Using Separation-of-Function Mutagenesis To Define the Full Spectrum of Activities Performed by the Est1 Telomerase Subunit in Vivo

Genetics: A Periodical Record of Investigations Bearing on Heredity and Variation

Johnathan W. Lubin^1¹  Timothy M. Tucey^1² 

[1] Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0130;Salk Institute for Biological Studies, La Jolla, California 92037-1099^1

关键词: Est1;    telomerase;    telomere;    separation-of-function;    mutagenesis;   

DOI  :  10.1534/genetics.117.300145

学科分类：医学（综合）

来源: Genetics Society of America

【 摘 要 】

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 -∆ 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.

【 授权许可】

CC BY