Analysis of Yeast Telomerase by Primer Extension Assays

i Summary/Abstract

Telomeres are specialized nucleoprotein structures located at eukaryotic chromosomal termini, which are required for chromosome stability and are maintained by a reverse transcriptase named telomerase. Budding yeast has served as an extremely useful model system for analyzing telomere maintenance because the organism offers a wide range of genetic and biochemical tools. Several milestones in telomerase research were reached through investigation of the yeast system. For example, the consequence of telomerase loss was first characterized in the budding yeast Saccharomyces cerevisiae (1). The catalytic component of telomerase (telomerase reverse transcriptase; TERT) was likewise initially cloned from this organism (2). Moreover, much of the current understanding of the structure and function of the telomerase complex was derived from yeast studies (3). In this chapter, we discuss one of the most useful tools for investigating yeast telomerase mechanisms and regulation: the primer extension assay. This assay can be used to examine the overall activity as well as the processivity of telomerase, which represents a unique aspect of telomerase enzymology (4,5). It can also be employed to analyze the mechanisms of telomerase regulatory proteins (6,7).

1. Introduction

Telomerase is a ribonucleoprotein required for the maintenance of chromosomal terminal repeats in most organisms (3,8). The reverse transcriptase protein subunit of telomerase (telomerase reverse transcriptase, TERT) catalyzes the addition of nucleotides onto the ends of telomeres in cooperation with an RNA subunit that provides the template (telomerase RNA, TER). The activity of telomerase was first characterized for the enzyme from Tetrahymena thermophila using a primer extension assay (9,10). The assay was based on the ability of telomerase to extend telomere-like oligodeoxynucleotides in the presence of dTTP and dGTP (the nucleotides present in the Tetrahymena telomere repeat). This assay was subsequently adapted to studies of telomerase in many organisms ranging from yeast (11) to humans (12). However, because of the low levels of telomerase in most cells, activity often cannot be detected in whole cell extracts using this assay. To overcome this difficulty, the TRAP (telomeric repeat amplification protocol) assay, which relies on PCR to amplify the initial telomerase-mediated products, was developed (13). Nevertheless, the TRAP assay cannot be used for detecting telomerases in yeast because these enzymes are generally non-processive (14), making it difficult to design appropriate PCR primers for amplifying the initial extension products. In addition, the telomere repeats in some yeast are degenerate. Telomerases from these organisms are thus expected to yield heterogeneous products, again making it difficult to design suitable primers for amplification (15). For these reasons, the study of yeast telomerase has continued to rely on the primer extension assay.

Because of the low levels of telomerase in yeast (16), a purification step following the preparation of whole cell extracts is necessary before the activity can be detected. DEAE anion-exchange chromatography was the first method developed for enriching yeast telomerase and for removing inhibitors (11,17). Subsequently, following the identification of telomerase protein genes, it became possible to fuse affinity tags to these proteins and employ affinity resins to purify telomerase from cell extracts (18,19). The DEAE and affinity purification methods, as well as the primer extension assay, have been applied successfully to the telomerase from S. cerevisiae (11,18), K. lactis (20), C. albicans (21,22), and S. pombe (17,23). In this chapter, we describe in detail our standard method for purifying yeast telomerase and analyzing its primer extension activity. We also outline our method for quantifying telomerase processivity, which allows us to determine the propensity of the enzyme to add the next nucleotide after each extension step. With suitable modifications, these methods can probably be adapted to the investigation of hitherto uncharacterized telomerases from other yeast.

2. Materials

2.1. Preparation of Yeast Extracts

1. YPD medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (v/v) glucose (see Note 1).

2. TMG buffer: 10 mM Tris⋅HCl (pH 8.0), 1.2 mM magnesium chloride, 0.1 mM EDTA, 0.1 mM EGTA, 10% (v/v) glycerol, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, 1 mM PMSF, 2 mM benzamidine (see Note 2).

