Functional Reconstitution of Human Telomerase Expressed inSaccharomyces cerevisiae

François Bachand,Chantal Autexier

doi:10.1074/jbc.274.53.38027

François Bachand, Chantal Autexier

Open Access

https://doi.org/10.1074/jbc.274.53.38027

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Abstract

Telomerase is a ribonucleoprotein enzyme complex that adds DNA repeats at the ends of chromosomes. In an effort to establish an in vivo heterologous expression system for active human telomerase, we expressed human telomerase reverse transcriptase (hTERT) in Saccharomyces cerevisiae and affinity-purified the protein as a fusion with glutathione S-transferase (GST). Addition of the GST moiety to the N terminus of hTERT did not interfere with telomerase activity when GST-hTERT was expressed in rabbit reticulocyte lysate (RRL) in the presence of the human telomerase RNA (hTR). Active human telomerase was immunoprecipitated from yeast lysates that co-expressed GST-hTERT and hTR. In addition, telomerase activity could be reconstituted in vitro by the addition of hTR to GST-hTERT that was immunoprecipitated from either RRL or S. cerevisiae lysates. The expression and reconstitution of human telomerase activity in yeast will provide powerful biochemical and genetic tools to study the various components required for the assembly and function of this enzyme.

Highlights

Telomerase is a ribonucleoprotein enzyme complex that adds DNA repeats at the ends of chromosomes
Reticulocyte Lysates—Since the cloning of the gene encoding the catalytic subunit of the human telomerase enzyme, reconstitution of enzymatic activity has been achieved by expressing human telomerase reverse transcriptase (hTERT) in the presence of human telomerase RNA (hTR) in rabbit reticulocyte lysates
To first determine whether the glutathione S-transferase (GST) moiety fused to the N terminus of hTERT interfered with telomerase function, the entire GSThTERT coding sequence from pEGKT-hTERT was cloned into a T7 expression vector

Summary

MATERIALS AND METHODS

Yeast Strain—Yeast strain cIABYS86 (MAT␣, leu, Ura, his, pra, prb, prc, cps) was used as a host strain for hTERT protein expression and as a source of yeast cell lysate [20]. Construction of Plasmids—Clone 712562, containing bases 1624 – 3399 of the hTERT coding sequence and 3Ј downstream sequences, was obtained from the IMAGE (Integrated Molecular Analysis of Genomes and their Expression) Consortium through Genome System Inc. and was used for construction of an hTERT mutation using the Quick Change site-directed mutagenesis kit from Stratagene [21]. The oligonucleotides 5Ј-CTCCTGCGTTTGGTTAACGATTTCTTGTTG-Ј3 and 5ЈCAACAAGAAATCGTTAACCAAACGCAGGAG-Ј3 were used to generate the D868N mutation and to create a HincII restriction site. This new construct (phTRTDNC) was sequenced using an Applied Biosys-

Reconstitution of Human Telomerase in Yeast

RESULTS AND DISCUSSION

François Bachand and Chantal Autexier