Identification of Kluyveromyces lactis telomerase: discontinuous synthesis along the 30-nucleotide-long templating domain.
Telomeres in the budding yeast Kluyveromyces lactis consist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactis telomerase RNA, encoded by the TER1 gene, is approximately 1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactis whole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactis telomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactis telomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactis telomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.
Fulton
T B
TB
Departments of Microbiology and Immunology & Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143-0414, USA.
Blackburn
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EH
D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics*; D012331:RNA, Fungal / Q000096:biosynthesis / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000235:genetics; D013698:Templates, Genetic
D017931:DNA Primers; D012331:RNA, Fungal; D019098:Telomerase