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. Y^WT, 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.

Full-length Cdc13p inhibits telomerase. (a) Coomassie-stained SDS–PAGE gel of purified Cdc13p (108 kDa with His₆ tag). (b) Cdc13p inhibits reconstituted yeast telomerase activity in a concentration dependent manner on TP1 primer. See Figure 1b for alignment of primer with telomerase RNA. Water, Cdc13p storage buffer and boiled Cdc13p negative controls are shown. Relative activity is listed below lanes. (c) Cdc13p (C) and its DBD (D) inhibit wild-type telomerase (Y^WT) purified from yeast extracts similarly to miniaturized reconstituted telomerase (M). Experimental conditions were the same as in Figure 1b.

Design of a single GxGT-containing mutant sequence, TEL*, that still permits binding by Cdc13(DBD) and full-length Cdc13p. (a) Like TEL, mutant TEL* binds to Cdc13(DBD), although with reduced affinity (see also Table 1). Left, schematic of mutations (bold) engineered into the canonical 11-nt Cdc13(DBD) binding site, TEL, to generate TEL*, which has only one remaining GxGT consensus site (indicated by gray bars). Right, representative membranes and quantified data from dot blot filter-binding experiments to determine the affinity of Cdc13(DBD) for the TEL and TEL* sequences. Note that filter binding was performed under high-salt (750 mM KCl) conditions (28) to reduce binding of Cdc13(DBD) to TEL, which is otherwise so tight (K_d = 3 pM at 75 mM monovalent salt) that it is technically difficult to measure accurately. (b) Like DBD, full-length Cdc13p also binds TEL* but with reduced affinity relative to TEL. A representative gel-shift using 5′-end labeled TEL* is shown (top). Cdc13p•TEL*, the protein–DNA complex in the nondenaturing gel; free probe is the unbound 5′-end labeled oligonucleotide. A graphical representation of the quantified gel shift signal is shown (bottom). All curves were fitted to a two-state binding model, as described in the Materials and Methods section.

SVP 3′ exonuclease assays show that Cdc13p and its DBD both bind specifically to the 5′ region of TEL*–GTGGG, but differ in binding site preference on longer oligonucleotides. (a) SVP digestion time course experiments show that Cdc13p DNA-binding protects the TEL region of a TEL–a₁₀ control and 11-nt TEL* sequence in TEL*–GTGGG. Time of incubation with SVP is indicated above. Correspondence of portions of the gel to relative position in the oligonucleotide sequence is indicated to the left: TEL or TEL* with tails of 10 adenosines or –GTGGG, a telomeric sequence complementary to a unique portion of the telomerase template. Totally digested represents the signal resulting from complete SVP digestion of the 5′-end labeled oligonucleotides. (b) Cdc13(DBD) and full length protein bind to TEL* when a GTGGG extension is appended, but Cdc13p selectively binds to a GTGTGTGGG extension. The molar ratios of DBD:DNA (lanes 3–9 and 18–24) are 0.5 : 1, 1 : 1, 1.5 : 1, 2 : 1, 3 : 1, 5 : 1, and 10 : 1. Molar ratios of Cdc13p : DNA (lanes 12–15 and 27–30) are 0.5 : 1, 1 : 1, 1.5 : 1, and 2 : 1.

A coordinated assay reveals that Cdc13p and its DBD inhibit telomerase similarly via distinct DNA interactions. (a) CoNuTe assay scheme used to generate data in b and c. (b) SVP assay shows that Cdc13(DBD) (D) preferentially protects TEL* even in the context of long 3′ telomeric appendages, while Cdc13p (C) protects the 3′ region of substrates with ⩾6-nt tails. Oligonucleotides tested are shown: TEL* region is boxed and vertical lines indicate complementarity to TLC1 RNA shown above, while gray arrows indicate the 7 TLC1-endoded nucleotides predicted to be added to each by telomerase (results for which are shown in c). SVP results are shown below. V-shaped arrowhead indicates completely undigested 5′-end labeled primer. TEL*, region of gel with SVP products 11 nucleotides or shorter. Arrowhead indicates full-length 5′-end labeled oligonucleotide. Totally digested, DNA not protected by binding to DBD or Cdc13p; serves as an indicator for fraction complex formation. Concentrations of Cdc13(DBD) were 0.15 µM, 0.3 µM and 0.6 µM and for Cdc13p were 0.3 µM, 0.6 µM and 1.2 µM. (c) Telomerase is consistently inhibited by the DBD and Cdc13p bound to TEL*-based oligonucleotides containing 2–9-nt appendages. Pre-labeled 5′-end-labeled primers, included in the reaction for the SVP experiment shown in (b), are indicated for the shortest primer and serves as a recovery and loading control for telomerase activity assays. The +1 and +7 telomerase products are indicated as in Figures 1 and 2 (gray arrows in b). See legend b for concentrations of DBD and Cdc13p.

‘Spacer’ tracts of deoxyadenosines separate the DBD- and Cdc13p-binding site (TEL) and a telomerase-extendible 3′-end, providing further insight into Cdc13p inhibitory mechanisms. (a) Cdc13p, like DBD, preferentially binds TEL, but it also loads towards the 3′-end at higher protein : DNA stoichiometry. SVP data are shown from CoNuTe assay [telomerase results from this CoNuTe experiment are shown in (b)]. Protein concentrations used here and in CoNuTe telomerase assay below are the same as in Figure 5. (b) Both Cdc13p and its DBD inhibit telomerase on TEL-A_n–TGTGGG oligonucleotides with 5, 8 or 11-nt spacers. See data in (a) for SVP results from this CoNuTe experiment, legend of Figure 5a for concentrations of DBD and Cdc13p. Primers a₁₁-a₁₁-TGTGGG, a₁₁-TGTGGG and a₅-TGTGGG serve as controls.

Models for the relationship between telomeric DNA binding and telomerase inhibition by Cdc13p. (a) In the absence of any telomere-binding protein, telomerase binds to its substrate and readily extends it via reverse transcription of its RNA template. (b) Complete occlusion model for inhibition of telomerase. The DNA-binding domain as well as full-length Cdc13p are capable of occluding telomerase access to the 3′-end of its substrate, even when additional 3′ nucleotides protrude beyond the Cdc13•DNA interaction interface. (c) Action at a distance inhibition. Although part of telomerase interaction with DNA is blocked by DBD or Cdc13p, some telomerase activity is achieved, presumably via interaction of the catalytic site and the very 3′-end. (d) Facilitated occlusion model. Cdc13p binds secondarily towards the 3′-end of the DNA primer in a concentration-dependent manner, leading to more potent inhibition. This is stepwise, effectively inhibiting first as indicated in c (action at a distance) and then, additionally, as shown in b (complete occlusion), when Cdc13p loads also on the 3′-end, facilitated by first loading at a more 5′ position.