Analysis of the Role of EST1 in Yeast Telomerase

Abstract

Abstract : My research proposal focuses on characterizing the roles of proteins that are responsible for mediating access of telomerase to the telomere in S. cerevisiae. I have previously shown that fusion of Cdcl3 to Estl results in substantial telomere elongation. Fusions consisting of mutant versions of Cdcl3 or Estl confer similar telomere elongation, indicating that close physical proximity can bypass telomerase-defective mutations in either protein. Fusing Cdcl3 directly to the catalytic core of telomerase (Est2) allows stable telomere maintenance in the absence of Estl. This study led to the proposal that Estl and Cdcl3 are co-mediators of telomerase access, and has further suggested that Estl may have a second role in telomere replication, based on the observation that the Cdcl3- Est2 fusion is unable to promote telomere elongation in the absence of Estl. Since Estl is a terminus-specific single-strand DNA binding protein, this second role for Estl may be to promote accessibility of the telomeric end to the telomerase active site. To explore this second role, I have isolated a set of alanine-scan mutations in Estl which retain association with telomerase but display telomere replication defects, and have tentatively identified an Estl mutant that is deficient in this second function.