Structural and Functional Characterization of a Telomerase-Associated Endonuclease
Final rept. 1 May 1999-30 Apr 2002
CORNELL UNIV MEDICAL COLL NEW YORK
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Telomerase is a ribonucleoprotein complex responsible for the maintenance of the terminal repeats of chromosomes. Specific activation of telomerase in most cancer cells confers these cells with unlimited replicative potential, and consequently promotes tumor growth. Telomerase is therefore an attractive target for anti-cancer therapeutics. Studies of telomerase in a variety of systems have revealed a tightly associated nuclease activity, whose physiologic function and molecular identity remain to be elucidated. We have recently shown that the telomerase complex from the budding yeast Saccharomyces cerevisiae also possesses a tightly associated nuclease activity. Consistent with results from other systems, we found that the yeast nuclease is single-strand specific and works via an endonucleolytic mechanism. In addition, the reverse transcriptase activity of telomerase can extend either the 5 or 3 fragment following cleavage. These results suggest that telomerase either possesses two distinct active sites for the nuclease and reverse transcriptase activity, or possesses two protomers, each containing a bi-functional active site. Careful analysis of a highly purified N-terminal domain of yeast TERT failed to reveal an autonomous nuclease activity. In addition, we identified a point mutation of TERT that exhibits a relative enhancement of the nuclease activity. This point mutation alters a residue in the vicinity of the reverse transcriptase active site. Taken together, these results suggest that the same active site of telomerase carries out both the polymerization and nucleolytic reaction.
- Medicine and Medical Research