Template Requirements for Telomerase Translocation in Kluyveromyces lactis

Abstract

Telomeres are synthesized by telomerase, a specialized reverse transcriptase, which contains a template in its intrinsic RNA component. In Kluyveromyces lactis, the repeats synthesized by the wild-type telomerase are 25 nucleotides (nt) in length and uniform in sequence. To determine the role of the 5-nt repeats defining the ends of the K. lactis telomerase RNA template in telomerase translocation, we have made mutations in and around them and observed their effects on telomere length and the sequence of newly made telomeric repeats. These template mutations typically result in telomeres that are shorter than those of wild-type cells. The mismatches between the telomerase template and the telomeric tip that occur after telomerase-mediated incorporation of the mutations are normally not removed. Instead, the mutations lead to the synthesis of aberrant repeats that range in size from 31 to 13 bp. Therefore, the specificity with which the telomeric tip aligns with the telomere is critical for the production of the uniform repeats seen in K. lactis. In addition, the region immediately 3′ of the template may play an important role in translocation of the enzyme.

This work was supported by the American Cancer Society, grants RPG-00-082-01 GMC and RPG-02-253-01 GMC, and NIH GM61645. D.H.U. was supported by an NIH training grant and University of Georgia fellowships.

We thank Jennifer Caffarella, Will McRae, Shobhana Natarajan, Myia Thomas, and Amin Hajiani for help in constructing and using the telomere rescue plasmids.