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Pif1p is the prototype member of a family of helicases that is highly conserved from yeast to humans. In yeast, Pif1p is involved in many aspects of the preservation of genome stability. In particular, Pif1p is involved in the maintenance of mitochondrial DNA and in the direct inhibition of telomerase at telomeres and double-stranded breaks. Here we describe methods to purify Pif1p and study in vitro its enzymatic properties and functional interaction with telomerase.
This chapter focuses on the characterization of the Saccharomyces cerevisiae Pif1p helicase. Pif1p is the prototype member of the PIF1 family of helicases, which is conserved from yeast to human (1, 2). Two isoforms of the enzyme are expressed in yeast, owing to alternative usage of two start codons from the same mRNA. Translation from the first start codon leads to the synthesis of a mitochondria-directed isoform, while translation from the second AUG codon leads to the synthesis of the nuclear isoform (3). Genetic studies have shown that the nuclear form of Pif1p plays an important role in counteracting the activity of telomerase, the specialized reverse transcriptase that elongates the end of eukaryotic chromosomes. Through this activity, Pif1p prevents gross chromosomal rearrangements that are due to the addition of telomerase-mediated de novo telomere addition at double strand breaks (4). In vivo and in vitro data suggest that this action is achieved through a direct interaction between Pif1p and telomerase (5, 6). Using oligonucleotide-based radiometric assays, Pif1p has been shown to unwind preferentially RNA–DNA hybrids over DNA substrates (7). This preference suggests that Pif1p inhibits telomerase by unwinding the RNA–DNA substrate formed by the telomerase RNA, TLC1, and the telomeric DNA end. Importantly, the interaction between Pif1p and telomerase is conserved in evolution, since Pif1p has been shown to interact with mouse and human telomerase (8–10). This chapter focuses on in vitro methods to purify recombinant yeast Pif1p and to characterize its activity by classical oligonucletide substrate-based radiometric assays. We also describe methods to study in vitro its functional interaction with yeast telomerase.
As telomerase activity is RNase-sensitive, all reagents, solutions, and equipment must be handled in an RNase-free environment (see Note 2).
All buffers and material should be RNase-free (see Note 2).
All steps should be performed at 4°C.
This protocol assumes the use of a yeast strain containing a 2-μm plasmid allowing the expression of EST2 and TLC1 ORFs under the control of GAL promoters. For example, we use a pESC plasmid (stratagene) containing the EST2 and TLC1 genes placed under the control of he GAL1 and GAL10 promoters, respectively. The yeast strain used for expression was the protease deficient strain BCY123 est1Δ type II survivor strain (described in (5)).
Since telomerase is a ribonucleoprotein, all steps of the purification should be performed at 4°C and in an RNase-free environment (see Note 2). With these considerations in mind, telomerase activity can be fractionated from yeast cells using the following method, adapted from (8).
Detailed methodological reviews describing radiometric assays and their application to helicase mechanistic studies exist (9–11). A favorite general reference that details synthesis of radiolabeled nucleic acids substrates and gel-based analysis of reaction products can be found in this book series (11). However, since optimal assay conditions vary among different helicases, we will briefly describe the assay system that we developed to analyze Pif1p helicase enzymatic properties using radiometric assays (5, 7).
Yeast telomerase activity can be monitored with an oligonucleotide extension assay, using an oligonucleotide whose sequence mimicks the end of a yeast telomere. Similarly to results from other labs, we find that telomerase extends efficiently short oligonucleotides (around 15 nucleotides in size) but displays poor polymerization efficiency on longer oligonucleotides (above 30 nucleotides). It is also advantageous that the 3′ end of the telomeric primer has with a unique sequence complementary to TLC1 RNA. We had the best success with the following sequence: TEL15: 5′-TGTGGTGTGTGTGGG-3′, which anneals on TLC1 at the template position 475C (8).
The telomerase reaction products are best resolved on a polyacrylamide-urea sequencing gel. We had the best results with 16% polyacryamide gels run on a STS45 IBI sequencing gel unit, but the method can be adapted for other equipment.
This work was supported by grants from the National Insitutes of Health to VAZ.
1Other type of IMAC resins can be adapted to fit this protocol, although binding, wash, and elution buffer components (pH, salt, and imidazole concentration) should be optimized for the specific resin.
