Contributions to telomerase anchor-site and template/primer alignment functions by yeast TERT residue E76

Abstract

This project explores the synthesis of heterogeneous G1-3T yeast telomere sequences by the enzyme telomerase. I demonstrate that a mutant in the essential N-terminal TEN domain of Est2p (telomerase reverse transciptase), glutamic acid 76 to lysine (est2-LTE76K), restricted possible alignments between the DNA primer and the RNA template and increased in vivo processivity. Within the context of telomerase, the Est2p TEN domain is thought to contribute to enzyme processivity by mediating an anchor-site interaction with the DNA primer. I showed that binding of the purified TEN domain (residues 1-161) to telomeric DNA is enhanced by the E76K mutation. These results suggest a novel role for the anchor-site in mediating primer/template alignment within the active site of yeast telomerase.