2.2. Isolation of Telomerase from Cell Extracts

1. TMG(n): TMG buffer with n millimolar concentration of sodium acetate.

2. DEAE-agarose (BioRad).

3. Immunoglobulin G (IgG)-Sepharose beads (GE healthcare).

4. Glycine buffer: 0.1 M glycine (pH 2.5).

2.3. Primer Extension Assay

1. Reaction buffer (3X): 150 mM Tris⋅HCl (pH 8.0), 3 mM magnesium chloride, 3 mM spermidine, 3mM DTT. Store at −20°C.

2. RNase solution: 10 mM Tris⋅HCl (pH 8.0), 20 mM EDTA (pH 8.0), 0.1 mg/mL RNase A.

3. Proteinase solution: 10 mM Tris⋅HCl (pH 8.0), 0.5% (v/v) SDS, 0.3 mg/mL proteinase K.

4. Denaturing polyacrylamide gel: 15% acrylamide/bisacrylamide solution (20:1), 7M urea, 1X TBE.

5. Gel loading solution: 80% (w/v) formamide, 0.1X TBE, 0.03% (w/v) xylene cyanol FF, 0.03% (w/v) bromophenol blue.

2.4. Quantitation of Telomerase RNA and Protein Levels

2.4.1. RT-PCR analysis of telomerase RNA

1. RNA preparation buffer: TMG (300), 0.5% (v/v) SDS, 200 μg/mL proteinase K, 30 μg/mL tRNA.

2. PCI: Phenol/chloroform/isoamyl alcohol (25:24:1).

3. RT-PCR 2X master mix (USB). Store at −20°C.

2.4.2. Western analysis of tagged telomerase protein components

1. 10% SDS-PAGE gel.

2. Loading solution (4X): 0.2 M Tris (pH 6.8), 7% (v/v) SDS, 1.426 M ß-mercaptoethanol.

3. Running buffer: 25 mM Tris, 192 mM glycine, 0.1% (v/v) SDS.

4. Transfer buffer: 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, 0.0075% (v/v) SDS.

5. Tris buffered saline (TBS): 20 mM Tris (ph 7.5), 200 mM sodium chloride.

6. TBST: TBS with 0.05% (v/v) Tween-20.

7. Blocking buffer: 2% (w/v) fraction V bovine serum albumen (BSA) in TBST.

8. Primary antibody solution: anti-protein A antibodies (Sigma Cat. No. P3775) (1:50,000-fold dilution) in TBST with 0.5% (w/v) BSA.

9. Secondary antibody: anti-rabbit IgG conjugated with alkaline phosphatase (Sigma Cat. No. A9919).

10. BCIP/NBT color development substrate (Promega): 10 mL alkaline phosphatase buffer, 66 μL NBT, and 33 μL BCIP.

3. Methods

The primer extension assay is based on the ability of telomerase to add nucleotides to DNA substrates. Using a specific primer whose 3’ end is complementary to telomerase RNA template, the telomerase reverse transcriptase can incorporate radiolabeled nucleotide tripohsphates to the 3’ end of the primer, which can then be detected by denaturing gel electrophoresis and autoradiography/PhosphorImager. However, due to the low levels of telomerase in yeast and the presence other activities and inhibitors, telomerase activity generally cannot be detected in whole cell extracts. Therefore, enrichment of telomerase from whole cell extracts is a crucial first step in the analysis of enzyme activity.

Two methods have been developed for the enrichment of yeast telomerase. The first method is DEAD anion-exchange chromatography. Because almost all yeast telomerases carry a large RNA subunit (generally > 1,000 nt) (24), the RNPs typically bind tightly to the DEAE resin and are released only with high salt. Active telomerase can thus be substantially purified by passage through a DEAE-agarose column. The second method is isolation of tagged telomerase through the high affinity interaction between protein A and IgG. The protein A tag [either alone or as part of the Tandem Affinity Purification (TAP) tag] can be fused to a telomerase component and the resulting RNP isolated from cell extracts using IgG-Sepharose. Notably, even though other protocols for yeast telomerase purification have been developed, they are all based on the principle of anion-exchange chromatography (e.g., mono-Q) or affinity isolation [e.g., streptavidin binding protein (SBP) tag and calmodulin binding protein (CBP) tag] (Hsu & Lue, unpublished, 25).

The processivity of telomerase has been quantified using a variety of methods. For telomerases that are capable of adding multiple repeat units, relative processivity is often assessed by plotting the levels of complete repeat addition products against the repeat number on a log scale (26). This procedure is not useful for most yeast telomerases, which are generally incapable of multiple repeat addition. Instead, we developed a protocol to calculate the propensity of telomerase to add the next nucleotide after each extension step (27). This protocol was based principles that had been used in the analysis of HIV-1 RT processivity (28).

