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Single-molecule real-time detection of telomerase extension activity
Filetype[PDF - 1.27 MB]


Details:
  • Pubmed ID:
    25263700
  • Pubmed Central ID:
    PMC4178293
  • Funding:
    1F30CA174323-01/CA/NCI NIH HHS/United States
    343 NIH 1 DP2 GM105453/DP/NCCDPHP CDC HHS/United States
    F30 CA174323/CA/NCI NIH HHS/United States
    R01 ES022944/ES/NIEHS NIH HHS/United States
    R01ES022944/ES/NIEHS NIH HHS/United States
  • Document Type:
  • Collection(s):
  • Description:
    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.