Analysis of yeast telomerase by primer extension assays

Abstract

Telomeres are specialized nucleoprotein structures located at eukaryotic chromosomal termini, which are required for chromosome stability and are maintained by a reverse transcriptase named telomerase. Budding yeast has served as an extremely useful model system for analyzing telomere maintenance because the organism offers a wide range of genetic and biochemical tools. Several milestones in telomerase research have been reached through investigation of the yeast system. For example, the consequence of telomerase loss was first characterized in the budding yeast Saccharomyces cerevisiae. The catalytic component of telomerase (telomerase reverse transcriptase; TERT) was likewise initially cloned from this organism. Moreover, much of the current understanding of the structure and function of the telomerase complex was derived from yeast studies. In this chapter, we discuss one of the most useful tools for investigating yeast telomerase mechanisms and regulation: the primer extension assay. This assay can be used to examine the overall activity as well as the processivity of telomerase, which represents a unique aspect of telomerase enzymology. It can also be employed to analyze the mechanisms of telomerase regulatory proteins.