3.1. Preparation of Yeast Extracts

1. Typically, 3 liters of S. cerevisiae or C. albicans cell cultures are grown in YPD or YPD + uri to an OD of 1.5–2.0.

2. Cells are harvested, washed with ice-cold water once, and resuspended with equal volume of TMG(0) buffer.

3. The cell suspension is transferred to a 50-mL round bottom centrifuge tube containing glass beads filled to about 1/5 of the total height of the tube, and lysed by vigorous vortexing. 120 s of vortexing at maximum speed is alternated with 180 s of cooling in ice-water bath for a total of 100 min (see Note 3).

4. Cell lysates are transferred to 30-mL PC Oak Ridge centrifuge tubes and spun in a T-865 rotor (Sorvall) at 32,000 r.p.m. (100,000 g) for 1 h.

5. The clear supernatant from each tube is collected, flash frozen in liquid nitrogen and stored at −80°C.

3.2. Isolation of Telomerase by DEAE Chromatography or IgG-Sepharose Pull Down

3.2.1. DEAE chromatography

1. Three mL of DEAE-agarose beads are equilibrated in TMG(0) and packed into a 1 cm X 10 cm Econo-column (BioRad).

2. The cell extract (15 mL) is loaded onto the column at a flow rate of 10 mL/h. The column is then washed successively with 15 mL of TMG(200) and 15 mL of TMG(400).

3. Subsequently, the bulk of telomerase activity is eluted with 15 mL of TMG(900). Typically 1-mL fractions are collected.

4. Peak protein fractions as determined by Bradford assays are pooled, flash frozen in liquid nitrogen and stored at −80°C.

3.2.2. IgG-Sepharose pull down

1. Extracts prepared from S. cerevisiae or C. albicans strains containing protein A or TAP-tagged telomerase subunits are needed for this isolation procedure (19,22). 45 μL of IgG-Sepharose beads are first equilibrated in glycine buffer and washed with TMG(0) until the pH is greater than 7.0. The beads are treated with 100 μg tRNA at 4°C for 30 min to block nonspecific binding.

2. Whole cell extracts (9.6 mg) are mixed with the IgG-Sepharose beads in 1.2 mL TMG(500) plus 0.05% Tween-20 and subjected to gentle rotation at 4°C for 2 h.

3. The beads are then washed five times in TMG(800) and twice in TMG(0) (see Note 4).

4. The beads are further divided into three equal aliquots. One aliquot is subjected to primer extension assay. The other aliquots are used for RT-PCR and Western blot analysis in order to quantify the levels of telomerase RNA and protein respectively.

3.3. Primer Extension Assays

The primer extension assay is preformed using a short oligonucleotide as the primer and a radiolabeled nucleotide triphosphate that can be incorporated by the telomerase RNP based on the RNA template sequence and the alignment between the primer and template (see Note 5).

1. The reaction mixture is prepared by adding sequentially the following components to a microcentrifuge tube: 10 μL 3X reaction buffer, 1 μg of primer, 15 μL of column fractions, and 30 μCi [α−³²P]dNTP (NEN, 3000 Ci/mmole) (The specific label used depends on the primer).

2. The reaction is allowed to proceed at room temperature for 30 min, and stopped by adding 80 μL of the RNase solution. Following incubation at room temperature for 10 min, the mixtures are further digested with 100 μL of the proteinase solution at 37°C for 20 min.

3. The nucleic acids in the reactions are precipitated with the addition of 100 μL 7.5 M ammonium acetate, 10 μg tRNA, and 750 μL absolute ethanol (see Note 6).

4. The pellets are resuspended with 10 μL of loading solution and analyzed on a 15% denaturing polyacrylamide gel. The ³²P-labeled products are visualized by using a PhosphorImager system and quantified using ImageQuant software (see Note 7).

3.4. Quantitation of Telomerase RNA and Protein Levels

3.4.1. RT-PCR analysis of telomerase RNA

1. Following incubation with extracts containing tagged telomerase, 15 μL of the IgG-Sepharose beads are treated with 330 μL of the RNA preparation buffer at room temperature for 30 min and at 37°C for another 30 min.

