An N-terminal Flag-tag impairs TPP1 regulation of telomerase function

Biochem Biophys Res Commun. 2019 Apr 30;512(2):230-235. doi: 10.1016/j.bbrc.2019.03.050. Epub 2019 Mar 15.

Abstract

The shelterin protein complex protects natural chromosome ends from being recognized as DNA damage sites and also regulates the synthesis of telomeric repeats by telomerase. TPP1, a shelterin subunit that is essential for telomerase extension of telomeres, has been studied intensively in recent years. Many such studies utilize epitope tagged TPP1, but it is unclear how the tags may affect the multiple cellular functions of TPP1. Here we analyzed the effect of adding a 3x Flag epitope tag to the N- or C-terminus of TPP1. While the position of the tag did not affect TPP1's interaction within the shelterin complex or its localization to telomeres, the N-terminal Flag tag on TPP1 impaired telomerase function, resulting in reduced telomerase processivity in vitro and a failure to stimulate telomere elongation in vivo. The C-terminally Flag-tagged TPP1, in contrast, behaved similarly to untagged TPP1 in all functional aspects examined. These findings suggest that caution is required when utilizing epitope tagged TPP1 to study its regulation of telomerase function.

Keywords: Shelterin complex; Telomerase function; Telomere length maintenance; Telomeric binding protein TPP1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminopeptidases / analysis
  • Aminopeptidases / metabolism*
  • Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / analysis
  • Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / metabolism*
  • HCT116 Cells
  • HeLa Cells
  • Humans
  • Protein Interaction Mapping / methods*
  • Protein Interaction Maps
  • Serine Proteases / analysis
  • Serine Proteases / metabolism*
  • Telomerase / metabolism*
  • Telomere Homeostasis
  • Telomere-Binding Proteins / metabolism

Substances

  • Telomere-Binding Proteins
  • shelterin, human
  • Telomerase
  • Serine Proteases
  • Aminopeptidases
  • Dipeptidyl-Peptidases and Tripeptidyl-Peptidases
  • tripeptidyl-peptidase 1