A unique pause pattern during telomere addition by the error-prone telomerase from the ciliate Paramecium tetraurelia

Telomeric DNA - the short, tandemly repeated sequences at the ends of chromosomes - is synthesized by telomerase, a ribonucleoprotein enzyme that copies a specific template sequence within its integral RNA moiety. The error-prone telomerase from the ciliate Paramecium tetraurelia stereotypically misincorporates TTP at telomerase RNA templating nucleotide C52, accounting for the 30% TTTGGG repeats randomly distributed in wild-type telomeres. Paramecium tetraurelia telomerase has been isolated from macronuclear extracts and characterized with respect to the extension of telomeric primers in vitro. Unlike telomerase activities from other species, the predominant pause during telomeric repeat synthesis by P. tetraurelia telomerase does not occur at the 5' end of the templating domain (templating nucleotide C49). Instead, the pause by P. tetraurelia telomerase is at templating nucleotide C53, immediately prior to incorporation of dGTP (or TTP) at C52. The configuration of the catalytic site at this template position during telomere synthesis is most likely responsible for the high incidence of misincorporation of TTP at C52. The gene for the P. tetraurelia telomerase catalytic subunit, telomerase reverse transcriptase (TERT), has been cloned and sequenced. A comparative analysis of the P. tetraurelia TERT with homologs from other species, including that from another Paramecium species that does not make a high percentage of misincorporation errors, has been initiated. This study may delineate those TERT structural elements that contribute to telomerase fidelity.