IDENTIFICATION OF KLUYVEROMYCES-LACTIS TELOMERASE - DISCONTINUOUS SYNTHESIS ALONG THE 30-NUCLEOTIDE-LONG TEMPLATING DOMAIN

Telomeres in the budding yeast Kluyveromyces lactis consist of perfect ly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats fou nd in many other eukaryotes. Telomeric DNA is synthesized by the ribon ucleoprotein telomerase, which uses a portion of its RNA moiety as a t emplate. K, lactis telomerase RNA, encoded by the TER1 gene, is simila r to 1.3 kb long and contains a 30-nucleotide templating domain, the l argest ever examined. To examine the mechanism of polymerization by th is enzyme, we identified and analyzed telomerase activity from K, lact is whole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactis telomerase to exami ne primer elongation within one round of repeat synthesis. Under all i n vitro conditions tested, K, lactis telomerase catalyzed only one rou nd of repeat synthesis and remained bound to reaction products. We dem onstrate that K. lactis telomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the templa te. Increasing the amount of primer DNA-template RNA complementarity r esults in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymeriz ation through particular regions of the template. We suggest that thes e observations provide an insight into the mechanism of telomerase and its regulation.