 min at 37°C with RNase A to degrade Mini-T RNA. Lane 12, 0.9 µM Cdc13(DBD) added after primer and telomerase were incubated on ice for 10 min. Lane 13, The first nucleotide encoded by the RNA template, dTTP (b), was omitted. LC, loading and recovery control: TP1 primer labeled with 32P at its 5′-end, which increases electrophoretic migration from increased negative charge. (d) DNA-binding-defective point mutant of Cdc13(DBD) does not inhibit telomerase in vitro. Standard deviation from three independent experiments is shown. Experiments were performed as in b and quantitated as described in Materials and Methods section. Primer concentration was 0.3 µM.