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

ABSTRACT: 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 ?M, indicating super-telomerase is as catalytically active as endogenous telomerase and is thus a good model for biochemical studies.

SUBMITTER: Xi L

PROVIDER: S-EPMC4117779 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Xi Linghe L Cech Thomas R TR

Nucleic acids research 20140702 13

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 estima ...[more]

PMID: 24990373

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Project description:BackgroundTelomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated.Methodology/principal findingsTo understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function.Conclusions/significanceWe provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.

Dataset Information

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

Publications

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

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