Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast.
Liao XH, Zhang ML, Yang CP, Xu LX, Zhou JQ
Biochemical Journal (2005)
Category: telomerase ¤ Added: Apr 12, 2005 ¤ Rating: ◊◊
Telomerase is a cellular reverse transcriptase that elongates the single-stranded chromosome ends and oligonucleotides in vivo and in vitro. In Saccharomyces cerevisiae, Est2p (telomerase catalytic subunit) and Tlc1 (telomerase RNA template subunit) constitute the telomerase core complex. We co-overexpressed GST-Est2p and Tlc1 in Saccharomyces cerevisiae, and reconstituted the telomerase activity. The GST-Est2p/Tlc1 complex was partially purified by ammonium sulfate fraction and affinity chromatography on Glutathione beads, and the partially purified telomerase didn't contain the other two subunits of telomerase holoenzyme, Est1p and Est3p. The purified recombinant GST-Est2p/Tlc1 telomerase core complex could specifically add nucleotides onto the single-stranded TG 1-3 primer in a processive manner, but could not translocate to synthesize more than one telomeric repeat. The purified telomerase 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 now allows for further mechanistic study on the functions of other subunits of telomerase holoenzyme as well as other telomerase regulation proteins.
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