2. The mixture is extracted with PCI and the RNA precipitated by ethanol, and resuspended with 20 μL of nuclease-free water (see Note 8).

3. The level of telomerase RNA is then assayed by semi-quantitative RT-PCR. A 20 μL reaction is carried out with 2 μL isolated RNA, 10 μL 2X RT-PCR master mix, and 1 μM primers designed to amplify a specific telomerase RNA fragment.

3.4.2. Western analysis of the tagged telomerase protein component

1. Electrophoresis and the transfer of protein to nitrocellulose membrane are performed according to standard procedures.

2. For detection of a protein A or TAP-tagged telomerase subunit, the membrane is first incubated with α-protein A antibody (Sigma Cat. No. P3775) in primary antibody solution (1:50,000-fold dilution) at room temperature for overnight, and then washed three times in TBST buffer.

3. The secondary antibody (α-rabbit IgG adsorbed with human IgG; Sigma Cat No. A9919) (see Note 9) is added to the membrane at 1:5000 dilution in TBST and the incubation continued for 40 min at room temperature. The membrane is then washed three times in TBST and twice in TBS buffer.

4. The BCIP/NBT color development substrate solution (Promega) is freshly prepared and added to the membrane until sufficient signals developed on the membrane.

3.5. Analysis of telomerase processivity

1. The reaction products from a telomerase primer extension assay are identified from a PhosphorImager scan. The relative signals of individual products are quantified using the ImageQuant software.

2. The signal for each band is normalized to the amount of transcript by dividing against the number of labeled residues. For example, if the sequence TGTGGTG was added to the primer and the labeled nucleotide in the reaction was dTTP (Fig. 2), then the +2 to +5 product signals should be divided by 2, and the +6 and +7 product signals divided by 3.

3. Processivity at each position is calculated using the following formula.

Fig. 2.

Multiple nucleotide addition using S. cerevisiae telomerase.

(Top) The RNA template for S. cerevisiae telomerase, a primer substrate, and the predicted sequence addition to the primer are illustrated.

(Bottom) Primer extension assays were performed using wild type telomerase (WT) and a C-terminal truncation mutant (ΔCT) with reduced processivity. Included in the reactions were 0.2 μM [α−32P]dTTP and 50 μM dGTP. The positions of the various extension products are indicated by horizontal bars to the right of the panel. Applying the method described in section 3.5, we found that the processivity of the wild type and mutant telomerase at the primer+2 position are ~0.5 and 0.2, respectively.

$$\text{Processivity}{\text{P}}_{\text{i}}\text{=}\sum _{\text{j=i+1}}^{\text{N}}{\text{(T}}_{\text{j}}\text{)/}\sum _{\text{j=i}}^{\text{N}}{\text{(T}}_{\text{j}}\text{),}$$

where T_i denotes the amount of transcripts calculated for the primer+i position and N is the highest number such that a visible signal can be discerned in the PhosphorImager file for the primer+N product.

Fig. 1.

Single nucleotide addition using C. albicans telomerase.

(A) C. albicans telomerase is expected to add one nucleotide to the indicated primer in the presence of dTTP, based on the alignment between the primer and telomerase RNA template.

(B) Candia extracts and DEAE fractions were prepared from the wild type (BWP17), a telomerase RNA knockout mutant (ter1-ΔΔ), and a reconstituted strain (ter1-ΔΔ/TER1), and subjected to primer extension assays in the presence of the indicated primer and ³²P-labeled dTTP. Deletion of the telomerase RNA resulted in complete loss of telomerase activity. Fractions were also pre-treated with RNase A before the assays to demonstrate the dependence of the activity on RNA. A 46-mer labeled oligonucleotide was added to each sample before ethanol precipitation as a control for sample recovery (LC).

(C) DEAE fractions prepared from BWP17 and a strain with TAP-tagged telomerase were subjected to IgG-Sepharose pull down. The pull down beads were analyzed directly by primer extension assays to measure telomerase activity. As predicted, higher levels of RNase-sensitive primer extension products were detected in the beads pre-treated with the extract containing TAP-tagged telomerase.