Using separation-of-function mutagenesis to define the full spectrum of activities performed by the est1 telomerase subunit in vivo

Johnathan W. Lubin, Timothy M. Tucey, Victoria Lundblad

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4 Citations (Scopus)


A leading objective in biology is to identify the complete set of activities that each gene performs in vivo. In this study, we have asked whether a genetic approach can provide an efficient means of achieving this goal, through the identification and analysis of a comprehensive set of separation-of-function (sof-) mutations in a gene. Toward this goal, we have subjected the Saccharomyces cerevisiae EST1 gene, which encodes a regulatory subunit of telomerase, to intensive mutagenesis (with an average coverage of one mutation for every 4.5 residues), using strategies that eliminated those mutations that disrupted protein folding/stability. The resulting set of sof- mutations defined four biochemically distinct activities for the Est1 telomerase protein: two temporally separable steps in telomerase holoenzyme assembly, a telomerase recruitment activity, and a fourth newly discovered regulatory function. Although biochemically distinct, impairment of each of these four different activities nevertheless conferred a common phenotype (critically short telomeres) comparable to that of an est1-Δ null strain. This highlights the limitations of gene deletions, even for nonessential genes; we suggest that employing a representative set of sof- mutations for each gene in future high- and low-throughput investigations will provide deeper insights into how proteins interact inside the cell.

Original languageEnglish
Pages (from-to)97-110
Number of pages14
Issue number1
Publication statusPublished - 1 Jan 2018


  • Est1
  • Mutagenesis
  • Separation-of-function
  • Telomerase
  • Telomere

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