The p23 molecular chaperone promotes functional telomerase complexes through DNA dissociation

Abstract

Telomeres are the composite of short DNA element tandem arrays and heterotypic protein components that protect and maintain chromosomal termini. As proper telomere maintenance requires a multitude of DNA extension events, it is important to understand the factors that modulate telomerase DNA association. Here, we show that the endogenous levels of the yeast p23 molecular chaperone Sba1p are required for telomere length maintenance and that Sba1p can modulate telomerase DNA binding and extension activities in vitro. Notably, telomere occupancy by telomerase and the extension rate of a shortened telomere fluctuated with changing Sba1 protein levels in vivo. In addition, we found that Sba1p displayed a cell cycle-dependent telomere interaction that paralleled telomerase binding; telomere association by Sba1p depended on its inherent chaperone activity. Taken together, our results support a model in which Sba1p modulates telomerase DNA binding activity for optimal function in vitro and in vivo.

Fig. 1.

Full-length Sba1p modulates telomerase DNA extension activity in vitro. Telomerase-dependent extension of an immobilized seven-base single-stranded 3′ overhang DNA substrate was examined. The DNA extension activities of WT, sba1Δ, or sba1Δ MonoQ fractions supplemented with varying levels of Sba1p or Sba1pΔ50 (0.1, 0.2, 0.4, 0.8, 1.6, or 3.2 μM) were tested as indicated. To serve as a loading control a polynucleotide kinase end-labeled 27-base oligonucleotide was added before the precipitation of all of the telomerase extension products. The position of +1 was determined by using terminal transferase-labeled DNA and α-ddATP (lanes 1 and 10).

Fig. 2.

Sba1p promotes telomerase DNA substrate exchange in vitro. (A) The ability of Sba1p or Sba1pΔ50 to promote telomerase transfer between DNA substrates was determined by challenging a bound 7-base substrate with increasing levels of a 15-base 3′ overhang telomeric DNA substrate. The position of +1 for the 7-base and 15-base substrates was determined by using terminal transferase end-labeled DNAs (lanes 1 and 2, respectively). As positive controls, extension reactions were performed by using sba1Δ MonoQ extract on the 7-base substrate, 15-base substrate, or the mixture of the 7- and 15-base substrates (2 pmol) (lanes 3, 4, and 5, respectively). The ability of either Sba1pΔ50 or Sba1p to promote telomerase transfer was examined by adding the Sba1 proteins (400 nM final) to prebound 7-base DNA (2 pmol, gray bar) followed by varying amounts of the 15-base telomeric substrate (2, 4, 6, 8, 10, 15, or 20 pmol). (B) Association of Sba1p and Hsp82p with DNA-bound telomerase was determined. WT MonoQ telomerase extract was incubated with immobilized DNA terminating with a G-rich single stranded seven-base 3′ overhang (G) in the absence or presence of RNase A or with DNA terminating with a C-rich sequence (C) as indicated. The presence of Sba1p or Hsp82p in the DNA bound (B) or free supernatant (F) fraction was detected by immunoblotting; as a control the total chaperones levels in the MonoQ extracts were determined (T).

Fig. 3.

Telomerase DNA binding is Sba1p-dependent in vitro. (A) Fluorescence anisotropy was used to detect DNA binding to a fluorescein-labeled telomeric oligonucleotide. The used MonoQ fractions were from WT (empty bars), sba1Δ (filled bars), est2Δ (striped bar), or tlc1Δ (hatched bar) yeast. The WT and sba1Δ reactions contain equivalent levels of TLC1 RNA and total protein; the tlc1Δ and est2Δ fractions contained equivalent total protein. (B) To measure the binding affinity between telomerase and a telomeric DNA WT (○) or sba1Δ (●) fractions were titrated into a reaction containing a fluorescein-labeled oligonucleotide (12.5 nM). (C) Purified recombinant full-length Sba1p or carboxyl-terminal truncations (Δ50 or Δ84) were titrated (0.1, 0.2, 0.4, 0.8, 1.6, or 3.2 μM) into anisotropy reactions containing sba1Δ MonoQ extract; for comparison, the binding activities of unsupplemented telomerase fractions with equivalent TLC1 and protein levels from WT (open bar) and sba1Δ (filled bar) yeast are shown.

Fig. 4.

In vivo levels of Sba1p affect telomere occupancy by telomerase. (A) Telomere association by telomerase was gauged by using the ChIP assay. The relative levels of telomerase-telomere binding were determined in the WT, WT overexpressing Sba1p (WT + Sba1p), and sba1Δ yeast. Telomerase residency at a population of telomeres was determined by using oligonucleotides selected for a subtelomeric Y′ element found at 11 chromosomal termini (Y′ elements) or at a single telomere using primers specific for a subtelomeric region of chromosome XV (Chr. XV); all values were normalized to the signal from an internal nontelomeric DNA. (B) The ability of the carboxyl-terminal Sba1p deletions to associate with telomeric DNA was addressed by using the ChIP assay and asynchronous sba1Δ yeast expressing the indicated proteins. All data represent average values (mean ± SD) from three independent assays.

Fig. 5.

Telomere length is Sba1p-dependent. (A) Genomic DNA was isolated from WT or yeast passaged one, three, or five times after SBA1 disruption. The asterisk indicates the approximate position of the Y′ subtelomeric fragment. As a migration control, the position of a nontelomeric DNA (control) was visualized by using a probe specific to an internal fragment of chromosome IV (15). (B) The effect of Sba1p overexpression on telomere length was examined by Southern blot analysis in WT yeast constitutively expressing Sba1p (GPD promoter) for the indicated passages. The asterisk indicates the approximate position of the Y′ subtelomeric fragment.

Fig. 6.

Disruption of SBA1 alters telomere extension rates in vivo. The elongation rates of an Flp-FRT-directed shortened telomere were monitored in WT and sba1Δ yeast (17, 18). Briefly, genomic DNA was isolated from cells growing exponentially in raffinose media (P), DNA was recovered after the FRT-encompassed telomere tract was removed by shifting the cells to galactose media (0), yeast were switched to glucose media, and genomic DNA samples were recovered after 3, 6, and 10 generations as indicated. Representative Southern blot results are shown (A), and the quantified data from five independent experiments are presented (B). In A, the asterisk indicates the position of the telomeric restriction fragment of the left arm of chromosome VII, and the arrow marks the residual uninduced nontelomeric URA3–ADH4.