title: Single-molecule real-time detection of telomerase extension activity.
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.
authors.affiliation: Bioengineering Department, University of Illinois.
authors.forename: Helen
authors.initials: H
authors.lastname: Hwang
authors.affiliation: Department of Environmental and Occupational Health, University of Pittsburgh.
authors.forename: Patricia
authors.initials: P
authors.lastname: Opresko
authors.affiliation: 1] Bioengineering Department, University of Illinois [2] Biophysics and Computational Biology, University of Illinois [3] Institute for Genomic Biology, University of Illinois [4] Physics Frontier Center (Center of Physics for Living Cells), University of Illinois.
authors.forename: Sua
authors.initials: S
authors.lastname: Myong
mesh_terms: D000818:Animals; D002875:Chromosomes / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D057057:Serine Proteases / Q000235:genetics* / Q000378:metabolism; D000089804:Shelterin Complex; D019098:Telomerase / Q000096:biosynthesis* / Q000235:genetics; D016615:Telomere / Q000235:genetics; D059505:Telomere Homeostasis / Q000235:genetics*; D034501:Telomere-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C495876:ACD protein, human; C440849:POT1 protein, human; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D019098:Telomerase; D057057:Serine Proteases