Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomyces pombe

The chromosome end-replicating enzyme telomerase is composed of a template- containing RNA subunit, a reverse transcriptase (TERT), and additional prot eins. The importance of conserved amino acid residues in Trt1p. the TERT of Schizosaccharomyces pombe, was tested. Mutation to alanine of the proposed catalytic aspartates in reverse transcriptase motifs A and C and of conser ved amino acids in motifs 1 and B' resulted in defective growth, progressiv e loss of telomeric DNA, and loss of detectable telomerase enzymatic activi ty in vitro. Mutation of the phenylalanine (F) in the conserved FYxTE of te lomerase-specific motif T had no phenotype in vivo or in vitro whereas muta tion of a conserved amino acid in RT motif 2 had an intermediate effect. In addition to identifying single amino acids of TERT required for telomere m aintenance in the fission yeast, this work provides useful tools for S. pom be telomerase research: a functional epitope-tagged version of Trt1p that a llows detection of the protein even in crude cellular extracts, and a conve nient and robust in vitro enzymatic activity assay based on immunopurificat ion of telomerase.