Catalysis-dependent inactivation of human telomerase and its reactivation by intracellular telomerase-activating factors (iTAFs)

Abstract

Edited by Xiao-Fan Wang Human telomerase maintains genome stability by adding telomeric repeats to the ends of linear chromosomes. Although previous studies have revealed profound insights into telomerase functions, the low cellular abundance of functional telomerase and the difficulties in quantifying its activity leave its thermodynamic and kinetic properties only partially characterized. Employing a stable cell line overexpressing both the human telomerase RNA component and the N-terminally biotinylated human telomerase reverse transcriptase and using a newly developed method to count individual extension products, we demonstrate here that human telomerase holoenzymes contain fast-and slow-acting catalytic sites. Surprisingly, both active sites became inactive after two consecutive rounds of catalysis, named single-run catalysis. The fast active sites turned off ϳ40fold quicker than the slow ones and exhibited higher affinities to DNA substrates. In a dimeric enzyme, the two active sites work in tandem, with the faster site functioning before the slower one, and in the monomeric enzyme, the active sites also perform single-run catalysis. Interestingly, inactive enzymes could be reactivated by intracellular telomerase-activating factors (iTAFs) from multiple cell types. We conclude that the single-run catalysis and the iTAFtriggered reactivation serve as an unprecedented control circuit for dynamic regulation of telomerase. They endow native telomerase holoenzymes with the ability to match their total number of active sites to the number of telomeres they extend. We propose that the exquisite kinetic control of telomerase activity may play important roles in both cell division and cell aging.

Soluble RAGE and skeletal muscle tissue RAGE expression profiles in lean and obese young adults across differential aerobic exercise intensities

“…Droplet digital PCR (BioRad) was used to quantify transcripts of RAGE, esRAGE, P65, and hypoxia inducible factor-1α (HIF-1α) as previously described ( 80 ). Primers were designed for each target using Roche’s universal probe library (UPL) assay design center (lifescience.roche.com).…”

Occurrence, functionality and abundance of the TERT promoter mutations

“…1,[31][32][33][34] With a limited amount of both the enzyme and substrates, telomerase extends telomeres in the late S-phase through stringent mechanisms "where any perturbation becomes causal for different telomere related diseases, insufficiency leading to stem cell and tissue failure syndromes and too much to cancer predisposition". [35][36][37][38][39][40][41] The recruitment and processivity of telomerase on telomeres are assisted by the shelterin components and terminated by the heterotrimeric CTC1-STN1-TEN1 (CST) complex, followed by a C-strand fill-in by DNA polymerase α-primase. 37,[42][43][44][45] Most human somatic tissues and adult stem cells do not express sufficient telomerase to maintain telomere length infinitely due to repression of the reverse transcriptase subunit upon differentiation in a histone deacetylase-dependent manner and through alternative splicing of TERT.…”

“…The ribonucleic protein telomerase, comprised of intricately interlocked catalytic reverse transcriptase subunit (TERT) and an RNA component (TERC) along with auxiliary elements including histone H2‐H2B dimer, counteracts telomere shortening to overcome the “end replication problem” and maintain genomic integrity in pluripotent stem cells, early embryonic tissues and cells that undergo divisions as a physiological requirement 1,31‐34 . With a limited amount of both the enzyme and substrates, telomerase extends telomeres in the late S‐phase through stringent mechanisms “where any perturbation becomes causal for different telomere related diseases, insufficiency leading to stem cell and tissue failure syndromes and too much to cancer predisposition” 35‐41 . The recruitment and processivity of telomerase on telomeres are assisted by the shelterin components and terminated by the heterotrimeric CTC1‐STN1‐TEN1 (CST) complex, followed by a C‐strand fill‐in by DNA polymerase α‐primase 37,42‐45 .…”

Occurrence, functionality, and abundance of theTERTpromoter mutations

“…The ribonucleic protein telomerase, comprised of intricately interlocked catalytic reverse transcriptase subunit (TERT) and an RNA component (TERC) along with auxiliary elements including histone H2-H2B dimer, counteracts telomere shortening to overcome the 'end replication problem' and maintain genomic integrity in pluripotent stem cells, early embryonic tissues, and cells that undergo divisions as a physiological requirement 1,[31][32][33][34] . With a limited amount of both the enzyme and substrates, telomerase extends telomeres in the late S-phase through stringent mechanisms "where any perturbation becomes causal for different telomere related diseases, insufficiency leading to stem cell and tissue failure syndromes and too much to cancer predisposition" [35][36][37][38][39][40][41] . The recruitment and processivity of telomerase on telomeres are assisted by the shelterin components and terminated by the heterotrimeric CTC1-STN1-TEN1 (CST) complex, followed by a Cstrand fill-in the engagement of DNA polymerase α-primase 37,[42][43][44][45] .…”