Biochemical reconstitution and analysis of the telomerase holoenzyme in Saccharomyces cerevisiae.
Nancy Laterreur (1), Sherif Abou Elela (2), Raymund J. Wellinger (2)
(1) 3001, 12th avenue N., Sherbrooke, Qc, Canada, J1H 5N4;
(2) Microbiology and Infectiology, University of Sherbrooke, 3001, 12th avenue N, Sherbrooke, Qc, J1H 5N4, Canada
Telomeres, the ends of eukaryotic chromosomes, are essential structures for genome integrity and cell viability. Maintenance of both their length and structure is crucial and the telomerase enzyme is a key player in this role. Telomerase has a catalytic core that consists of a reverse transcriptase protein and an integral RNA component that serves as template for nucleotide addition by the telomerase onto G-rich strand of telomeres. For S. cerevisiae telomerase, Est2p is the catalytic subunit and TLC1 encodes the RNA component of the enzyme. These two are the only subunits required for in vitro telomerase activity. However, the proteins Est1p, Est3p and Cdc13p are also required to form an active telomerase holoenzyme in vivo. In this project, we intend to characterize and biochemically analyze the yeast telomerase holoenzyme. By using a combination of conventional and affinity-based chromatography, the holoenzyme will be purified to homogeneity in order to analyze the different interactions between the subunits (only known genetically for now). We also want to characterize the enzymatic properties of telomerase in order to get a better understanding of its action on telomere maintenance. Hopefully, we will get better insight into how the telomerase does its work and how its different known and unknown partners regulate it. The capacity to purify the holoenzyme will provide us with a tool to examine the mode of action of telomerase and analyze a variety of mutants.
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