Mechanisms of nucleotide selection by telomerase

Abstract

Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.

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 .…”

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

“…Given the remarkably low IC 50 for 6-thio-dGTP, we examined the catalytic efficiency of 6-thio-dGTP incorporation versus dGTP using the Tribolium castaneum ( tc TERT) model of the telomerase catalytic core 42 . The ability to purify sufficient quantities of tc TERT enables characterization of the catalytic nucleotide addition by pre-steady-state single turnover kinetics using a defined DNA–RNA primer–template substrate 43 . The catalytic efficiency of incorporating a single dNTP is measured by dividing the observed nucleotide incorporation rate constant, k pol , by the equilibrium dissociation constant for dNTP binding to the tc TERT–primer–template complex ( K d ) 44 .…”

“… Incoming nucleotide k pol (s −1 ) K d (μM) Catalytic efficiency ( k pol / K d ) (μM −1 s −1 ) Fold change dGTP a 1.1 ± 0.03 18.1 ± 3.78 0.058 ± 0.012 1.000 6-thio-dGTP 3.1 ± 0.23 92.4 ± 12.94 0.034 ± 0.005 0.579 a Values from ref. 43 . …”

“…tc TERT was expressed and purified as described with some modifications 42 , 43 . An Epiphyte3 LEX bioreactor was used to grow tc TERT in BL-21(DE3) pLysS cells at 37 °C until they reached an OD 600 of 0.6–0.8, after which protein expression was induced with 1 mM isopropyl β- d -1-thiogalactopyranoside (IPTG) and the temperature was decreased to 30 °C for 4-5 hours of protein induction.…”