Telomeres and Telomerase pp 97-106
Analysis of Yeast Telomerase by Primer Extension Assays
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 were reached through investigation of the yeast system. For example, the consequence of telomerase loss was first characterized in the budding yeast Saccharomyces cerevisiae (Lundblad and Szostak, Cell 57:633–643, 1989). The catalytic component of telomerase (telomerase reverse transcriptase; TERT) was likewise initially cloned from this organism (Lendvay et al., Genetics 144:1399–1412, 1996). Moreover, much of the current understanding of the structure and function of the telomerase complex was derived from yeast studies (Autexier and Lue, Annu Rev Biochem 75:493–517, 2006). 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 (Lue et al., Mol Cell Biol 23:8440–8449, 2003; Bosoy and Lue, Nucleic Acids Res 32:93–101, 2004). It can also be employed to analyze the mechanisms of telomerase regulatory proteins (Zappulla et al., Nucleic Acids Res 37:354–367, 2009; DeZwaan and Freeman, Proc Natl Acad Sci USA 106, 17337–17342, 2009).
Key wordsTelomerase DEAE chromatography IgG-Sepharose pull down Primer extension assay TRAP assay
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