RECONSTITUTION OF HUMAN TELOMERASE ACTIVITY IN-VITRO

Telomerase is a ribonucleoprotein enzyme complex that adds single-stra nded telomere DNA to chromosome ends [1]. The RNA component of telomer ase contains the template for telomeric DNA addition and is essential for activity [1,2]. Telomerase proteins have been identified in ciliat es, yeast and mammals [3-12]. In Saccharomyces cerevisiae, the Est2 pr otein is homologous to the 123 kDa reverse transcriptase subunit of Eu plotes telomerase, and is essential for telomerase activity [8]. In hu mans, telomerase activity is associated with the telomerase RNA hTR [1 3], the telomerase RNA-binding protein TP1/TLP1 [5,12] and the TP2 pro tein encoded by the human EST2 homolog [12] (also known as TRT1, hEST2 or TCS1 [9-11]). The minimal complex sufficient for activity is, howe ver, unknown. We have reconstituted human telomerase activity in retic ulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro. Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10-159 of hTR were sufficient f or telomerase activity. Telomerase activity immunoprecipitated from th e reticulocyte lysate contained hTR and recombinant TP2, Substitution of conserved amino acid residues in the reverse transcriptase domain o f TP2 completely abolished telomerase activity. We suggest that TP2 an d hTR might represent the minimal catalytic core of human telomerase. (C) Current Biology Ltd.