Inventory of telomerase components in human cells reveals multiple subpopulations of hTR and hTERT

• Published in: Nucleic acids research 2014-07, Vol.42 (13), p.8565-8577
• Main Authors: Xi, Linghe, Cech, Thomas R
• Format: Article
• Language: English
• Subjects: Blotting, Western - methods; Cell Line; HEK293 Cells; HeLa Cells; Humans; Immunoprecipitation; Nucleic Acid Enzymes; RNA - metabolism; Telomerase - analysis; Telomerase - isolation & purification; Telomerase - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gku560

Telomerase is the ribonucleoprotein (RNP) enzyme that elongates telomeric DNA to compensate for the attrition occurring during each cycle of DNA replication. Knowing the levels of telomerase in continuously dividing cells is important for understanding how much telomerase is required for cell immortality. In this study, we measured the endogenous levels of the human telomerase RNP and its two key components, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). We estimate ∼ 240 telomerase monomers per cell for HEK 293T and HeLa, a number similar to that of telomeres in late S phase. The subunits were in excess of RNPs (e.g. ∼ 1150 hTR and ∼ 500 hTERT molecules per HeLa cell), suggesting the existence of unassembled components. This hypothesis was tested by overexpressing individual subunits, which increased total telomerase activity as measured by the direct enzyme assay. Thus, there are subpopulations of both hTR and hTERT not assembled into telomerase but capable of being recruited. We also determined the specific activity of endogenous telomerase and of overexpressed super-telomerase both to be ∼ 60 nt incorporated per telomerase per minute, with Km(dGTP) ∼ 17 μM, indicating super-telomerase is as catalytically active as endogenous telomerase and is thus a good model for biochemical studies.