2To prevent RNase contamination, wear latex gloves at all times and change them regularly. All glassware should be DEPC-treated. Work surfaces should be cleaned with an RNase inhibitor solution, e.g., RNaseZAP (Ambion). All buffers should be DEPC-treated and autoclaved prior to use. Tris-containing buffers can not be DEPC-treated as DEPC will react with primary amines. Therefore, RNase-free Tris and DEPC-treated stock solutions should be used to make these buffers.
3The presence of excess unlabeled oligonucleotide prevents reannealing of the unwound strand to its complementary strand.
4To prevent confusion between the loading control and the addition products, we use a 60-mer single-stranded oligonucleotide of random sequence as a loading control.
5Although the pET system is convenient and has become a widely used standard, other inducible systems for heterologous expression in bacteria can be used.
6Given the low level of Pif1p overexpression, we usually induce large volumes (5–10 L). The volume of LB in each flask should be no more than a third of the flask volume. For example, use a 6 L flask for a 2 L culture.
7Induction at lower temperatures greatly increases Pif1p solubility. The optimal induction temperature and time will depend on the expression system and should be determined experimentally.
8To prevent heating of the supernatant, sonication should be paused every minute for 30 s to cool the probe. One round of 100 pulses is usually enough to sonicate 100 mL of supernatant.
9Optimal column volume will depend on the culture volume and on the level of Pif1p expression in the system used. We find that 5-mL columns give reproducible yields and quality in Pif1p purified from 10-L cultures.
10As a rule of thumb, 5 column volumes of washing buffer should be enough to remove proteins interacting non-specifically with the Talon resin. If another resin is used for affinity chromatography, optimal volumes for washing the column before elution should be determined experimentally.
11We find that the enzyme is stable at −80°C for a year. It is not recommended to freeze/thaw the enzyme as it makes the helicase activity decrease rapidly. If an aliquot is thawed, it can be kept at −20°C for 2–3 months without a significant drop in activity. However, we have observed precipitation at −20°C. Therefore, aliquots kept at −20°C should be checked for precipitation and protein concentration should be recalculated before each use.
12Since liquid N2 can cause serious burns, safety glasses and protective gloves should be worn at all times during this procedure.
13Frozen lysates can take a long time to thaw (> 30 min for a 20-mL lysate). Thawing can be initiated by warming the lysate between the hands and then on a rotating wheel at 4°C until thawing is complete.
14Similarly to what has been reported for other helicases, we find that the Pif1p concentration necessary to observed efficient unwinding exceeds several fold the concentration of the substrate. We routinely perform Pif1p helicase assays using 100 nM enzyme and 1 nM substrate.
15For a control reaction, set up a reaction using Pif1p storage buffer instead of the helicase. This is the “no enzyme” control. Another control is the heat denatured substrate, which is achieved by heating the reaction mix containing the labeled substrate and no enzyme at 95°C for 2 min.
16The final polyacrylamide percentage of the gel and the optimal electrophoresis time depends on the sizes of the intact nucleic acids substrate and the unwound radiolabeled product. These conditions should be optimized depending on the size of the substrate used.
17Use of DEAE paper is recommended when the gel is going to be dried before quantification, since, unlike 3 MM paper, it will bind and retain small nucleic acids.
18Use a 5-mm well-dented comb, not a sequencing shark-tooth comb.
19To visualize the displacement of telomerase, a variation of this protocol can be performed. During the course of the reaction, an excess of a “chasing” telomeric oligonucleotide of different size is incorporated in the reaction. For example, we use a 30-mer oligonucleotide containing the TEL15 sequence extended by 15 random nucleotides from its 5′ end. This oligonucleotide is utilized more efficiently than a 30-mer oligonucleotide containing only telomeric sequence, as discussed in Section 3.5.1. The reaction is started as described, but a 10-fold excess chasing oligonucleotide is added after 15 min into the reaction. Since yeast telomerase stays associated with its product (12), telomerase will only elongate the chasing oligonucleotide if telomerase is released from its elongation product.
20The effect of Pif1p on telomerase activity can be calculated in term of telomerase nucleotide processivity, defined as the probability P with which telomerase adds more than one nucleotide without dissociating from its product. Telomerase nucleotide processivity is then defined for each product of size +n by P+n = (Σ(x > n) Ix)/T. This calculation assumes that in the presence of Pif1p, an already elongated product is not re-elongated by secondary association with telomerase, provided that TEL15 primer is present in large excess compared to telomerase.
21Alternatively, quantification of telomerase core enzyme in each fraction can be achieved by detection of the TLC1 RNA, either by qRT-PCR or Northern blotting.