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

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  • Additional Information
    • Source:
      Publisher: Oxford University Press Country of Publication: United States NLM ID: 0374636 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1943-2631 (Electronic) Linking ISSN: 00166731 NLM ISO Abbreviation: Genetics Subsets: MEDLINE
    • Publication Information:
      Publication: 2021- : [Oxford] : Oxford University Press
      Original Publication: Austin, Tex. [etc.]
    • Subject Terms:
      Mutagenesis*; Protein Subunits/*genetics ; Telomerase/*genetics; Amino Acid Sequence ; Models, Biological ; Mutation ; Phenotype ; Proteasome Endopeptidase Complex/metabolism ; Protein Binding ; Proteolysis ; Telomerase/chemistry ; Telomere/genetics ; Telomere/metabolism ; Telomere-Binding Proteins/genetics ; Telomere-Binding Proteins/metabolism
    • Abstract:
      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.
      (Copyright © 2018 by the Genetics Society of America.)
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    • Grant Information:
      P30 CA014195 United States CA NCI NIH HHS; R01 AG011728 United States AG NIA NIH HHS; T32 GM007240 United States GM NIGMS NIH HHS
    • Contributed Indexing:
      Keywords: Est1; mutagenesis; separation-of-function; telomerase; telomere
    • Accession Number:
      0 (Protein Subunits)
      0 (Telomere-Binding Proteins)
      EC 2.7.7.49 (Telomerase)
      EC 3.4.25.1 (Proteasome Endopeptidase Complex)
    • Publication Date:
      Date Created: 20171201 Date Completed: 20181002 Latest Revision: 20201107
    • Publication Date:
      20221213
    • Accession Number:
      PMC5753878
    • Accession Number:
      10.1534/genetics.117.300145
    • Accession Number:
      29187507