Purification And Characterization Of Telomerase Core Enzyme Of Saccharomyces Cerevisiae

Telomerase is a specialized reverse transcriptase that elongates the single-stranded chromosome ends and oligonucleotides in vivo and in vitro. It is believed that telomerase catalytic subunit and RNA subunit constitute the core enzyme, which is essential for the telomerase activity in vitro. We co-overexpressed GST fused yeast telomerase catalytic subunit Est2p and yeast RNA subunit Tlc1 in Saccharomyces cerevisiae, and reconstituted the telomerase activity. The GST-Est2p/Tlc1 complex was partially purified by ammonium sulfate fraction and affinity purification methods, and the purified complex sample did not contain the endogenous other two subunits of telomerase holoenzyme, Est1p and Est3p, analyzed by mass spectrometry and Western blot. The purified Est2p/Tlc1 complex could specifically add nucleotides onto the single-stranded TG1-3 primer in a processive manner, but could not translocate to synthesize the second telomeric repeat. The purified core complex exhibited different activities when primers were paired with Tlc1 template at different positions. The procedure of reconstitution and purification of telomerase core enzyme we developed allows for further mechanistic study on the functions of other subunits of telomerase holoenzyme as well as other...