Single-molecule real-time detection of telomerase extension activity

Sci Rep. 2014 Sep 29:4:6391. doi: 10.1038/srep06391.

Abstract

The ends of eukaryotic chromosomes are capped by telomeres which consist of tandem G-rich DNA repeats stabilized by the shelterin protein complex. Telomeres shorten progressively in most normal cells due to the end replication problem. In more than 85% of cancers however, the telomere length is maintained by telomerase; a reverse transcriptase that adds telomeric TTAGGG repeats using its integral RNA template. The strong association between telomerase activity and malignancy in many cancers suggests that telomerase activity could serve as a diagnostic marker. We demonstrate single-molecule, real-time telomerase extension activity observed digitally as the telomeric repeats are added to a substrate. The human telomerase complex pulled down from mammalian cells displays extension activity dependent on dNTP concentration. In complex with the processivity factor, POT1-TPP1, telomerase adds repeats at an accelerated rate and yields longer products. Our assay provides a unique detection platform that enables the study of telomerase kinetics with single molecule resolution.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosomes / genetics
  • DNA Replication / genetics
  • HEK293 Cells
  • Humans
  • Neoplasms / genetics*
  • Neoplasms / pathology
  • Serine Proteases / genetics*
  • Serine Proteases / metabolism
  • Shelterin Complex
  • Telomerase / biosynthesis*
  • Telomerase / genetics
  • Telomere / genetics
  • Telomere Homeostasis / genetics*
  • Telomere-Binding Proteins / genetics*
  • Telomere-Binding Proteins / metabolism

Substances

  • ACD protein, human
  • POT1 protein, human
  • Shelterin Complex
  • Telomere-Binding Proteins
  • Telomerase
  • Serine Proteases